ZEUS ELISA HSV GG-2 IGG TEST SYSTEM

K103603 · Zeus Scientific, Inc. · MYF · May 20, 2011 · Microbiology

Device Facts

• Record ID: K103603
• Device Name: ZEUS ELISA HSV GG-2 IGG TEST SYSTEM
• Applicant: Zeus Scientific, Inc.
• Product Code: MYF · Microbiology
• Decision Date: May 20, 2011
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3305
• Device Class: Class 2

Indications for Use

The ZEUS ELISA HSV gG-2 IgG Test System is intended for the qualitative detection of type specific IgG class antibodies to Herpes Simplex Virus Type 2 (HSV-2) in human serum. The test is intended for testing sexually active individuals or pregnant women for aiding in the presumptive diagnosis of HSV-2 infection. The predictive value of positive or negative results depends on the population's prevalence and the pretest likelihood of HSV-2. The test is not intended for donor screening or for self testing. The performance of this assay has not been established for use in a pediatric population, neonates, or immunocompromised patients.

Device Story

ZEUS ELISA HSV gG-2 IgG Test System is an enzyme-linked immunosorbent assay (ELISA) for qualitative detection of IgG antibodies to HSV-2 glycoprotein G (gG-2) in human serum. Device uses polystyrene 96-well plates sensitized with affinity-purified HSV-2 native antigen. Patient serum is incubated in wells; antigen-specific IgG binds to immobilized antigen. After washing, horseradish peroxidase-conjugated goat anti-human IgG (Fc-specific) is added. Following further incubation and washing, peroxidase substrate (TMB) is added, causing color change proportional to bound antibody. Reaction is stopped and optical density measured via spectrophotometer. Results are interpreted against a cutoff value (Index Value <= 0.90 negative; 0.91-1.09 equivocal; >= 1.10 positive). Used in clinical laboratories by trained personnel to aid in HSV-2 diagnosis. Output assists clinicians in evaluating patient infection status based on clinical history and epidemiological data.

Clinical Evidence

No clinical data provided in the summary document; substantial equivalence is based on analytical performance characteristics and comparison to the predicate device.

Technological Characteristics

Immunoassay (ELISA) using affinity-purified HSV-2 native antigen on polystyrene 96-well plates. Detection via horseradish peroxidase-conjugated goat anti-human IgG (Fc-specific) and TMB substrate. Requires spectrophotometer for optical density measurement. Qualitative interpretation based on index values. No automated software algorithm; manual or instrument-assisted photometric reading.

Indications for Use

Indicated for sexually active individuals or pregnant women to aid in the presumptive diagnosis of HSV-2 infection. Not for pediatric, neonatal, or immunocompromised populations.

Regulatory Classification

Identification

Herpes simplex virus serological assays are devices that consist of antigens and antisera used in various serological tests to identify antibodies to herpes simplex virus in serum. Additionally, some of the assays consist of herpes simplex virus antisera conjugated with a fluorescent dye (immunofluorescent assays) used to identify herpes simplex virus directly from clinical specimens or tissue culture isolates derived from clinical specimens. The identification aids in the diagnosis of diseases caused by herpes simplex viruses and provides epidemiological information on these diseases. Herpes simplex viral infections range from common and mild lesions of the skin and mucous membranes to a severe form of encephalitis (inflammation of the brain). Neonatal herpes virus infections range from a mild infection to a severe generalized disease with a fatal outcome.

Special Controls

*Classification.* Class II (special controls). The device is classified as class II (special controls). The special control for the device is FDA's revised guidance document entitled “Class II Special Controls Guidance Document: Herpes Simplex Virus Types 1 and 2 Serological Assays.” For availability of the guidance revised document, see § 866.1(e).

Predicate Devices

• HerpeSelect 1 and 2 Immunoblot IgG (K000238)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number: K103603
B. Purpose for Submission: Clearance of New Device
C. Measured: Herpes Simplex Virus HSV-2 type specific IgG antibodies to the affinity purified HSV glycoprotein G (gG) 2 native antigen
D. Type of Test: Enzyme-linked Immunosorbent Assay (ELISA)
E. Applicant: Zeus Scientific, Inc.
F. Proprietary and Established Names: ZEUS ELISA HSV gG-2 IgG Test System

## G. Regulatory Information:

1. Regulation section: 21 CFR 866.3305. Herpes Simplex Virus Serological Reagents
2. Classification: Class II
3. Product code: MYF, Enzyme linked immunosorbent assay, Herpes Simplex Virus, HSV-2
4. Panel: Microbiology (83)

## H. Intended Use:

1. Intended use(s):

The ZEUS ELISA HSV gG-2 IgG Test System is intended for the qualitative detection of type specific IgG class antibodies to Herpes Simplex Virus Type 2 (HSV-2) in human serum. The test is intended for testing sexually active individuals or pregnant women for aiding in the presumptive diagnosis of HSV-2 infection.

The predictive value of positive or negative results depends on the population's prevalence and the pretest likelihood of HSV-2. The test is not intended for donor screening or for self testing.

The performance of this assay has not been established for use in a pediatric population, neonates, or immunocompromised patients.

2. Indication(s) for use:

Same as Intended Use

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3. Special conditions for use statement(s):

For prescription use only

4. Special instrument requirements:

Spectrophotometer

I. Device Description:

The ZEUS ELISA HSV gG-2 IgG Test System is an immunoassay for the qualitative detection of IgG antibodies to HSV glycoprotein G (gG) 2 in human serum. The test system consists of affinity purified HSV-2 native antigen and horse radish peroxidase conjugated goat anti-human IgG (Fc chain specific) to detect IgG class antibodies to HSV-2 in human sera.

J. Substantial Equivalence Information:

1. Predicate device name(s): Reference Method for clinical evaluation

HerpeSelect® 1 and 2 Immunoblot IgG (Focus Diagnostics)

2. Predicate Numbers (s): K000238

Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | ZEUS ELISA HSV gG-2 IgG | Focus HerpeSelect 1 and 2 Immunoblot IgG |
| Intended Use | The ZEUS ELISA HSV gG-2 IgG Test System is intended for the qualitative detection of type specific IgG class antibodies to HSV-2 in human serum. The test is intended for testing sexually active individuals or pregnant women for aiding in the presumptive diagnosis of HSV infection. The predictive value of positive or negative results depends on the population's prevalence and the pretest likelihood of HSV-2. The test is not intended for donor screening or for self testing. The performance of this assay has not been established for use in a pediatric population, neonates, or immunocompromised patients. | Focus Diagnostics' HerpeSelect 1 and 2 Immunoblot IgG test is intended for qualitatively detecting the presence or absence of human IgG class antibodies to HSV-1 and HSV-2 in human sera. The test is indicated for testing sexually active adults or pregnant women for aiding in the presumptive diagnosis of HSV-1 and HSV-2 infection. The predictive value of a positive or negative result depends on the population's prevalence and the pretest likelihood of HSV-1 and HSV-2 infection. The performance of this assay has not been established for use in a pediatric population, for neonatal screening, for testing of immunocompromised patients, for use by a point of care facility or for use with automated equipment. |

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| Assay | Immunoassay | Immunoassay |
| --- | --- | --- |
| Sample Matrix | Human Serum | Human Serum |
| Analyte | Human IgG | Human IgG |
| Conjugate | Goat anti-human IgG; Fc chain specific | Goat anti-human IgG; Fc chain specific |
| Controls | 2 (Negative and Positive) | 2 (Negative and Positive) |
| Differences | | |
| Item | ZEUS ELISA HSV gG-2 IgG | Focus HerpeSelect 1 and 2 Immunoblot IgG |
| Detection Method | Colorimetric | Visual |
| Solid Phase | Polystyrene 96 well plate | Nitrocellulose membrane |
| Substrate | TMB | Bromo-chloro-indolyl phosphate and nitroblue tetrazolium/A |
| Conjugate Label | Horseradish peroxidase | Alkaline phosphatase |
| Calibrators | Includes Calibrator (human serum) | Cutoff/Positive control |
| Reading | Read the color change (optical density) of the wells | Visually compare each band on a strip relative to the control band four bands per strip |
| Detection HSV type | HSV-2 IgG only | HSV-1 IgG and HSV-2 IgG |
| Antigen Used | HSV gG-2 native antigen | Recombinant and native HSV gG-1 and gG2 antigen |
| Interpretation Criteria | Negative is <= 0.90, Positive is >= 1.10 and Equivocal is 0.90 - 1.09 | If patient band equal or darker than control band, result is positive. If band is lighter than control band, result is negative |

# K. Standard/Guidance Documents Referenced (if applicable):

1. Class II Special Controls Guidance Document: Herpes Simplex Virus Types 1 &amp; 2 Serological Assays, September 28, 2010. http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm227411.htm
2. CLSI EP5: Evaluation of Precision Performance of Clinical Chemistry Devices-Second Edition, Villanova PA
3. CLSI EP7-A2: Interference Testing in Clinical Chemistry; Approved Guideline, 2nd Ed. (2005).

# L. Test Principle:

The ZEUS ELISA HSV gG-2 test system is designed to detect IgG class antibodies to HSV-2 in human sera. Wells of plastic microwell strips are sensitized by passive adsorption with affinity purified HSV-2 native antigen. The test procedure involves three incubation steps:

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1. Test sera (properly diluted) are incubated in antigen coated microwells. Any antigen specific antibody in the sample will bind to the immobilized antigen. The plate is washed to remove unbound antibody and other serum components.
2. Peroxidase Conjugated goat anti-human IgG (Fc chain specific) is added to the wells and the plate is incubated. The Conjugate will react with IgG antibody immobilized on the solid phase in step 1. The wells are washed to remove non-reactive Conjugate.
3. The microwells containing immobilized peroxidase Conjugate are incubated with peroxidase Substrate Solution. Hydrolysis of the Substrate by peroxidase produces a color change. After a period of time, the reaction is stopped and the color intensity of the solution is measured photometrically. The color intensity of the solution will be greater than the cutoff value if anti-HSV-2 IgG is present in the serum being tested.

# M. Performance Characteristics (if/when applicable):

# 1. Analytical performance:

# a. Precision/Reproducibility:

Precision was evaluated internally at Zeus Scientific. The study was conducted as follows: twelve samples were identified and/or prepared (by Zeus Scientific, Inc.) for use in the study based upon their activity on the ZEUS ELISA HSV gG-2 kit. Two samples each were selected that were negative, high negative, near cut-off, low positive, moderate positive and high positive. On each day of testing, the samples were aliquoted in duplicate and tested. This was repeated in a second run on the same day by a different technologist for a total of twenty days (2 replicates x 2 runs/day x 20 days = 80 replicates per sample). The precision data were analyzed according to the principles described in the Clinical Laboratory Standards Institute guidance EP5-A2, revised November 2004. The standard deviation (SD) and percent coefficient of variation (%CV) were calculated. Results are shown in Table 1.

Table 1. Summary of In-house Precision HSV gG-2 IgG

| Panel Member | Sample N | Mean IV | Within-Run | | Within-Day | | Between-Run | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| High Positive | 80 | 9.096 | 0.22 | 2.4% | 0.34 | 3.7% | 0.29 | 3.2% | 0.41 | 4.5% |
| High Positive | 80 | 5.290 | 0.12 | 2.3% | 0.19 | 3.5% | 0.15 | 2.9% | 0.25 | 4.6% |
| Mod Positive | 80 | 2.512 | 0.06 | 2.5% | 0.11 | 4.3% | 0.10 | 4.1% | 0.15 | 5.9% |
| Mod Positive | 80 | 2.308 | 0.07 | 3.0% | 0.10 | 4.3% | 0.09 | 3.9% | 0.12 | 5.3% |
| Low Positive | 80 | 1.377 | 0.06 | 4.5% | 0.07 | 5.2% | 0.04 | 3.3% | 0.08 | 6.0% |
| Low Positive | 80 | 1.288 | 0.05 | 4.0% | 0.07 | 5.1% | 0.04 | 3.3% | 0.09 | 6.7% |
| Near Cut-off | 80 | 1.028 | 0.03 | 3.0% | 0.05 | 5.0% | 0.05 | 4.7% | 0.06 | 6.0% |
| Near Cut-off | 80 | 0.992 | 0.03 | 2.9% | 0.05 | 4.6% | 0.04 | 3.9% | 0.06 | 6.1% |
| High Negative | 80 | 0.702 | 0.02 | 3.3% | 0.03 | 4.6% | 0.02 | 3.4% | 0.04 | 5.8% |
| High Negative | 80 | 0.663 | 0.03 | 3.9% | 0.03 | 5.3% | 0.02 | 3.8% | 0.05 | 7.5% |
| Negative | 80 | 0.205 | 0.01 | 5.2% | 0.02 | 7.6% | 0.01 | 6.6% | 0.02 | 10.2% |

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Reproducibility was evaluated internally and at two external clinical sites. The study was conducted as follows: twelve samples were identified and/or prepared (by Zeus Scientific, Inc.) for use in the study based upon their activity on the ZEUS ELISA HSV gG-2 IgG test system. Two samples each were selected that were negative, high negative, near cut-off, low positive, moderate positive and high positive. To assess reproducibility, on each day of testing, each sample was aliquoted in duplicate, each aliquot was tested in triplicate in two runs by two operators resulting in twelve results per day. This was repeated for five days at each site and the resulting data used for analysis (3 replicates x 4 runs/day x 5 days x 3 sites = 180 replicates per sample). Results are shown in Table 2.

Table 2. Summary of Multi-Site Reproducibility HSV gG-2 IgG

| Panel Member | Sample N | Mean IV | Within-Run | | Within-Day | | Between-Run | | Between-Site | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| High Positive | 180 | 8.270 | 0.33 | 3.9% | 0.38 | 4.5% | 0.24 | 2.8% | 0.43 | 5.2% | 0.68 | 8.3% |
| High Positive | 180 | 5.263 | 0.26 | 4.9% | 0.32 | 6.1% | 0.22 | 4.3% | 0.36 | 6.8% | 0.41 | 7.8% |
| Mod Positive | 180 | 2.535 | 0.15 | 5.7% | 0.17 | 6.8% | 0.11 | 4.2% | 0.21 | 8.3% | 0.25 | 10.0% |
| Mod Positive | 180 | 2.237 | 0.10 | 4.6% | 0.12 | 5.5% | 0.08 | 3.6% | 0.14 | 6.3% | 0.18 | 8.1% |
| Low Positive | 180 | 1.350 | 0.08 | 5.8% | 0.09 | 6.8% | 0.06 | 4.3% | 0.10 | 7.5% | 0.11 | 8.4% |
| Low Positive | 180 | 1.320 | 0.08 | 5.9% | 0.10 | 7.5% | 0.07 | 5.6% | 0.11 | 8.0% | 0.14 | 10.7% |
| Near Cut-off | 180 | 1.020 | 0.06 | 5.9% | 0.07 | 7.2% | 0.05 | 4.9% | 0.09 | 9.0% | 0.11 | 10.4% |
| Near Cut-off | 180 | 0.969 | 0.06 | 6.5% | 0.08 | 7.9% | 0.05 | 5.2% | 0.08 | 8.8% | 0.10 | 10.1% |
| High Negative | 180 | 0.668 | 0.05 | 7.0% | 0.05 | 8.2% | 0.03 | 4.9% | 0.06 | 9.3% | 0.07 | 10.3% |
| High Negative | 180 | 0.653 | 0.05 | 8.4% | 0.06 | 9.7% | 0.04 | 5.8% | 0.07 | 11.1% | 0.08 | 12.1% |
| Negative | 180 | 0.352 | 0.03 | 9.3% | 0.04 | 11.3% | 0.03 | 7.5% | 0.05 | 14.0% | 0.05 | 15.5% |
| Negative | 180 | 0.320 | 0.03 | 10.5% | 0.04 | 12.4% | 0.02 | 6.8% | 0.05 | 15.0% | 0.05 | 16.4% |
| Non-Reactive Control | 180 | 0.245 | 0.03 | 12.6% | 0.04 | 14.8% | 0.02 | 7.4% | 0.04 | 17.3% | 0.05 | 18.7% |
| Reactive Control | 180 | 8.869 | 0.51 | 6.0% | 0.59 | 6.8% | 0.34 | 3.9% | 0.73 | 8.3% | 0.87 | 9.9% |

b. Linearity/assay reportable range: N/A
c. Traceability, Stability, Expected values (controls, calibrators, or methods): There is no standard available for measuring HSV antibody in serum
d. Detection limit: Not applicable. This device is for qualitative HSV-2 antibody detection.

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# e. Analytical specificity:

Cross Reactivity: A study was conducted to assess cross reactivity with the ZEUS ELISA HSV gG-2 IgG test system using sero-positive serum to EBV VCA IgG, ANA, Rubella, VZV IgG, CMV, Measles, Treponema pallidum, Gonorrhea, HPV, Chlamydia, rheumatoid factor (RF), Toxoplasma gondii, and HSV-1 IgG. To demonstrate the assay only detects HSV-2 IgG antibody, HSV-2 IgM and IgG sero-positive samples were characterized by FDA cleared HSV-2 IgM ELISA test system from which total IgG was removed. Micro-particle and ELISA immunoassay test systems manufactured by various companies for commercial distribution were used to determine the sero-positivity of the samples. Ten samples for each possible cross-reactant were tested. In total, 140 samples were tested for possible cross reactivity with 14 analytes. None of the samples showed cross-reactivity with any of the analytes tested. The results of this study are summarized in Table 3.

Table 3. Cross Reactivity Summary

| HSV gG-2 Cross reactivity Study | |
| --- | --- |
| Analyte | positive/tested |
| EBV VCA IgG | 0/10 |
| ANA | 0/10 |
| Measles | 0/10 |
| Rubella | 0/10 |
| CMV | 0/10 |
| VZV | 0/10 |
| T. pallidum | 0/10 |
| Gonorrhea | 0/10 |
| HPV | 0/10 |
| Chlamydia | 0/10 |
| RF | 0/10 |
| T. gondii | 0/10 |
| HSV gG-1 | 0/10 |
| HSV-2 IgM | 0/10 |

Specimens known to contain potentially cross reactive antibodies to Candida albicans have not been tested with this device, therefore it is unknown if there is any cross reactivity with these antibodies. This is indicated in the limitation section of the package insert.

Interfering Substances: The effect of potentially interfering substances on sample results generated using the ZEUS ELISA HSV gG-2 IgG test system was evaluated with the following possible interfering substances: albumin, bilirubin, cholesterol, hemoglobin, triglycerides and intralipids.

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The level of each potentially interfering substance is as follows:

Bilirubin: 1mg/dL (low), 15 mg/dL (high)

Albumin: 3.5 g/dL (low), 5 g/dL (high)

Cholesterol: 150 mg/dL (low), 250 mg/dL (high)

Triglycerides: 150 mg/dL (low), 500 mg/dL (high)

Hemoglobin: 10 g/dL (low), 20 g/dL (high)

Intralipid: 300 mg/dL (low), 750 mg/dL (high)

Three samples for HSV gG-2 were chosen based on their performance on the investigational device: positive, borderline and negative. The possible interfering substances were added to the samples. Test and control samples were evaluated in replicates of ten. All positive samples showed a change of signal less than 20%. All borderline samples showed a change of signal less than 20% with the exception of the high spike of intralipid (75.5%). The negative sample showed a change of signal (&gt;20%) with the low spike (42.9%) and high spike of albumin (78.6%), the high spike of hemoglobin (245.2), the low spike of intralipid (78.6%), and the low and high spikes of bilirubin (272.7% and 318.2%, respectively). The negative sample results in each instance stayed below the cut-off and the change in signal did not affect the qualitative result.

f. Assay cut-off:

The cut off for this assay was established against a panel of 25 confirmed negative specimens as well as nine clinically characterized positive samples. The mean and standard deviation were established for the negative population. Using a mathematical calculation based on these data, a theoretical cut-off was established and validated with the characterized specimens. Based upon the results of this testing, the manufacturer established the following guidelines for interpretation of patient samples.

Interpretations:

Index Values or OD ratios are interpreted as follows:

| Result | Index Value or OD Ratio |
| --- | --- |
| Negative | ≤ 0.90 |
| Equivocal | 0.91 to 1.09 |
| Positive | ≥ 1.10 |

1. An OD ratio ≤ 0.90 indicates no detectable IgG antibody to HSV-2.
2. Specimens with OD ratio values in the equivocal range (0.91 – 1.09) should be retested in duplicate. If on retesting one of the two samples remains equivocal, the samples should be tested by an alternate serological procedure

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such as Westen Blot or re-evaluated by drawing another sample one to three weeks later.

3. An OD ratio $\geq 1.10$ indicates that HSV-2 IgG antibodies were detected.
4. The numeric value of the final result above the cutoff is not indicative of the amount of anti-HSV-2 IgG antibody present.
5. Test results should be interpreted in conjunction with the clinical history, epidemiological data and other information available to the attending physician in evaluating the patient.
6. False positive test results may occur. Repeat testing or testing with a different device may be indicated in some settings e.g., patients with low likelihood of HSV infection.

## 2. Comparison studies:

a. Method comparison with predicate device: Comparative studies were performed using a total of 788 samples at three clinical sites to demonstrate the equivalence of the ZEUS ELISA HSV gG-2 test system to the reference method (a commercially distributed HSV-1 and 2 immunoblot test system). Five hundred and eighty-eight samples were from the Intended Use populations of sexually active individuals (n=336) and pregnant women (n=252) with an HSV test requested. One hundred additional samples from a low prevalence population of 17-19 year old individuals (n=100) were tested as well as 100 HSV samples acquired from the CDC (CDC panel). The clinical sites and quantity of samples tested are summarized in Table 4.

| Populations | Number of Samples Tested at Each Site | | | |
| --- | --- | --- | --- | --- |
| | Site 1 | Site 2 | Site 3 | Total |
| Sexually Active Individuals | 100 | 136 | 100 | 336 |
| Pregnant women | 33 | 155 | 64 | 252 |
| Low Prevalence Population | 33 | 33 | 34 | 100 |
| CDC Panel | 33 | 25 | 42 | 100 |

Results of this comparative study in the three sites combined are presented below. The data was analyzed counting any discordant equivocal results between the comparator and the investigational device against the performance of the investigational device.

## Performance in the Intended Use Population of Sexually Active Individuals: A total of 336 prospective, unselected samples from sexually active individuals with an HSV-2 test ordered were tested with the ZEUS ELISA HSV gG-2 IgG Test System and compared with a commercially available immunoblot test. The samples were submitted for HSV-2 antibody

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testing, sequentially numbered, de-identified and archived. Results are presented in Table 5.

| | Table 5. Sexually Active Individuals | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Immunoblot | | | | | | |
| | | Positive | Equivocal | Negative | Site Total | | % Agreement* | 95% CI |
| ZEUS ELISA | | | | | | | | |
| | Positive Equivocal Negative Site Total | 70 | 0 | 1 | 71 | PPA | 93.3% (70/75) | 86.9%-98.5% |
| | | 0 | 0 | 0 | 0 | | | |
| | | 1 | 4 | 260 | 265 | NPA | 99.6% (260/261) | 97.9%-100% |
| | | 71 | 4 | 261 | 336 | | | |

*Equivocal results in one test but not the other were treated as discrepant and included in the calculations.

Performance in the Intended Use Population of Pregnant Women: A total of 252 prospective, unselected samples from pregnant women with an HSV-2 test ordered were tested with the ZEUS ELISA HSV gG-2 IgG Test System and compared with a commercially available immunoblot test. One hundred and twenty-one samples were from women in the first trimester, 64 from women in the second trimester and 67 from women in the third trimester. The samples were submitted for HSV-2 antibody testing, sequentially numbered, de-identified and archived. Results are presented in Table 6.

| | Table 6. Pregnant Women | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Immunoblot | | | | | | |
| | | Positive | Equivocal | Negative | Site Total | | % Agreement* | 95% CI |
| ZEUS ELISA HSV gG-2 | | | | | | | | |
| | Positive Equivocal Negative Site Total | 70 | 1 | 1 | 72 | PPA | 98.6% (70/71) | 92.5%-100% |
| | | 0 | 0 | 0 | 0 | | | |
| | | 1 | 0 | 179 | 180 | NPA | 98.9% (179/181) | 96.1%-99.9% |
| | | 71 | 1 | 180 | 252 | | | |

* Equivocal re*Equivocal results in one test but not the other were treated as discrepant and included in the calculations.

Performance in a Low Prevalence Population: A total of 100 samples requested to be collected from 17-19 year old individuals in a non-STD setting were tested and compared with a commercially available immunoblot test. These samples were purchased from a New York vendor. The results are shown in Table 7.

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| | Table 7. Low Prevalence Population | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Immunoblot | | | | | | |
| | | Positive | Equivocal | Negative | Site Total | | % Agreement* | 95% CI |
| ZEUS ELISA HSV gG-2 | | | | | | | | |
| | Positive | 0 | 0 | 0 | 0 | PPA | N/A | N/A |
| | Equivocal | 0 | 0 | 2 | 2 | | | |
| | Negative | 0 | 0 | 98 | 98 | NPA | 98.0% (98/100) | 93.0%-99.8% |
| | Site Total | 0 | 0 | 100 | 100 | | | |

*Equivocal results in one test but not the other were treated as discrepant and included in the NPA calculations.

CDC HSV-2 IgG Panel: A total of 100 samples were obtained from the CDC for analysis. The performance of the ZEUS ELISA HSV gG-2 IgG Test System was assessed using a masked, well characterized HSV serum panel from the CDC. The panel consists of 46 HSV-2 IgG positive and 54 HSV-2 IgG negative samples. Of the 46 HSV-2 IgG positive samples, 24 samples were HSV-1 IgG and HSV-2 IgG dual-positive samples. The results are presented to convey further information on the performance of the test kit and do not imply endorsement of the assay by the CDC.

| | Table 8. CDC HSV Panel | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | CDC HSV-2 Results | | | | | | |
| | | Positive | Equivocal | Negative | Site Total | | % Agreement* | 95% CI |
| ZEUS ELISA HSV gG-2 | | | | | | | | |
| | Positive | 46 | 0 | 0 | 46 | PPA | 100.0% (46/46) | 93.7%-100% |
| | Equivocal | 0 | 0 | 0 | 0 | | | |
| | Negative | 0 | 0 | 54 | 54 | NPA | 96.9% (54/54) | 94.6%-100% |
| | Site Total | 46 | 1 | 54 | 100 | | | |

*Equivocal results in one test but not the other were treated as discrepant and included in the NPA calculations.

b. Matrix Comparison: N/A

3. Clinical studies:

a. Clinical Sensitivity: N/A
b. Clinical specificity: N/A
c. Other clinical supportive data (when a. and b. are not applicable):

4. Clinical cut-off: N/A

5. Expected values/Reference range:

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The observed prevalence with the Intended Use populations was assessed internally and externally at two sites with unselected, masked and archived sera. With the sexually active individuals $(n = 336)$ the observed prevalence with the ZEUS ELISA HSV gG-2 was $21.4\%$ (72/336).

In the population of pregnant women $(n = 252)$ , the observed prevalence with the ZEUS ELISA HSV gG-2 was $28.7\%$ (72/251). One sample was excluded from this analysis due to unknown age.

Values for the prevalence observed with the investigational device in each age group for the two populations are summarized in tables 9 and 10.

Table 9. Observed Prevalence with Sexually Active Individuals

| Age | Sex | HSV gG-2 | | total | Observed % Prevalence HSV gG-2 |
| --- | --- | --- | --- | --- | --- |
| | | Positive | Negative | | |
| 15-19 | Male | 1 | 10 | 11 | 9.1% |
| | Female | 2 | 23 | 25 | 8.0% |
| 20-29 | Male | 2 | 31 | 34 | 5.9% |
| | Female | 19 | 69 | 88 | 21.6% |
| 30-39 | Male | 2 | 18 | 20 | 10.0% |
| | Female | 17 | 43 | 60 | 28.3% |
| 40-49 | Male | 7 | 22 | 29 | 24.1% |
| | Female | 5 | 16 | 21 | 23.8% |
| 50-59 | Male | 7 | 20 | 27 | 25.9% |
| | Female | 5 | 5 | 10 | 50.0% |
| 60-69 | Male | 3 | 6 | 9 | 33.3% |
| | Female | 1 | | 1 | 100% |
| 70 + | Male | | 1 | 1 | 0.0% |
| | Female | | | 0 | 0.0% |
| Sub-total | Male | 23 | 108 | 131 | 17.6% |
| | Female | 49 | 156 | 205 | 23.9% |
| | Total | 71 | 264 | 336 | 21.1% |

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Table 10. Observed Prevalence with Pregnant women

| | HSV gG-2 | | | Observed % Prevalence |
| --- | --- | --- | --- | --- |
| Age | Positive | Negative | total | HSV gG-2 |
| 15-19 | 2 | 22 | 24 | 8.3% |
| | | | | |
| 20-29 | 46 | 103 | 149 | 30.9% |
| | | | | |
| 30-39 | 15 | 48 | 63 | 23.8% |
| | | | | |
| 40-49 | 9 | 6 | 15 | 60.0% |
| | | | | |
| Total | 72 | 179 | 251* | 28.7% |

*one negative sample submitted with age unknown was excluded

The hypothetical predictive values for the two populations are shown in the table below. The calculations are based on the ZEUS ELISA HSV gG-2 IgG having:

1. A positive percent agreement of 93.3% and a negative percent agreement of 99.6% in sexually active adults
2. A positive percent agreement of 98.6% and a negative percent agreement of 98.9% in pregnant women

Table 11. Prevalence vs. Hypothetical Predictive Values

| Prevalence | HSV gG-2 IgG | | | |
| --- | --- | --- | --- | --- |
| | Sexually Active Individuals | | Pregnant Women | |
| | PPV | NPV | PPV | NPV |
| 50% | 99.6% | 93.7% | 98.9% | 98.6% |
| 40% | 99.4% | 95.7% | 98.4% | 99.1% |
| 30% | 99.1% | 97.2% | 97.5% | 99.4% |
| 25% | 98.8% | 97.8% | 96.8% | 99.5% |
| 20% | 98.4% | 98.3% | 95.7% | 99.6% |
| 15% | 97.8% | 98.8% | 94.1% | 99.8% |
| 10% | 96.5% | 99.3% | 90.9% | 99.8% |
| 5% | 92.9% | 100.0% | 82.5% | 100.0% |

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.