BIOPLEX 2200 SYPHILIS IGG KIT ON THE BIOPLEX 2200 MULTI-ANALYTE DETECTION SYSTEM
Device Facts
| Record ID | K063866 |
|---|---|
| Device Name | BIOPLEX 2200 SYPHILIS IGG KIT ON THE BIOPLEX 2200 MULTI-ANALYTE DETECTION SYSTEM |
| Applicant | Bio-Rad Laboratories |
| Product Code | LIP · Microbiology |
| Decision Date | Mar 19, 2007 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3830 |
| Device Class | Class 2 |
Intended Use
The Bio-Rad Syphilis IgG kit is a multiplex flow immunoassay intended for the qualitative detection of Treponema pallidum IgG antibodies in human serum. This test system is also indicated for use for confirming reactive test results from non-treponemal based screening assays.
Device Story
The BioPlex 2200 Syphilis IgG kit is a multiplex flow immunoassay used on the BioPlex 2200 System. It utilizes three populations of dyed beads coated with recombinant T. pallidum proteins (15kD, 17kD, 47kD). The system mixes patient serum, diluent, and beads in a reaction vessel; after incubation and washing, phycoerythrin-conjugated anti-human IgG is added. The system detects bead identity via dye fluorescence and antibody binding via PE fluorescence, outputting results as relative fluorescence intensity (RFI) and antibody index (AI). It includes internal standard, serum verification, and reagent blank beads for quality control. Operated by laboratory personnel, the system provides automated, simultaneous detection of multiple antibodies. Results assist clinicians in diagnosing syphilis when used alongside non-treponemal assays and for confirming reactive screening tests. It benefits patients by providing rapid, multiplexed serological evidence of T. pallidum infection.
Clinical Evidence
Clinical performance evaluated using 1,750 serum samples across various populations (pregnant women, patients with syphilis orders, known HIV-positive, and CDC-characterized samples). Sensitivity and specificity were compared against reference methods (RPR, TPPA, and treponemal EIA). In the CDC panel (N=140), clinical sensitivity was 91.4%. Agreement with reference assays in clinical populations was high, with positive agreement ranging from 93.8% to 100% depending on the cohort.
Technological Characteristics
Multiplex flow immunoassay using fluoromagnetic beads coated with recombinant T. pallidum antigens (15kD, 17kD, 47kD). Energy source: two-laser excitation. Connectivity: standalone, automated analyzer. Software: BioPlex 2200 System Software v2.0. Calibration: 30-day stability, lot-specific value assignment via CD-ROM. Controls: Internal Standard, Serum Verification, and Reagent Blank beads.
Indications for Use
Indicated for qualitative detection of Treponema pallidum IgG antibodies in human serum to provide serological evidence of infection or confirm reactive non-treponemal screening results. Not for blood or plasma donor screening. Positive results may indicate prior treated infection; negative results may not exclude early primary or incubating disease.
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
- Trep-Check Anti-Treponema IgG EIA (k001552)
Related Devices
- K170413 — BioPlex 2200 Syphilis Total & RPR, BioPlex 2200 Syphilis Total & RPR Calibrator Set, BioPlex 2200 Syphilis Total & RPR Control Set · Bio-Rad Laboratories · May 11, 2017
- K093837 — ATHENA MULTI-LYTE TREPONEMA PALLIDUM IGG PLUS TEST SYSTEM · Zeus Scientific, Inc. (Zeus) · Oct 1, 2010
- K120439 — BIOPLEX 2200 EBV IGG AND SYPHILIS IGG · Bio-Rad Laboratories, Inc. · Mar 14, 2012
Submission Summary (Full Text)
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K063866
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MAR 1 9 2007
# BIOPLEX 2200 SYPHILIS IgG 510(k) SUMMARY
This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.
| 510(k) Number | 510(k) Summary Report Date |
|---------------|----------------------------|
| K063866 | March 8, 2007 |
# MANUFACTURER INFORMATION
| Manufacturer | |
|-----------------------------------------------------|----------------------------------------------------------------------------------------------------------|
| Manufacturer Address | Bio-Rad Laboratories, Inc.<br>Clinical Systems Division<br>4000 Alfred Nobel Drive<br>Hercules, CA 94547 |
| Telephone | (510) 724-7000 |
| Establishment Registration No. | 2915274 |
| Owner / Operator | Bio-Rad Laboratories, Inc.<br>4000 Alfred Nobel Drive<br>Hercules, CA 94547 |
| Owner / Operator No. | 9929003 |
| Official Correspondent for the BioPlex 2200 EBV IgG | |
| Official Correspondent Address | Bio-Rad Laboratories<br>6565 185th Ave NE<br>Redmond, WA 98052 |
| Telephone | 425-881-8300 |
| Establishment Registration No. | 3022521 |
| Owner / Operator | Bio-Rad Laboratories<br>6565 185th Ave NE<br>Redmond, WA 98052 |
| Official Correspondent | Mr. Christopher Bentsen |
| Telephone | (425) 498-1709 |
| Fax | (425) 498-1651 |
# CLASSIFICATION INFORMATION
| Classification Name | Treponema Pallidum Treponemal Test Reagents |
|----------------------|---------------------------------------------------------------------------------|
| Common Name: | Multi-Analyte Detection System Syphilis IgG |
| Product Trade Name | BioPlex 2200 Syphilis IgG on the BioPlex 2200 Multi-Analyte<br>Detection System |
| Device Class | Class II |
| Classification Panel | Microbiology |
| Regulation Number | 866.3830 |
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## LEGALLY MARKETED EQUIVALENT (SE) DEVICES
| Comparative FDA Cleared<br>PREDICATE DEVICE | 510(k) Number | Decision<br>Date |
|--------------------------------------------------------|---------------|------------------|
| Macro-Vue RPR Card (Becton Dickenson) | Pre-Amendment | N/A |
| Serodia TPPA (Fujirebio Inc.) | 971502 | 11/13/1997 |
| Trep-Chek Anti-Treponemal EIA (Phoenix Bio-Tech Corp.) | 001552 | 10/19/2000 |
## DEVICE DESCRIPTION
The Syphilis IgG kit uses multiplex flow immunoassay, a methodology that greatly resembles traditional EIA, but permits simultaneous detection and identification of many antibodies in a single tube.19 Three (3) different populations of dyed beads are coated with recombinant proteins associated with T. pallidum (15kD, 17kD and 47kD). 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°C. After a wash cycle, anti-human IgG antibody, conjugated to phycoerythrin (PE), is added to the dyed beads, and this mixture is incubated at 37°C. 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 of the dyes, and the amount of antibody captured by the antigen is determined by the fluorescence of the attached PE. Raw data is calculated in relative fluorescence intensity (RFI).
Three additional dyed beads, Internal Standard Bead (ISB), Serum Verification Bead (SVB) and a Reagent Blank Bead (RBB) are present in each reaction mixture to verify detector response, the addition of serum or plasma to the reaction vessel and the absence of significant non-specific binding in serum or plasma. Refer to the BioPlex 2200 System Operation Manual for more information.
The system is calibrated using a set of four (4) distinct calibrators vials, supplied separately by Bio-Rad Laboratories. Four (4) vials representing two (2) or three (3) different antibody concentrations are used for calibration. Results are calculated for each of the three (3) antibodies and are compared against their own respective cut-off and are expressed as an antibody index (AI). A single result is reported after completing a composite analysis of all the antibodies (the highest Al value is reported).
# KIT COMPONENTS
Syphilis IgG (665-1450). The reagent pack contains supplies sufficient for 100 tests.
| Vial | Description |
|----------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Bead Set | One (1) 10 mL vial, containing dyed beads coated with affinity-purified E.<br>coli derived recombinant proteins to T. pallidum (15kD, 17kD, and 47kD),<br>an Internal Standard (ISB), a Serum Verification (SVB), and a Reagent<br>Blank (RBB), with Glycerol and protein stabilizers (bovine) in a MOPS (3-<br>[NMorpholino] propanesulfonic acid) buffer. ProClin® 300 (0.3%) and sodium<br>azide (<0.1%) as preservatives. |
| Conjugate | One (1) 5 mL vial, containing murine monoclonal anti-human<br>IgG/phycoerythrin conjugate and anti-human FXIII/phycoerythrin conjugate,<br>with protein stabilizers (bovine) in a phosphate buffer. ProClin® 300 (0.3%)<br>and sodium azide (<0.1%) as preservatives. |
| Sample Diluent | One (1) 10 mL vial, containing protein stabilizers (bovine and murine) in a<br>triethanolamine buffer. ProClin® 300 (0.3%) and sodium azide (<0.1%) as<br>preservatives. |
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### ADDITIONAL REQUIRED ITEMS, AVAILABLE FROM BIO-RAD
| Catalog # | Description |
|-----------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 663-1400 | BioPlex 2200 Syphilis IgG Calibrator Set: Four (4) 500 µL vials, containing<br><i>T. pallidum</i> (15kD, 17kD, and 47kD) antibodies in a human serum matrix<br>made from defibrinated plasma. ProClin® 300 (0.3%) as a preservative for<br>all calibrators. |
| 663-1430 | BioPlex 2200 Syphilis IgG Control Set: Two (2) 1.5 mL vials of Positive<br>Control containing antibodies to <i>T. pallidum</i> 17kD and two (2) 1.5 mL vials<br>of Positive Control containing antibodies to <i>T. pallidum</i> 15kD and 47kD in a<br>human serum matrix made from defibrinated plasma, and two (2) 1.5 mL<br>vials of Negative Control in a human serum matrix made from defibrinated<br>plasma. ProClin® 300 (0.3%) as a preservative for all controls. |
| 660-0817 | BioPlex 2200 System Sheath Fluid: Two (2) 4 L bottles containing<br>Phosphate Buffered Saline (PBS). ProClin® 300 (0.3%) and Sodium azide<br>(0.1%) as preservatives. |
| 660-0818 | BioPlex 2200 System Wash Solution: One (1) 10 L bottle containing<br>Phosphate Buffered Saline (PBS) and Tween 20. ProClin® 300 (0.3%) and<br>Sodium azide (0.1%) as preservatives. |
| 660-0000 | BioPlex 2200 Instrument and Software. |
#### INTENDED USE / INDICATIONS FOR USE
The Bio-Rad Syphilis IgG kit is a multiplex flow immunoassay intended for the qualitative detection of Treponema pallidum IgG antibodies in human serum. The test system, when used in conjunction with non-treponemal based assays, provides serological evidence of infection with T. pallidum. This test system is also indicated for use in confirming reactive test results from nontreponemal based screening assays.
The Syphilis IgG kit is intended for use with the Bio-Rad BioPlex 2200 System.
The BioPlex 2200 Syphilis IgG kit is not intended for use in screening blood or plasma donors.
Warning: A positive result is not useful for establishing a diagnosis of Syphilis. In most situations, such a result may reflect prior treated infection; a negative result can exclude a diagnosis of syphilis except for incubating or early primary disease.
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# TECHNOLOGICAL CHARACTERISTICS
The following tables summarize similarities and differences between the BioPlex 2200 Syphilis IgG Kit and the predicate devices used in comparative studies with the BioPlex 2200 Syphilis IgG Kit.
# A. BioPlex 2200 EBV IgG Assay vs. Predicate RPR Test
Table 1: Similarities between reagents and materials
| Similarities between<br>Components / Materials | BioPlex 2200 Syphilis IgG Kit | Predicate RPR Test |
|------------------------------------------------|-------------------------------------------------------------------------------------------------|-------------------------------------------------------------------|
| Reagents | The Bead Set reagent containing<br>dyed antigen coated beads reacts with<br>the patient sample. | The Antigen Suspension reagent<br>reacts with the patient sample. |
Table 2: Similarities between reagents with regard to function and use
| Similarities between<br>Function and Use | BioPlex 2200 Syphilis IgG Kit | Predicate RPR Test |
|------------------------------------------|----------------------------------|----------------------------------|
| Intended Use | Aid in the diagnosis of Syphilis | Aid in the diagnosis of Syphilis |
Table 3: Differences between reagents and materials
| Differences between<br>Components / Materials | BioPlex 2200 Syphilis IgG Kit | Predicate RPR Test |
|-----------------------------------------------|--------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------|
| Solid Phase | Bead Set reagent - dyed antigen<br>coated beads. | None. |
| Reagents | Conjugate, Sample Diluent, Wash<br>Buffer. | Antigen Suspension. |
| Sheath Fluid | Sheath Fluid is used to suspend the<br>bead reagent and introduce it into the<br>detector. | Not similar; not utilized in RPR<br>tests. |
| Calibrators | Calibrators. | None. |
| Control | Negative Control and multi-analyte<br>Positive Control. | User provided controls with<br>established patterns of graded<br>activity (optional Control Card Sets<br>available) |
Table 4: Differences between reagents with regard to function and use
| Differences between<br>Function and Use | BioPlex 2200 Syphilis IgG Kit | Predicate RPR Test |
|-----------------------------------------|------------------------------------------------------------------------------------------|------------------------------------------------------------------------------|
| Analyte Detection | Multi-analyte qualitative detection of<br>human IgG antibodies to Treponema<br>pallidum. | Non-treponemal qualitative and<br>quantitative detection of human<br>reagin. |
| Matrices | Serum. | Serum and Plasma. |
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# B. BioPlex 2200 EBV IgG Assay vs. Predicate TPPA Test
## Table 5: Similarities between reagents and materials
| Similarities between<br>Components / Materials | BioPlex 2200 Syphilis IgG Kit | Predicate TPPA |
|------------------------------------------------|--------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------|
| Reagents | Sample Diluent | Sample Diluent |
| Controls | Negative Control and multi-analyte<br>Positive Control | Non-reactive Control Serum and<br>Positive Control Serum (containing<br>rabbit antibodies to Treponema<br>pallidum) |
Table 6: Similarities between reagents with regard to function and use
| Similarities between<br>Function and Use | BioPlex 2200 Syphilis IgG Kit | Predicate TPPA |
|------------------------------------------|----------------------------------|----------------------------------|
| Intended Use | Aid in the diagnosis of Syphilis | Aid in the diagnosis of Syphilis |
Table 7: Differences between reagents and materials
| Differences between<br>Components / Materials | BioPlex 2200 Syphilis IgG Kit | Predicate TPPA |
|-----------------------------------------------|--------------------------------------------------------------------------------------------|--------------------------------------------------|
| Solid Phase | Bead reagent - dyed antigen coated<br>beads. | None. |
| Reagents | Conjugate, Wash Buffer. | Sensitized Particles, Unsensitized<br>Particles. |
| Sheath Fluid | Sheath Fluid is used to suspend the<br>bead reagent and introduce it into the<br>detector. | Not similar; not utilized in TPPA<br>tests. |
| Calibrators | Calibrators. | None. |
Table 8: Differences between reagents with regard to function and use
| Differences between<br>Function and Use | BioPlex 2200 Syphilis IgG Kit | Predicate TPPA |
|-----------------------------------------|------------------------------------------------------------------------------|---------------------------------------------------------|
| Analyte Detection | Multi-analyte detection of human<br>IgG antibodies to Treponema<br>pallidum. | Detection of human antibodies to<br>Treponema pallidum. |
| Matrices | Serum. | Serum and Plasma. |
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# C. BioPlex 2200 EBV IgG Assay vs. Predicate EIA Test
| Similarities between<br>Components / Materials | BioPlex 2200 Syphilis IgG Kit | Predicate EIA |
|------------------------------------------------|-----------------------------------------------------|---------------------------------------|
| Reagents | Wash Buffer, Sample Diluent | Sample Diluent, Wash Buffer |
| Calibrator(s) | Calibrators | Calibrator |
| Controls | Negative Control and multi-analyte Positive Control | Negative Control and Positive Control |
# Table 9: Similarities between reagents and materials
Table 10: Similarities between reagents with regard to function and use
| Similarities between<br>Function and Use | BioPlex 2200 Syphilis IgG Kit | Predicate EIA |
|------------------------------------------|-------------------------------------------------------------------|-------------------------------------------------------------------|
| Intended Use | Qualitative detection of IgG<br>antibodies to Treponema pallidum. | Qualitative detection of IgG<br>antibodies to Treponema pallidum. |
#### Table 11: Differences between reagents and materials
| Differences between<br>Components / Materials | BioPlex 2200 Syphilis IgG Kit | Predicate EIA |
|-----------------------------------------------|--------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------|
| Solid Phase | Bead reagent - dyed antigen coated<br>beads. | 96 well microplate - antigen coated<br>microwells. |
| Reagents | Conjugate: Anti-human IgG /<br>Phycoerythrin. | Conjugate: goat anti-human IgG<br>horseradish peroxidase, Substrate<br>(TMB), Stop Solution. |
| Sheath Fluid | Sheath Fluid is used to suspend the<br>bead reagent and introduce it into the<br>detector. | Not similar; not utilized in EIA's. |
# Table 12: Differences between reagents with regard to function and use
| Differences between<br>Function and Use | BioPlex 2200 Syphilis IgG Kit | Predicate EIA |
|-----------------------------------------|------------------------------------------------------------------------------|-------------------------------------------------------------|
| Analyte Detection | Multi-analyte detection of human<br>IgG antibodies to Treponema<br>pallidum. | Detection of human IgG antibodies<br>to Treponema pallidum. |
| Matrices | Serum. | Serum and Plasma. |
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## PERFORMANCE SUMMARY
## A. Expected Values
#### Prevalence
Expected values for the Syphilis IgG kit are presented by age and gender in the following tables for serum samples from unselected hospitalized patients (N=300), serum samples from patients who had a syphilis test ordered (N=500), and serum samples from unselected pregnant women (N=497). For all analytes, results of ≤0.8 Ai are nonreactive, 0.9 and 1.0 Al are equivocal, and ≥1.1 Al are reported as reactive.
| Age | Gender | Reactive | | Equivocal | | Nonreactive | | Total |
|--------------------|--------|----------|-------|-----------|------|-------------|-------|-------|
| | | N | % | N | % | N | % | N |
| 0-9 years of age | F | 0 | 0.0% | 0 | 0.0% | 0 | 0.0% | 0 |
| | M | 0 | 0.0% | 0 | 0.0% | 1 | 100% | 1 |
| 10-19 years of age | F | 0 | 0.0% | 0 | 0.0% | 3 | 100% | 3 |
| | M | 0 | 0.0% | 0 | 0.0% | 9 | 100% | 9 |
| 20-29 years of age | F | 1 | 5.3% | 0 | 0.0% | 18 | 94.7% | 19 |
| | M | 0 | 0.0% | 0 | 0.0% | 16 | 100% | 16 |
| 30-39 years of age | F | 1 | 8.3% | 0 | 0.0% | 11 | 91.7% | 12 |
| | M | 3 | 10.0% | 0 | 0.0% | 27 | 90.0% | 30 |
| 40-49 years of age | F | 3 | 10.7% | 0 | 0.0% | 25 | 89.3% | 28 |
| | M | 4 | 9.3% | 0 | 0.0% | 39 | 90.7% | 43 |
| 50-59 years of age | F | 2 | 7.4% | 0 | 0.0% | 25 | 92.6% | 27 |
| | M | 7 | 16.3% | 0 | 0.0% | 36 | 83.7% | 43 |
| 60-69 years of age | F | 1 | 8.3% | 0 | 0.0% | 11 | 91.7% | 12 |
| | M | 4 | 19.0% | 0 | 0.0% | 17 | 81.0% | 21 |
| 70-79 years of age | F | 4 | 36.4% | 0 | 0.0% | 7 | 63.6% | 11 |
| | M | 2 | 18.2% | 0 | 0.0% | 9 | 81.8% | 11 |
| 80-89 years of age | F | 0 | 0.0% | 0 | 0.0% | 6 | 100% | 6 |
| | M | 0 | 0.0% | 0 | 0.0% | 3 | 100% | 3 |
| > 89 years of age | F | 1 | 25.0% | 0 | 0.0% | 3 | 75.0% | 4 |
| | M | 0 | 0.0% | 0 | 0.0% | 1 | 100% | 1 |
| Total | | 33 | 11.0% | 0 | 0.0% | 267 | 89.0% | 300 |
Table 13. Samples From Unselected Hospitalized Patients (N=300)
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| Age | Gender | Reactive | | Equivocal | | Nonreactive | | Total |
|--------------------|--------|----------|-------|-----------|------|-------------|-------|-------|
| | | N | % | N | % | N | % | N |
| < 10 years of age | F | 0 | 0.0% | 0 | 0.0% | 0 | 0.0% | 0 |
| < 10 years of age | M | 0 | 0.0% | 0 | 0.0% | 1 | 100% | 1 |
| 10-19 years of age | F | 0 | 0.0% | 0 | 0.0% | 31 | 100% | 31 |
| 10-19 years of age | M | 0 | 0.0% | 0 | 0.0% | 1 | 100% | 1 |
| 20-29 years of age | F | 3 | 4.0% | 0 | 0.0% | 72 | 96.0% | 75 |
| 20-29 years of age | M | 4 | 14.8% | 0 | 0.0% | 23 | 85.2% | 27 |
| 30-39 years of age | F | 3 | 6.8% | 0 | 0.0% | 41 | 93.2% | 44 |
| 30-39 years of age | M | 5 | 10.9% | 2 | 4.3% | 39 | 84.8% | 46 |
| 40-49 years of age | F | 3 | 10.3% | 0 | 0.0% | 26 | 89.7% | 29 |
| 40-49 years of age | M | 23 | 21.5% | 0 | 0.0% | 84 | 78.5% | 107 |
| 50-59 years of age | F | 3 | 10.0% | 0 | 0.0% | 27 | 90.0% | 30 |
| 50-59 years of age | M | 18 | 26.5% | 1 | 1.5% | 49 | 72.1% | 68 |
| 60-69 years of age | F | 2 | 14.3% | 0 | 0.0% | 12 | 85.7% | 14 |
| 60-69 years of age | M | 3 | 18.8% | 0 | 0.0% | 13 | 81.3% | 16 |
| 70-79 years of age | F | 1 | 33.3% | 0 | 0.0% | 2 | 66.7% | 3 |
| 70-79 years of age | M | 3 | 50.0% | 0 | 0.0% | 3 | 50.0% | 6 |
| 80-89 years of age | F | 0 | 0.0% | 0 | 0.0% | 1 | 100% | 1 |
| 80-89 years of age | M | 0 | 0.0% | 0 | 0.0% | 1 | 100% | 1 |
| Total | | 71 | 14.2% | 3 | 0.6% | 426 | 85.2% | 500 |
Table 14. Serum Samples From Patients Who Had a Syphilis Test Ordered (N=500)
Table 15. Serum Samples From Unselected Pregnant Women (N=497)
| Age | Reactive | | Equivocal | | Nonreactive | | Total |
|--------------------|----------|------|-----------|------|-------------|-------|-------|
| | N | % | N | % | N | % | |
| 10-19 years of age | 0 | 0.0% | 0 | 0.0% | 42 | 100% | 42 |
| 20-29 years of age | 3 | 1.4% | 0 | 0.0% | 208 | 98.6% | 211 |
| 30-39 years of age | 3 | 1.5% | 0 | 0.0% | 193 | 98.5% | 196 |
| 40-49 years of age | 0 | 0.0% | 0 | 0.0% | 47 | 100% | 47 |
| Unknown | 0 | 0.0% | 0 | 0.0% | 1 | 100% | 1 |
| Total | 6 | 1.2% | 0 | 0.0% | 491 | 98.8% | 497 |
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### B. Reproducibility Studies
A reproducibility panel, consisting of seven (7) panel members for each of the three (3) recombinant proteins associated with T. pallidum (15kD, 17kD and 47kD) was prepared by Bio-Rad Laboratories. Two (2) of the seven (7) members for each analyte had high positive levels of antibodies, and two (2) had positive antibody levels near the cutoff, additionally, there were two (2) high negative panel members and one (1) low negative panel member for each analyte. In addition, one positive control (antibody positive for 15kD and 47kD), one positive control antibody positive for 17kD) and a negative control (antibody negative for all analytes) were also tested. Reproducibility testing was performed at each of three (3) US testing facilities on a total of three (3) lots of the Syphilis IgG kit, three (3) lots of the Syphilis IgG Calibrator Set, and three (3) lots of the Syphilis IgG Control Set. Each testing facility evaluated reproducibility using one (1) kit lot of the Syphilis IgG kit with matched calibrators and controls. The panels were provided to each of the testing sites. Each of the seven (7) panel members and positive and negative controls was tested in duplicate (x2) on two (2) runs per day for three (3) days at each of three (3) US testing facilities using one (1) lot of BioPlex 2200 Syphilis IgG Reagent Pack and one (1) lot of Syphilis IgG Calibrator Set (2 times x 2 runs x 3 days x 3 sites = 36 replicates per panel member and controls). The data were analyzed for intra-assay and inter-assay reproducibility according to the principles described in the Clinical Laboratory Standards Institute guidance EP5-A2, revised November 2004 and ISO/TR 22971:2205. The standard deviation (SD) and percent coefficient of variation (%CV) were calculated. Positive results can be found in Table 16.
| | Syphilis IgG<br>Panel Members | Sample<br>N | Grand<br>Mean<br>AI | Within-Run | | Between-Run | | Between-Day | | Between-Site* | | Total | |
|---------------------------------|-------------------------------|-------------|---------------------|------------|------|-------------|------|-------------|-------|---------------|-------|-------|-------|
| | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| T. pallidum<br>recombinant 15kD | High Positive 1 | 36 | 3.0 | 0.1 | 3.3% | 0.1 | 4.4% | 0.1 | 2.3% | 0.3 | 10.4% | 0.4 | 12.0% |
| | High Positive 2 | 36 | 3.3 | 0.1 | 2.2% | 0.1 | 2.4% | 0.2 | 4.7% | 0.2 | 7.2% | 0.3 | 9.2% |
| | Low Positive 1 | 36 | 1.4 | 0.0 | 3.0% | 0.1 | 4.7% | 0.1 | 10.1% | 0.2 | 15.5% | 0.3 | 19.3% |
| | Low Positive 2 | 36 | 1.3 | 0.0 | 2.2% | 0.0 | 3.6% | 0.0 | 3.1% | 0.2 | 14.0% | 0.2 | 15.0% |
| | Positive Control | 36 | 2.2 | 0.1 | 2.9% | 0.1 | 2.2% | 0.0 | 0.0% | 0.5 | 23.3% | 0.5 | 23.6% |
| T. pallidum<br>recombinant 17kD | High Positive 1 | 36 | 3.2 | 0.1 | 3.1% | 0.2 | 4.8% | 0.0 | 0.0% | 0.3 | 7.9% | 0.3 | 9.7% |
| | High Positive 2 | 36 | 2.8 | 0.1 | 2.8% | 0.1 | 4.2% | 0.1 | 2.5% | 0.2 | 8.2% | 0.3 | 9.9% |
| | Low Positive 1 | 36 | 1.3 | 0.0 | 3.7% | 0.0 | 1.9% | 0.1 | 5.5% | 0.1 | 6.2% | 0.1 | 9.3% |
| | Low Positive 2 | 36 | 1.3 | 0.0 | 2.5% | 0.0 | 3.7% | 0.1 | 6.0% | 0.1 | 5.6% | 0.1 | 9.4% |
| | Positive Control | 36 | 2.3 | 0.1 | 3.2% | 0.0 | 1.5% | 0.1 | 3.5% | 0.3 | 13.3% | 0.3 | 14.2% |
| T. pallidum<br>recombinant 47kD | High Positive 1 | 36 | 3.8 | 0.1 | 2.3% | 0.1 | 1.9% | 0.2 | 5.2% | 0.3 | 7.8% | 0.4 | 9.9% |
| | High Positive 2 | 36 | 3.6 | 0.1 | 2.4% | 0.1 | 2.3% | 0.0 | 0.0% | 0.3 | 7.5% | 0.3 | 8.2% |
| | Low Positive 1 | 36 | 1.2 | 0.0 | 3.8% | 0.0 | 2.4% | 0.1 | 5.6% | 0.1 | 11.1% | 0.2 | 13.2% |
| | Low Positive 2 | 36 | 1.3 | 0.0 | 2.9% | 0.0 | 3.2% | 0.1 | 4.1% | 0.2 | 11.9% | 0.2 | 13.3% |
| | Positive Control | 36 | 2.3 | 0.1 | 4.0% | 0.0 | 0.0% | 0.0 | 0.0% | 0.5 | 19.5% | 0.5 | 19.9% |
Table 16. Reproducibility: BioPlex 2200 Syphilis IgG
*Between site variance includes between lot variance.
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# C. Precision Studies
A precision panel, consisting of nine (9) panel members for each of the three (3) recombinant proteins associated with T. pallidum 5kD, 17kD and 47kD) was prepared by Bio-Rad Laboratories. Two (2) of the nine (9) panel members had high reactive levels of the ntibodies, two (2) had low reactive levels of the antibodies, and two (2) had antibody levels near the cut-off; additionally there were two (2) high negative panel members and one (1) low negative panel member for each analyte. Precision testing was performed at Bio-Rad Laboratories on one lot of the Syphilis IgG kit, one lot of the Syphilis IgG Calibrator Set and one lot of the Syphilis IgG Control Set. Each of the nine (9) panel members was tested in duplicate (x2) on two (2) runs per day for twenty (20) days using one (1) lot of Syphilis IgG kit, one (1) lot of Syphilis IgG Calibrator Set and one (1) lot of Syphilis IgG Control Set (2 times x 2 runs x 20 days = 80 replicates per panel member). The data were analyzed for intra-assay and inter-assay precision according to the principles described in the Clinical Laboratory Standards Institute guidance EP5-A2, revised November 2004 and ISO/TR 22971:2205. The standard deviation (SD) and percent coefficient of variation (%CV) were calculated. Results can be found in Tables 17 - 19.
| Syphilis 15kD<br>Panel Members | Sample<br>N* | Mean | Within-Run | | Between-Day | | Between-Run | | Total | |
|--------------------------------|--------------|------|------------|------|-------------|------|-------------|------|-------|-------|
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Negative | 80 | 0.0 | 0.02 | N/A | 0.00 | N/A | 0.00 | N/A | 0.02 | N/A |
| High Negative 1 | 80 | 0.5 | 0.01 | 2.3% | 0.03 | 6.6% | 0.03 | 6.6% | 0.04 | 8.3% |
| High Negative 2 | 80 | 0.5 | 0.02 | 4.0% | 0.03 | 6.7% | 0.00 | 0.0% | 0.05 | 9.6% |
| Near Cut-off 1 | 80 | 1.0 | 0.04 | 3.5% | 0.07 | 7.0% | 0.03 | 3.1% | 0.09 | 8.4% |
| Near Cut-off 2 | 80 | 1.0 | 0.04 | 3.8% | 0.06 | 6.5% | 0.03 | 3.3% | 0.08 | 7.8% |
| Low Positive 1 | 80 | 1.4 | 0.05 | 3.9% | 0.12 | 8.6% | 0.05 | 4.0% | 0.14 | 10.4% |
| Low Positive 2 | 80 | 1.3 | 0.05 | 3.5% | 0.10 | 7.9% | 0.05 | 4.1% | 0.13 | 9.6% |
| High Positive 1 | 80 | 3.1 | 0.11 | 3.7% | 0.22 | 7.3% | 0.11 | 3.6% | 0.27 | 9.0% |
| High Positive 2 | 80 | 3.4 | 0.07 | 2.1% | 0.17 | 5.0% | 0.18 | 5.2% | 0.26 | 7.6% |
Table 17. Precision Results; BioPlex 2200 Syphilis IgG 15kD
N/A = No precision specification established for samples below cut-off.
| | Table 18. Precision Results; BioPlex 2200 Syphilis IgG 17kD | | | |
|--|-------------------------------------------------------------|--|--|--|
|--|-------------------------------------------------------------|--|--|--|
| Syphilis 17kD | Sample | Mean | Within-Run | | Between-Day | | Between-Run | | Total | |
|-----------------|--------|------|------------|------|-------------|------|-------------|------|-------|-------|
| Panel Members | N | | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Negative | 80 | 0.0 | 0.00 | N/A | 0.00 | N/A | 0.00 | N/A | 0.00 | N/A |
| High Negative 1 | 80 | 0.5 | 0.04 | 7.0% | 0.03 | 6.0% | 0.03 | 6.0% | 0.06 | 10.8% |
| High Negative 2 | 80 | 0.5 | 0.03 | 6.0% | 0.04 | 8.4% | 0.03 | 6.0% | 0.06 | 11.5% |
| Near Cut-off 1 | 80 | 1.0 | 0.03 | 3.3% | 0.07 | 6.9% | 0.03 | 3.1% | 0.08 | 7.9% |
| Near Cut-off 2 | 80 | 1.0 | 0.03 | 3.1% | 0.05 | 5.3% | 0.04 | 4.3% | 0.08 | 7.3% |
| Low Positive 1 | 80 | 1.3 | 0.06 | 4.3% | 0.09 | 7.5% | 0.04 | 3.5% | 0.12 | 9.4% |
| Low Positive 2 | 80 | 1.3 | 0.04 | 3.3% | 0.08 | 6.3% | 0.03 | 2.4% | 0.10 | 7.4% |
| High Positive 1 | 80 | 3.2 | 0.11 | 3.3% | 0.26 | 8.2% | 0.08 | 2.4% | 0.30 | 9.1% |
| High Positive 2 | 80 | 2.9 | 0.07 | 2.5% | 0.20 | 7.1% | 0.09 | 3.1% | 0.23 | 8.2% |
N/A = No precision specification established for samples below cut-off.
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| | | | | Within-Run | | Between-Day | | Between-Run | | Total | |
|--------------------------------|-------------|------|------|------------|------|-------------|------|-------------|------|-------|--|
| Syphilis 47kD<br>Panel Members | Sample<br>N | Mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV | |
| Negative | 80 | 0.0 | 0.00 | N/A | 0.00 | N/A | 0.00 | N/A | 0.00 | N/A | |
| High Negative 1 | 80 | 0.6 | 0.01 | 1.9% | 0.03 | 5.4% | 0.00 | 0.0% | 0.04 | 5.9% | |
| High Negative 2 | 80 | 0.6 | 0.03 | 5.2% | 0.00 | 0.0% | 0.03 | 5.1% | 0.05 | 7.4% | |
| Near Cut-off 1 | 80 | 1.1 | 0.03 | 2.4% | 0.04 | 4.3% | 0.03 | 3.0% | 0.06 | 5.7% | |
| Near Cut-off 2 | 80 | 1.1 | 0.04 | 3.1% | 0.05 | 4.8% | 0.03 | 2.8% | 0.07 | 6.4% | |
| Low Positive 1 | 80 | 1.2 | 0.05 | 4.1% | 0.08 | 6.8% | 0.03 | 2.6% | 0.10 | 8.5% | |
| Low Positive 2 | 80 | 1.4 | 0.05 | 3.4% | 0.09 | 6.5% | 0.07 | 5.2% | 0.12 | 8.8% | |
| High Positive 1 | 80 | 3.4 | 0.06 | 1.8% | 0.21 | 6.2% | 0.08 | 2.5% | 0.23 | 7.0% | |
| High Positive 2 | 80 | 3.8 | 0.06 | 1.7% | 0.18 | 4.7% | 0.14 | 3.9% | 0.24 | 6.3% | |
Table 19. Precision Results; BioPlex 2200 Syphilis IgG 47kD
N/A = No precision specification established for samples below cut-off.
# D. Comparative Testing
Comparative Testing
#### Comparison of Syphilis IgG and Non-treponemal (RPR) and TPPA test
The performance of the Syphilis IgG kit was evaluated against corresponding reference (RPR and TPPA) assays. Results were interpreted according to Table 20 below. Discordant samples were further tested by a treponemal EIA kit to determine the final result.
| Non-treponemal<br>Result (RPR) | Treponemal<br>Result (TPPA) | Reference Assays Result | Treponemal (EIA)<br>Result | Final Reference<br>Assays Result |
|--------------------------------|-----------------------------|-------------------------|----------------------------|----------------------------------|
| Nonreactive | Nonreactive | Negative | N/A | Negative |
| Nonreactive | Reactive | Discordant | Negative | Negative |
| | | | Equivocal | Equivocal |
| | | | Positive | Positive |
| Reactive | Nonreactive | Discordant | Negative | Negative |
| | | | | Equivocal |
| | | | | Positive |
| Reactive | Reactive | Positive | N/A | Positive |
Table 20. Reference Syphilis Testing Algorithm
A total of 1,750 serum samples were evaluated at three (3) U.S. clinical testing sites. The results can be observed in Tables 21 - 27 for banked serum samples from patients who had a syphilis test ordered; from unselected pregnant women; from serum samples requested to be RPR/TPPA reactive; from pregnant women requested to be treponemal assay positive; from pregnant women requested to be RPR/TPPA nonreactive; and from patients medically diagnosed with syphilis. For the purposes of percent agreement, reference assay equivocal results were assigned to the opposite result than that of the corresponding BioPlex 2200 Syphilis IgG result. Results are shown in Tables 21 - 27.
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·
:
| | | BioPlex 2200 Syphilis IgG | | | | | | | |
|----------------------------|-----------|---------------------------|-----------|-------------|-------|-----------------------------|-------------------------------|-----------------------------|-------------------------------|
| | | Reactive | Equivocal | Nonreactive | Total | Positive (+)<br>% Agreement | 95%<br>Confidence<br>Interval | Negative (-)<br>% Agreement | 95%<br>Confidence<br>Interval |
| Reference Assays<br>Result | Positive | 46 | 0 | 0 | 46 | 100%<br>(46/46) | 92.0 - 100% | 93.8%<br>(426/454) | 91.2 - 95.7% |
| | Equivocal | 3* | 0 | 0 | 3 | | | | |
| | Negative | 22** | 3 | 426 | 451 | | | | |
| | Total | 71 | 3 | 426 | 500 | | | | |
Table 21. Serum Samples From Patients Who Had a Syphilis Test Ordered (N=500)
* Three (3) BioPlex 2200 Syphills IgG reactive samples were RPR nonreactive, TPPA reactive and treponemal EIA equivocal.
** Of 22 samples with reactive results on the BioPlex 2200 Syphilis IgG assay, 16 were RPR and TPPA nonreactive, and 6 samples were RPR reactive, TPPA nonreactive and treponemal EIA negative.
| Table 22. Unselected Serum Samples From Pregnant Women (N=497) | | | | |
|----------------------------------------------------------------|-----------------------------------------|---------------------------------------------------------------------------------------------------------------------|--|--|
| | THE LAND SHEET AND FORMICAL CAR LEASERS | "WITH A A A A B A C A C A C A L A L A L E S A C S A C S A C S A C A C A C A C A C A C A C A C A C A C A . A . A . A | | |
| Reference Assays Result | | Reactive | Equivocal | Nonreactive | Total | Positive (+) % Agreement | 95% Confidence Interval | Negative (-) % Agreement | 95% Confidence Interval |
|-------------------------|----|----------|-----------|-------------|-------|--------------------------|-------------------------|--------------------------|-------------------------|
| Positive | | 5 | 0 | 0 | 5 | | | | |
| Equivocal | | 0 | 0 | 0 | 0 | 100%<br>(5/5) | 56.5 - 100% | 99.8%<br>(491/492) | 98.9 - 100.% |
| Negative | 1* | 0 | 491 | 492 | | | | | |
| Total | | 6 | 0 | 491 | 497 | | | | |
* One (1) sample with a BioPlex 2200 reactive result was RPR and TPPA nonreactive.
| Reference Assays<br>Result | Reactive | Equivocal | Nonreactive | Total | Positive (+)<br>% Agreement | 95%<br>Confidence<br>Interval |
|----------------------------|----------|-----------|-------------|-------|-----------------------------|-------------------------------|
| Positive | 249 | 0 | 0 | 249 | 100%<br>(249/249) | 98.5 - 100% |
| Equivocal | 0 | 0 | 0 | 0 | | |
| Negative | 0 | 0 | 1 | 1 | | |
| Total | 249 | 0 | 1 | 250 | | |
:
Table 23. Serum Samples Requested to be RPR and TPPA Reactive (N=250)
:
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Table 24. Serum Samples From Pregnant Women Requested to be Treponemal Assay Positive (N=183)
| | | BioPlex 2200 Syphilis IgG | | | | | |
|----------------------------|-----------|---------------------------|-------------|-------|-----------------------------|-------------------------------|-----------|
| | Reactive | Equivocal | Nonreactive | Total | Positive (+)<br>% Agreement | 95%<br>Confidence<br>Interval | |
| Reference Assays<br>Result | Positive | 166 | 0 | 0 | 166 | | |
| | Equivocal | 4* | 0 | 0 | 4 | 100%<br>(166/166) | 97.7-100% |
| | Negative | 8** | 0 | 5 | 13 | | |
| | Total | 178 | 0 | 5 | 183 | | |
* Four ( 4) BioPlex 2200 Syphilis IgG reactive samples were RPR nonreactive, TPPA reactive and treponemal EIA equivocal.
** Of eight (8) BioPlex 2200 Syphilis IgG reactive samples, 1 sample was RPR and TPPA nonreactive and 7 were RPR reactive, TPPA nonreactive and treponemal EIA negative.
| | | | | | Table 25. Serum Samples From Pregnant Women Requested to be RPR/TPPA Nonreactive |
|---------|--|--|--|--|----------------------------------------------------------------------------------|
| (N=250) | | | | | |
| Reference Assays<br>Result | | BioPlex 2200 Syphilis IgG | | | | | |
|----------------------------|----------|---------------------------|-------------|-------|-----------------------------|-------------------------------|--|
| | Reactive | Equivocal | Nonreactive | Total | Negative (+)<br>% Agreement | 95%<br>Confidence<br>Interval | |
| Positive | 0 | 0 | 0 | 0 | | 96.5 -99.6% | |
| Equivocal | 0 | 0 | 0 | 0 | 98.8% | | |
| Negative | 3* | 0 | 247 | 250 | (247/250) | | |
| Total | 3 | 0 | 247 | 250 | | | |
* Three (3) BioPlex 2200 Syphilis IgG reactive samples were RPR and TPPA nonreactive.
・
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Table 26. Combined Serum Samples From 183 Pregnant Women Requested to be Treponemal Assay Positive and 250 Pregnant Women Requested to be RPR/TPPA Nonreactive (N=433)
| | | BioPlex 2200 Syphilis IgG | | | | | | | |
|----------------------------|-----------|---------------------------|-----------|-------------|-------|-----------------------------|-------------------------------|-----------------------------|-------------------------------|
| | | Reactive | Equivocal | Nonreactive | Total | Positive (+)<br>% Agreement | 95%<br>Confidence<br>Interval | Negative (-)<br>% Agreement | 95%<br>Confidence<br>Interval |
| Reference Assays<br>Result | Positive | 166 | 0 | 0 | 166 | 100%<br>(166/166) | 97.7 -<br>100.0% | 94.4%<br>(252/267) | 90.9 - 96.6% |
| | Equivocal | 4* | 0 | 0 | 4 | | | | |
| | Negative | 11** | 0 | 252 | 263 | | | | |
| | Total | 181 | 0 | 252 | 433 | | | | |
* Four (4) BioPlex 2200 Syphilis IgG reactive samples were RPR reactive, TPPA nonreactive and treponemal EIA equivocal.
** Of 11 BioPlex 2200 Syphilis IgG reactive samples, 4 samples were RPR and TPPA nonreactive, and 7 samples were RPR nonreactive, TPPA reactive and treponemal EIA negative.
| | BioPlex 2200 Syphilis IgG | | | | | | |
|----------------------------|---------------------------|-----------|-------------|-------|-----------------------------|-------------------------------|-------------|
| | Reactive | Equivocal | Nonreactive | Total | Positive (+)<br>% Agreement | 95%<br>Confidence<br>Interval | |
| Reference Assays<br>Result | Positive | 63 | 0 | 0 | 63 | 100.0%<br>(63/63) | 94.2 - 100% |
| | Equivocal | 1* | 0 | 0 | 1 | | |
| | Negative | 3** | 0 | 3 | 6 | | |
| | Total | 67 | 0 | 3 | 70 | | |
Table 27. Serum Samples From Patients Medically Diagnosed With Syphilis (N=70)
* One (1) sample with BioPlex 2200 Syphilis IgG reactive results was RPR nonreactive, TPPA reactive and treponemal EIA equivocal.
** Three (3) samples with BioPlex 2200 Syphilis IgG reactive results were RPR and TPPA nonreactive.
As can be seen in the table above, 95.7% (67/70) of the patients medically diagnosed with syphilis infection were reactive when tested with the BioPlex 2200 Syphilis IgG test, in comparison to 90% (63/70) by reference assay testing.
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## Correlation with Known HIV-1 Positive Samples
Performance of the BioPlex 2200 Syphilis IgG kit was evaluated in two hundred twenty (220) banked known HIV-1 positive serum samples. Results were compared to reference assay results for RPR/TPPA and if applicable, treponemal EIA.
Of the 220 known HIV-1 positive serum samples, one hundred eighty-one (181) were negative by reference assay testing and one hundred twenty-three (123) samples were negative by BioPlex 2200 Syphilis IgG testing, for 68.0% (123/181) negative agreement with a 95% confidence interval of 60.8-74.3%. Of the one hundred eighty-one (181) samples were equivocal by reference assay testing (and were considered negative for the purposes of percent agreement calculations) and nineteen (19) samples were equivocal by BioPlex 2200 Syphilis IgG testing (and were considered positive for the purposes of percent agreement calculations).
Of the 220 known HIV-1 positive serum samples, thirty-nine (39) were positive by reference assay testing and by BioPlex 2200 Syphilis IgG testing, for 100% (39/39) positive agreement with a 95% confidence interval of 91.0-100%.
## Correlation with Disease Stages and Clinical Sensitivity
The performance of the Syphilis IgG kit was evaluated at one clinical testing site using a CDC panel of banked frozen characterized sera (N=140), with known disease state and treatment status. Panel samples were from treated and untreated patients with primary, secondary or latent infections. Performance of the Syphilis IgG kit for the CDC panel was compared to reference assay results using commercially available (RPR, TPPA and if required treponemal EIA) kits by known clinical status. Results are shown in Tables 28 - 30 below.
| | | Reference Assays Result | | | | | | | | | |
|-----------------------------|-----------|------------------------------------------|-----|----|-------------------------------------------|-----|----|------------------------------------------|-----|----|-------|
| Known<br>Clinical<br>Status | | Positive<br>BioPlex 2200<br>Syphilis IgG | | | Equivocal<br>BioPlex 2200<br>Syphilis IgG | | | Negative<br>BioPlex 2200<br>Syphilis IgG | | | Total |
| | | R | EQV | NR | R | EQV | NR | R | EQV | NR | |
| Untreated | Primary | 10 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 12 |
| | Secondary | 10 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 10 |
| | Latent | 8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 13 |
| Treated | Primary | 15 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 16 |
| | Secondary | 36 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 36 |
| | Latent | 49 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 53 |
| Total | | 128 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 12 | 140 |
# Table 28. BioPlex 2200 Syphilis IgG vs. Reference Assays: Comparison by Known Clinical Status
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| Known Clinical Status | N | BioPlex 2200 Syphilis IgG | | | | Reference Assays Result | | | | |
|-----------------------|-----------|---------------------------|-----------|-------------|------------------------|-------------------------|-----------|----------|------------------------|--------------------|
| | | Reactive | Equivocal | Nonreactive | Clinical Sensitivity % | Positive | Equivocal | Negative | Clinical Sensitivity % | |
| Untreated | Primary | 12 | 10 | 0 | 2 | 83.3%<br>(10/12) | 10 | 0 | 2 | 83.3%<br>(10/12) |
| | Secondary | 10 | 10 | 0 | 0 | 100%<br>(10/10) | 10 | 0 | 0 | 100%<br>(10/10) |
| | Latent | 13 | 8 | 0 | 5 | 61.5%<br>(8/13) | 8 | 0 | 5 | 61.5%<br>(8/13) |
| Treated | Primary | 16 | 15 | 0 | 1 | 93.8%<br>(15/16) | 15 | 0 | 1 | 93.8%<br>(15/16) |
| | Secondary | 36 | 36 | 0 | 0 | 100%<br>(36/36) | 36 | 0 | 0 | 100%<br>(36/36) |
| | Latent | 53 | 49 | 0 | 4 | 92.5%<br>(49/53) | 49 | 0 | 4 | 92.5%<br>(49/53) |
| Total | | 140 | 128 | 0 | 12 | 91.4%<br>(128/140) | 128 | 0 | 12 | 91.4%<br>(128/140) |
Table 29. Bio-Rad Syphilis IgG vs. a CDC Panel of Characterized Sera (N=140)
| | Table 30. BioPlex 2200 Syphilis IgG vs. Reference Assays: Percent Agreement by Known |
|-----------------|--------------------------------------------------------------------------------------|
| Clinical Status | |
| | Known Clinical Status | N | Positive Agreement | 95% Confidence Interval |
|-----------|-----------------------|-----|--------------------|-------------------------|
| Untreated | Primary | 12 | (10/10) | 72.2 - 100% |
| | Secondary | 10 | (10/10) | 72.2 - 100% |
| | Latent | 13 | (8/8) | 67.5 - 100% |
| Treated | Primary | 16 | (15/15) | 79.6 - 100% |
| | Secondary | 36 | (36/36) | 90.3 - 100% |
| | Latent | 53 | (49/49) | 91.8 - 100% |
| Total | | 140 | (128/128) | 97.1 - 100% |
The performance of the Syphilis IgG kit was further evaluated at one clinical testing site using banked prospectively collected serum samples from treated and untreated patients with primary and secondary syphilis infections (N=10). Performance of the Syphilis IgG kit in the 10 samples was compared to a corresponding composite result using commercially available (RPR, TPPA and if required treponemal EIA) kits by known clinical status. Results are shown in Tables 31 - 33 below.
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Table 31. BioPlex 2200 Syphilis IgG vs. Reference Assays: Comparison by Known Clinical Status
| | | Reference Assays Result | | | | | | | | | |
|-----------------------------|-----------|------------------------------------------|-----|----|-------------------------------------------|-----|----|------------------------------------------|-----|----|-------|
| Known<br>Clinical<br>Status | | Positive<br>BioPlex 2200<br>Syphilis IgG | | | Equivocal<br>BioPlex 2200<br>Syphilis IgG | | | Negative<br>BioPlex 2200<br>Syphilis IgG | | | Total |
| | | R | EQV | NR | R | EQV | NR | R | EQV | NR | |
| Untreated | Primary | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 |
| | Secondary | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Treated | Primary | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
| | Secondary | 7 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 7 |
| Total | | 9 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 10 |
Table 32. Bio-Rad Syphilis IgG vs. Prospectively Collected Serum Samples With Known Clinical Status (N=10)
| Known Clinical<br>Status | N | BioPlex 2200 Syphilis IgG | | | Clinical Sensitivity % | 95% Confidence Interval | Reference Assays Result | | | Clinical Sensitivity % | 95% Confidence Interval | |
|--------------------------|-----------|---------------------------|-----------|-------------|------------------------|-------------------------|-------------------------|-----------|----------|------------------------|-------------------------|--------------|
| | | Reactive | Equivocal | Nonreactive | | | Positive | Equivocal | Negative | | | |
| Untreated | Primary | 1 | 1 | 0 | 0 | 100%<br>(1/1) | 20.6 - 100% | 0 | 0 | 1 | 0.0%<br>(0/1) | N/A* |
| | Secondary | 0 | 0 | 0 | 0 | N/A* | N/A* | 0 | 0 | 0 | N/A* | N/A* |
| Treated | Primary | 2 | 2 | 0 | 0 | 100%<br>(2/2) | 34.2 - 100% | 2 | 0 | 0 | 100%<br>(2/2) | 34.2 - 100% |
| | Secondary | 7 | 7 | 0 | 0 | 100%<br>(7/7) | 64.5 - 100% | 7 | 0 | 0 | 100%<br>(7/7) | 64.5 - 100% |
| Total | | 10 | 10 | 0 | 0 | 100%<br>(10/10) | 72.2 - 100% | 9 | 0 | 1 | 90.0%<br>(9/10) | 59.5 - 98.2% |
* In cases where agreement resulted in a numerator of zero (0), 95% confidence interval could not be calculated; in cases where agreement resulted in (0/0) samples, percent and 95% confidence interval could not be calculated.
Table 33. BioPlex 2200 Syphilis IgG vs. Reference Assays: Percent Agreement by Known Clinical Status
| | Known Clinical Status | N | Positive Agreement | 95% Confidence Interval | |
|-----------|-----------------------|---------|--------------------|-------------------------|-------------|
| | Untreated | Primary | 1 | (0/0) | N/A* |
| Secondary | | 0 | (0/0) | N/A* | |
| Treated | Primary | 2 | (2/2) | 100% | 34.2 - 100% |
| | Secondary | 7 | (7/7) | 100% | 64.5 - 100% |
| | Total | 10 | (9/9) | 100% | 70.1 - 100% |
* In cases where agreement resulted in (0/0) samples percent and 95% confidence intervals could not be calculated.
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The results from testing the CDC panel of samples from patients with treated and untreated primary, secondary and latent syphilis infections (N=140); and the banked prospectively collected samples from treated and untreated patients with primary and secondary syphilis infections (N=10), were combined and results are presented in Tables 34 - 36 below.
| | | | | Table 34. BioPlex 2200 Syphilis IgG vs. Reference Assays: Combined Comparison By Known | |
|-----------------|--|--|--|-----------------------------------------------------------------------------------------|--|
| Clinical Status | | | | | |
| Known<br>Clinical<br>Status | | Positive<br>BioPlex 2200<br>Syphilis IgG | | | Equivocal<br>BioPlex 2200<br>Syphilis IgG | | | Negative<br>BioPlex 2200<br>Syphilis IgG | | | Total |
|-----------------------------|-----------|------------------------------------------|-----|----|-------------------------------------------|-----|----|------------------------------------------|-----|----|-------|
| | | R | EQV | NR | R | EQV | NR | R | EQV | NR | |
| Untreated | Primary | 10 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 2 | 13 |
| | Secondary | 10 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 10 |
| | Latent | 8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 13 |
| Treated | Primary | 17 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 18 |
| | Secondary | 43 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 43 |
| | Latent | 49 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 53 |
| Total | | 137 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 12 | 150 |
| | Table 35. BioPlex 2200 Syphilis IgG vs. Combined CDC Panel and Prospectively Collected | | | | |
|--|----------------------------------------------------------------------------------------|--|--|--|--|
| | Samples With Known Clinical Status (N=150) | | | | |
| Known Clinical<br>Status | | N | BioPlex 2200 Syphilis IgG | | | | | Reference Assays Result | | | | |
|--------------------------|-----------|-----|---------------------------|-----------|-------------|---------------------------|-------------------------------|-------------------------|-----------|-------------|---------------------------|-------------------------------|
| | | | Reactive | Equivocal | Nonreactive | Clinical<br>Sensitivity % | 95%<br>Confidence<br>Interval | Reactive | Equivocal | Nonreactive | Clinical<br>Sensitivity % | 95%<br>Confidence<br>Interval |
| Untreated | Primary | 13 | 11 | 0 | 2 | 84.6%<br>(11/13) | 57.8 - 95.7% | 10 | 0 | 3 | 76.9%<br>(10/13) | 49.7 - 91.8% |
| | Secondary | 10 | 10 | 0 | 0 | 100%<br>(10/10) | 72.2 - 100% | 10 | 0 | 0 | 100%<br>(10/10) | 72.2 - 100% |
| | Latent | 13 | 8 | 0 | 5 | 61.5%<br>(8/13) | 35.5 - 82.3% | 8 | 0 | 5 | 61.5%<br>(8/13) | 35.5 - 82.3% |
| Treated | Primary | 18 | 17 | 0 | 1 | 94.4%<br>(17/18) | 74.2 - 99.0% | 17 | 0 | 1 | 94.4%<br>(17/18) | 74.2 - 99.0% |
| | Secondary | 43 | 43 | 0 | 0 | 100%<br>(43/43) | 91.8 - 100% | 43 | 0 | 0 | 100%<br>(43/43) | 91.8 - 100% |
| | Latent | 53 | 49…
