The EUROIMMUN Anti-West Nile Virus ELISA (IgM) is intended for the qualitative detection of IgM antibodies to West Nile virus in human serum and plasma (K+EDTA, Li+-heparin). This test is intended as an aid in the presumptive laboratory diagnosis of West Nile virus infection in patients with clinical symptoms consistent with meningitis/encephalitis, in conjunction with other laboratory and clinical findings. Positive results must be confirmed by the plaque reduction test (PRNT) or by using the current CDC guidelines for diagnosis of this disease.

The assay characteristics have not been established for testing cord blood, neonates, prenatal screening, and general population screening of patients without symptoms of meningoencephalitis. This assay is not FDA cleared or approved for testing blood or plasma donors.

Warning: Cross-reactivity with IgM to Dengue virus, Malarialanti-Plasmodium falciparum and Parvovirus B19 has been observed with the EUROIMMUN Anti-West Nile Virus ELISA (IgM). Reactive results must be reported with a caution statement regarding possible IgM cross-reactivity with other flaviviruses.

Device Description

Patient serum or plasma samples are diluted 1:101 in sample buffer and incubated for 10 minutes at room temperature to allow IgG/RF separation. 100 ul of each diluted patient sample and pre-diluted controls and calibrator are added to the antigen coated microtiter wells and incubated for 60 minutes at +37°C. After incubation the microtiter well strips are washed 3 times with wash buffer to remove unbound antibodies and 100 ul of the anti-human IgM enzyme conjugate reagent is added to each microtiter well. After an additional 30 minutes incubation at room temperature, the microtiter wells are again washed 3 times with wash buffer to remove any unbound enzyme conjugate and 100 ul of the chromogen substrate is added. The strips are incubated for 15 minutes at room temperature and 100 ul stop solution is added. The microtiter placed in an ELISA reader and read at a wavelength of 450 nm and a reference wavelength of between 620 nm and 650 nm within 30 minutes.

AI/ML Overview

Here's an analysis of the provided text regarding the acceptance criteria and study for the EUROIMMUN Anti-West Nile Virus ELISA (IgM):

Note: This document describes an in vitro diagnostic (IVD) device, not a typical AI/ML device. Therefore, the concepts of "test set," "training set," "experts to establish ground truth," "adjudication method," and "MRMC comparative effectiveness study" do not directly apply in the same way they would to an AI-powered image analysis or diagnostic tool. Instead, the performance is evaluated against a predicate device and/or a reference standard like PRNT (Plaque Reduction Neutralization Test), and clinical studies involve comparisons of the device's results with these established methods.

Acceptance Criteria and Reported Device Performance

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for this device, being a diagnostic test, are generally based on sensitivity and specificity relative to a reference standard or a predicate device. The document explicitly states the ranges for these measures.

Acceptance Criteria Category	Acceptance Criteria (Implicit from Clinical Studies)	Reported Device Performance (Range)
Sensitivity	High sensitivity (e.g., >80-90%)	84.0% (89.9% in Clinical Study III)
Specificity	High specificity (e.g., >90-95%)	97.3% (from Assay Cut-off section for healthy donors); 99.7% in Clinical Study II
Positive Agreement	High agreement with predicate device (e.g., >85%)	85.7% (Clinical Study I); 90.9% (Clinical Study II); 89.9% (Clinical Study III)
Negative Agreement	High agreement with predicate device (e.g., >95%)	97.3% (Clinical Study I); 99.7% (Clinical Study II)
Repeatability (CV%)	Low Coefficient of Variation (e.g., <15%)	Range: 6.8% (for mean ratio 4.9) to 14.1% (for mean ratio 0.4)
Reproducibility (CV%)	Low Coefficient of Variation (e.g., <15%)	Range: 8.5% (for mean ratio 4.9) to 15.1% (for mean ratio 0.0)
Cross-Reactivity	Minimal cross-reactivity with other diseases	95.2% Overall Negative Rate with non-WNV seropositive specimens
Interference	No significant interference from common sample types	Hemolytic, lipemic, icteric samples showed no influence up to specified conc.
Matrix Equivalence	Equivalence between serum and plasma	Mean %recovery: 101% (EDTA plasma), 98% (Li-heparin plasma)

Study Details (Applicable sections)

For IVD devices like this, the "study" typically refers to the analytical and clinical performance evaluations.

2. Sample size used for the test set and the data provenance:

Clinical Study I (Prospective):
- Sample Size: 155 samples from patients suspected of West Nile Virus infection.
- Provenance: Collected at hospitals and clinics across the US in 2015 (prospective).
Clinical Study II:
- Sample Size: 398 serum samples.
- Provenance: Collected prospectively from patients suspected of West Nile virus infection at a clinical laboratory in the midwest (US).
Clinical Study III:
- Sample Size: 99 clinically collected serum samples, positive for WNV IgM.
- Provenance: Public health agency/laboratory in Canada.
Analytical Specificity/Cross-Reactivity Study:
- Sample Size: 692 serologically characterized seropositive specimens.
- Provenance: Not explicitly stated, but these are "serologically characterized seropositive specimens from patients with diseases other than WNV."
Assay Cut-off Establishment:
- Sample Size: 18 sera from clinically characterized positive WNV patients and 150 sera from normal healthy blood donors.
- Provenance: Normal healthy blood donors from a "non-endemic region." Clinical characterization implies real patient data.
Expected Values (US Studies):
- Sample Size: 553 samples.
- Provenance: Prospectively collected from US population.
Matrix Comparison:
- Sample Size: 20 determinations for EDTA plasma, 20 for Li-heparin plasma.
- Provenance: Created from 5 different sets of normal blood donor samples (serum, EDTA plasma, Li-heparin plasma) that were spiked with positive serum samples.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g., radiologist with 10 years of experience):

This concept doesn't apply directly to an IVD device like an ELISA. The "ground truth" for clinical samples is established by:
- Clinical diagnosis/symptoms: "patients with clinical symptoms consistent with meningitis/encephalitis" (as per Indications for Use).
- Reference standard tests: Plaque Reduction Neutralization Test (PRNT) or current CDC guidelines for diagnosis are mentioned as confirmatory methods.
- Serological characterization: For cross-reactivity studies, specimens are "serologically characterized."
- Predicate device results: The predicate device (Focus Diagnostics West Nile Virus IgM DxSelect™ ELISA) serves as a comparative "truth" in many agreements.

4. Adjudication method (e.g., 2+1, 3+1, none) for the test set:

Not applicable in the way it's used for AI studies. For the clinical studies comparing with the predicate device, it states: "Average of three results for each clinical specimen tested at three sites was considered to calculate the positive percent and negative percent agreement between the EUROIMMUN Anti-West Nile Virus ELISA (IgM) vs reference assay." This implies an averaging or consensus approach for the new device results across sites, but not an adjudication of ambiguous ground truth. The ground truth itself (PRNT or predicate result) is the established reference.

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

Not applicable as this is an in vitro diagnostic device, not an AI-assisted diagnostic tool that involves human readers interpreting AI output.

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

The device being an ELISA assay inherently operates in a "standalone" fashion (algorithm/assay only) once the sample is processed, generating quantitative results that are interpreted by predefined cut-offs. There isn't an "algorithm" in the AI sense, but rather a biochemical reaction leading to a detectable signal.

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

The ground truth or reference standard for validation was:

Clinical diagnosis and symptoms: For assessing the aid in diagnosis for patients with meningitis/encephalitis symptoms.
Plaque Reduction Neutralization Test (PRNT): A gold standard method for arbovirus serology, explicitly mentioned for confirmation and in Clinical Study I.
Current CDC guidelines for diagnosis of this disease: Another established reference.
Predicate device (Focus Diagnostics West Nile Virus IgM DxSelect™ ELISA): Used as a comparative reference for positive and negative agreement.
Serologically characterized seropositive specimens: For the cross-reactivity study.

8. The sample size for the training set:

For IVD devices, there isn't a "training set" in the machine learning sense. Instead, the device's design, reagent formulations, and cut-off values are established through development studies.
The "Assay Cut-off" was established using:
- 18 sera from clinically characterized positive West Nile virus patients.
- 150 sera from normal healthy blood donors from a non-endemic region.
- This set of 168 individuals could be considered analogous to a development/calibration set used to define the diagnostic thresholds.

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

Again, not a "training set" in the ML context. For the samples used to establish the assay cut-off:
- The 18 samples were from "clinically characterized positive West Nile virus patients." This implies diagnosis through established clinical and laboratory methods (likely including PRNT or CDC guidelines).
- The 150 samples were from "normal healthy blood donors," implying they were confirmed negative for WNV.

Summary

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Image /page/0/Picture/1 description: The image is a seal for the Department of Health & Human Services - USA. The seal is circular and contains the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter. In the center of the seal is an abstract image of an eagle with three heads.

August 12, 2016

Public Health Service

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

EUROIMMUN US Inc. Michael Locke Director of Regulatory Affairs 1 Bloomfield Ave. Mountain Lakes, New Jersey 07046

Re: K153308

Trade/Device Name: EUROIMMUN Anti-West Nile Virus ELISA (IgM) Regulation Number: 21 CFR 866.3940 Regulation Name: West Nile Virus Serological Reagents Regulatory Class: II Product Code: NPO Dated: July 8, 2016 Received: July 12, 2016

Dear Mr. Locke:

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 (800) 638 2041 or (301) 796-7100 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 (800) 638-2041 or (301) 796-7100 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) K153308

Device Name

EUROIMMUN Anti-West Nile Virus ELISA (IgM)

Indications for Use (Describe)

Type of Use (Select one or both, as applicable)

|×| Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

PLEASE DO NOT WRITE BELOW THIS LINE - CONTINUE ON A SEPARATE PAGE IF NEEDED.

FOR FDA USE ONLY

Concurrence of Center for Devices and Radiological Health (CDRH) (Signature)

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510(k) SUBSTANTIAL EQUIVALENCE

510(k) Number

K153308

Applicant

EUROIMMUN US INC. 1 Bloomfield Ave., Mountain Lakes, New Jersey 07046 Phone: 800-913-2022 Fax: 973-656-1098

Contact:

Michael Locke Director of Regulatory Affairs & Quality Management 1 Bloomfield Ave., Mountain Lakes, New Jersey 07046 Phone: 800-913-2022 Fax: 973-656-1098

Product Name

EUROIMMUN Anti-West Nile Virus ELISA (IgM)

Device Identification

Regulation: 21 CFR 866.3940 (West Nile virus serological reagents) Classification: Class II Product Code: NOP Panel: Microbiology

Substantial Equivalence Information:

Comparison to Predicate Device

Similarities
Item	New Device	Predicate Device
Intended use	EUROIMMUN Anti-West Nile Virus ELISA (IgM) (K153308)	Focus Diagnostics West Nile Virus IgM DxSelectTM ELISA (K040854)
	Detection of IgM class antibodies against West Nile virus	Same
Assay format	Qualitative	Same
Technology	ELISA	Same
Assay platform	96-well microtiter plates	Same
Calibrators and Controls	1 calibrator (cut-off)2 controls: 1 positive; 1 negative	Same
Substrate	TMB	Same
Wash buffer	10x concentrate	Same
Serum sample dilution	1:101	Same
Procedure	Sample incubation with micro-well antigen coated plate, followed by a wash step, incubation with an anti-human IgM enzyme conjugate; wash step, incubation with substrate; stopping of the reaction with stop solution, photometric reading.	Same

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Differences
Item	New Device	Predicate Device
	(K153308) EUROIMMUN Anti-West NileVirus ELISA (IgM)	(K040854) Focus Diagnostics WestNile Virus IgM DxSelect™ ELISA
Antigen	Detergent-extracted glycoprotein E from themembrane fraction of human cells, inactivated;coated on microtiter plate	Recombinant West Nile virusantigen; lyophilized; not coated onmicrotiter plate
Conjugate	Anti-human IgM (rabbit) labelled withhorseradish peroxidase	Anti-flavivirus (mouse monoclonal)labelled with horseradish peroxidase
Stop solution	0.5 M / 1N sulphuric acid	1 M sulfuric acid
Reagent preparation	All reagents, calibrator and controls are readyto use, except for the wash buffer.	Calibrator and controls requiredilution before use.
Sample matrix	Serum or plasma (EDTA, Li-heparin)	Serum
Procedure	IgG/RF removal by incubation with samplebuffer containing IgG/RF-Absorbent.	Capture-Technology: Samples reactwith wells coated with ati-humanIgM; antigen specific for West Nilevirus added in an additionalincubation/wash step.
Reported results	Ratio	Index
Cut-off levels	RatioResult	IndexResult
	<0.8negative	< 0.90negative
	≥0.8 to <1.1borderline	0.91 to < 1.09equivocal
	≥1.1positive	≥ 1.10positive

Intended Use

The EUROIMMUN Anti-West Nile Virus ELISA (IgM) is intended for the qualitative detection of IgM antibodies to West Nile virus in human serum and plasma (K -EDTA, Li -heparin). This test is intended as an aid in the presumptive laboratory diagnosis of West Nile virus infection in patients with clinical symptoms consistent with meningitis/encephalitis, in conjunction with other laboratory and clinical findings. Positive results must be confirmed by the plaque reduction neutralization test (PRNT) or by using the current CDC guidelines for diagnosis of this disease.

Warning: Cross-reactivity with IgM to Dengue virus, Malaria/anti-Plasmodium falciparum and Parvovirus B19 has been observed with the EUROIMUN Anti-West Nile Virus ELISA (IgM). Reactive results must be reported with a caution statement regarding possible IgM cross-reactivity with other flaviviruses.

Device Description

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Test Principle

The test kit contains 12 microtier strips each with 8 break-off reagent wells coated with West Nile virus antigen. In the first reaction step, diluted patient samples, calibrators and controls are incubated in the wells. Anti-West Nile virus antibodies will bind to the antigens coated in the microtiter wells are washed to remove any unbound proteins and non-specific antibodies. In a second reaction step, goat anti-human IgM HRP enzyme conjugate is added to each well. The enzyme conjugate will bind to any wells that have human IgM binding to the West Nile virus antigen. The wells are washed to remove any unbound HRP enzyme conjugate. 3,3,5,5 tetramethylbenzidine (TMB) enzyme substrate is added. If the HRP enzyme is present in the well (positive reaction), the HRP enzyme will react with the TMB substrate and produce a blue color. After an additional incubation time to allow the color development, a stop solution is added which turns the blue color yellow and inhibits further color development to allow for a stable spectrophotometric reading. The test strips are placed in a microplate reader and the optical density of the color is measured. The amount of antigen specific bound antibody is proportional to the color intensity.

Performance Characteristics

Analytical Performance

Repeatability/Reproducibility

Repeatability: The repeatability of the EUROIMMUN Anti-West Nile Virus ELISA (IgM) was investigated by testing of a panel of 8 members prepared using native/natural patient samples seropositive at different concentrations. The inter-assay repeatability is based on 42 determinations per sample performed in 14 different runs on 7 different days (with 2 runs per day and 3 replicates per run). The data from the repeatability study is presented in the table below.

No.	MeanRatio	Within-Run		Within Day		Between Days		Total
1	0.1	0.01	6.9%	0.01	10.5%	0.0	3.3%	0.02	13.0%
2	0.4	0.02	5.2%	0.02	5.2%	0.05	13.1%	0.06	14.1%
3	0.9	0.05	5.2%	0.06	7.2%	0.03	3.8%	0.07	8.2%
4	1.0	0.03	2.8%	0.05	5.5%	0.05	5.4%	0.07	7.7%
5	1.1	0.04	3.8%	0.09	8.0%	0.10	8.9%	0.13	11.9%
6	2.4	0.05	2.3%	0.20	8.6%	0.00	0.0%	0.20	8.6%
7	3.9	0.06	1.5%	0.28	7.3%	0.00	0.0%	0.28	7.3%
8	4.9	0.08	1.7%	0.33	6.8%	0.00	0.0%	0.33	6.8%

Repeatability

Reproducibility was investigated using native/natural samples seropositive at different concentrations, which are assayed in 60 determinations per sample performed at 3 different sites (in-house: and 2 laboratories in the north-east) for 5 days with 2 runs per day and 2 replicates per run according to the package insert. Acceptance criterium was that all qualitative results (positive, borderline, negative) of the samples be in line with the expected result. The following results were obtained:

No.	MeanRatio	Within-Run		Within Day		Between Days		Between Sites		Total
		SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
1	0.0	0.01	10.3%	0.003	5.2%	0.00	0.0%	0.00	3.9%	0.01	15.1%
2	0.7	0.08	11.7%	0.000	0.0%	0.00	0.0%	0.04	5.9%	0.09	14.1%
3	0.9	0.06	6.7%	0.046	5.1%	0.03	3.0%	0.00	0.0%	0.09	10.2%
4	1.2	0.11	9.2%	0.044	3.6%	0.00	0.0%	0.00	0.0%	0.14	11.8%
5	1.3	0.06	4.9%	0.048	3.8%	0.00	0.0%	0.06	4.8%	0.13	9.9%
6	2.3	0.11	4.6%	0.070	3.1%	0.00	0.0%	0.10	4.3%	0.20	9.0%
7	4.9	0.26	5.3%	0.223	4.6%	0.15	2.9%	0.00	0.0%	0.42	8.5%

Reproducibility

Analytical Specificity/Cross Reactivity

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Cross Reactivity was investigated using 692 serologically characterized seropositive specimens from patients with diseases other than WNV. Each of the specimens included in the study was characterized with respect to disease state prior to analysis of the specimens with the EUROIMMUN Anti-West Nile Virus ELISA (IgM). Cross-reactivity across the flavivirus group is common (i.e., St. Louis encephalitis, Dengue 1, 2, 3 & 4; Murray Valley encephalitis, Japanese encephalitis, Yellow fever viruses and Zika virus); as well as persons vaccinated for flaviviruses.

No.	Panel	n	Anti-West Nile Virus ELISA (IgM)
			Positive	Negative	% Negative
1	Anti-Barmah Forest virus	20	0	20	100.0%
2	Anti-Borrelia burgdorferi	50	0	50	100.0%
3	Anti-Chikungunya virus	59	1	58	98.3%
4	Anti-CMV	16	0	16	100.0%
5	Anti-Dengue virus	57	14	43	75.4%
6	Anti-EBV	56	2	54	96.4%
7	Anti-Hanta virus	4	0	4	100.0%
8	Anti-Hepatitis virus	12	0	12	100.0%
9	Anti-HSV-1	29	0	29	100.0%
10	Anti-Leptospira	17	0	17	100.0%
11	Malaria/anti-Plasmodium falciparum	10	4	6	60.0%
12	Anti-Measles virus	18	1	17	94.4%
13	Anti-Mumps virus	14	0	14	100.0%
14	Anti-Parvovirus B19	7	2	5	71.4%
15	Anti-Polio virus	21	0	21	100.0%
16	Anti-Ross River virus	20	0	20	100.0%
17	Anti-Rubella virus	10	0	10	100.0%
18	Anti-TBE virus	31	3	28	90.3%
19	Anti-Toxoplasma gondii	13	1	12	92.3%
20	Anti-VZV	32	0	32	100.0%
21	Anti-West Nile Virus IgG	13	0	13	100.0%
22	Yellowfever virus immunization	31	2	29	93.5%
23	Anti-Zika virus	47	2	44	93.6%
24	Rheumatoid arthritis/polyarthritis/anti-CCP	16	0	16	100.0%
25	Anti-Rheumatoid factor	39	0	39	100.0%
26	Anti-nuclear autoantibodies	20	0	20	100.0%
27	ANCA-associated small vessel vasculitides/ANCA	6	0	6	100.0%
28	Celiac disease/anti-endomysium	10	0	10	100.0%
29	Plasma cell myeloma	14	0	14	100.0%
	Total	692	32	659	95.2%

Cross Reactivity

Interference: Hemolytic, lipemic and icteric samples showed no influence on the result up to a concentration of 1000 mg/dl for hemoglobin, 2000 mg/dl for triglycerides and 40 mg/dl for bilirubin in testing with the EUROIMMUN Anti-West Nile Virus ELISA (IgM). Interferences to high protein (albumin), cholesterol, and intralipids were not investigated.

Assay Cut-off:

The recommended assay cut-off is based on OD results of 18 sera from clinically characterized positive West Nile virus patients and of 150 sera from normal healthy blood donors from a non-endemic region (100 men, 50 women; mean age 39.9 years, age range 18 to 68 years). The samples were investigated using the EUROIMMUN Anti-West Nile Virus ELISA (IgM) and a ROC analysis was performed using the OD's obtained. The ROC analysis demonstrated optimal sensitivity (100.0%) and specificity (97.3%) at the OD value of 0.217. The calibrator was established at this cut-off OD.

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The borderline range of ratio 0.8 to ratio 1.1 was established to cover at least 95% of the negative samples (143 of 150 samples) in the negative range.

Using the cut-off of ratio 1.0 and borderline range of ratio 0.8 to 1.1 with the positive groups mentioned above, the EUROIMMUN Anti-West Nile Virus ELISA (IgM) showed a sensitivity of 100.0% (95% C.I.: 81.5 - 100.0%) with a specificity of 95.3% (95% C.I.: 90.6-98.1%).

Clinical Study I:

A prospective clinical study was performed with 155 samples from patients suspected of West Nile Virus infection collected at hospitals and clinics across the US in 2015. The panel consisted of 83 men and 72 women, age ranged from 6 to 85 years with a mean age of 49 years. Each specimen was tested at one internal and two external sites with the EUROIMMUN Anti-West Nile Virus ELISA (IgM) in parallel with the predicate assay. Average of three results for each clinical specimen tested at three sites was considered to calculate the positive percent and negative percent agreement between the EUROIMMUN Anti-West Nile Virus ELISA (IgM) vs reference assay. The following results were obtained.

Serumn = 155	Predicate
	Positive	Borderline	Negative
EUROIMMUN Anti- West NileVirus ELISA (IgM)	Positive	36	1	0
	Borderline	4	1	2
	Negative	2	0	109
Positive agreement	85.7% (36/42)	95% C.I.	71.5-94.6%

Negative agreement	97.3% (109/112)	95% C.I.	92.4-99.4%
--------------------	-----------------	----------	------------

Of the 42 presumptive positives by the predicate device, 25 patients were further tested by PRNT (Plaque Reduction Neutralization Test) and the EUROIMMUN Anti-West Nile Virus ELISA (IgM). The results are shown below.

Serumn = 25		PRNT Results
		Positive	Borderline	Negative
EUROIMMUN Anti- West NileVirus ELISA (IgM)	Positive	21	0	0
	Borderline	3	0	0
	Negative	1	0	0

Sensitivity 95% C.I. 63.9-95.5% 84.0% (21/25)

Clinical Study II:

A study was performed at a clinical laboratory in the midwest, with 398 serum samples collected prospectively from patients suspected of west nile virus infection. The serum panel consisted of 193 men and 205 women, age ranged from 3 to 102 years with a mean age of 47 years. The samples were tested with the EUROIMMUN Anti-West Nile Virus ELISA (IgM) in parallel with the Predicate ELISA.

Serumn = 398			Predicate
			Positive	Borderline	Negative
EUROIMMUN Anti-West NileVirus ELISA (IgM)	Positive	30	0	1
	Borderline	1	0	0
	Negative	2	0	364
Positive agreement		90.9% (30/33)	95% C.I.	75.7-98.1%
Negative agreement		99.7% (364/365)	95% C.I.	98.5-100.0%

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Clinical Study III:

A study was performed in cooperation with the public health agency/laboratory in Canada with 99 clinically collected serum samples, positive for WNV IgM. The samples were tested by the predicate and the EUROIMMUN Anti-West Nile Virus ELISA (IgM). The results are shown below.

Serumn = 99	Predicate
	Positive	Borderline	Negative
EUROIMMUN Anti- West NileVirus ELISA (IgM)	Positive	89	0	0
	Borderline	4	0	0
	Negative	6	0	0

Positive agreement	89.9% (89/99)	95% C.I.	82.2-95.0%
---------------------------	---------------	----------	------------

Expected Values

Euroimmun assessed reactivity with 553 samples prospectively collected from US population. The samples consisted of 50% females, and 50% males The range of positivity of different populations from the US prospective studies with the EUROIMMUN Anti-West Nile Virus ELISA (IgM) test kit are presented below.

US Studies

Age	n	Negative	Borderline	Positive	% Positive	95% C.I.
0-9	16	16	0	0		0.0 - 20.6%
10-19	32	28	0	4	12.5% (4/32)	3.5 - 29.0%
20-29	66	65	0	1	1.5% (1/66)	0.0 - 8.2%
30-39	86	81	1	4	4.7% (4/86)	1.3 - 11.5%
40-49	89	79	0	10	11.2% (10/89)	5.5 - 19.7%
50-59	100	83	2	15	15.0% (15/100)	8.7 - 23.5%
60+	164	125	5	34	20.7% (34/164)	14.8 - 27.7%
total	553	477	8	68	12.3% (68/553)	9.7 - 15.3%

Note: It is recommended that each laboratory determine its own normal range based on the population and equipment used.

Matrix Comparison (Serum vs Plasma)

The usability of plasma was investigated using sample sets each of serum and corresponding plasma (EDTA, Li-heparin). As no real plasma sample sets from patients containing anti-West Nile virus antibodies were available, the samples were created from 5 different sets of normal blood donor sample sets (serum, EDTA plasma, Li-heparin plasma) that were (after drawing) spiked with 5 different positive serum samples. After spiking, the sample sets were further diluted and processed according to the package insert.

Passing-Bablok regression was calculated for the comparison of serum to plasma. The regression equation is near the ideal correlation (intercept 0; slope 1.0) indicating equivalence of concentrations between serum and the corresponding plasma matrices. Coefficients of determination were found to be above 0.970 and % recovery compared to serum was in the range of 83 to 123% (serum = 100%).

{10}------------------------------------------------

Matrix Comparison (serum vs plasma)

	EDTA plasma	Li-heparin plasma
Determinations (n)	20	20
Concentration range (serum)	Ratio 0.3 - 2.6	Ratio 0.3 - 2.6
Concentration range (plasma)	Ratio 0.3 - 2.5	Ratio 0.3 - 2.4
Regression equation	y = -0.01 + 1.00x	y = 0.02 + 0.92x
(y = plasma, x = serum)
95% C.I. of intercept	-0.05 - 0.07	-0.05 - 0.12
95% C.I. of slope	0.99 - 1.05	0.89 - 1.10
Coefficient of determination R2	0.9787	0.9707
Mean %recovery	101%	98%
Range of %recovery	86 - 116%	83 - 123%

Conclusion

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

Regulation Number and Section

§ 866.3940 West Nile virus serological reagents.

(a)
Identification. West Nile virus serological reagents are devices that consist of antigens and antisera for the detection of anti-West Nile virus IgM antibodies, in human serum, from individuals who have signs and symptoms consistent with viral meningitis/encephalitis. The detection aids in the clinical laboratory diagnosis of viral meningitis/encephalitis caused by West Nile virus.(b)
Classification. Class II (special controls). The special control is FDA's guidance entitled “Class II Special Controls Guidance Document: Serological Reagents for the Laboratory Diagnosis of West Nile Virus.” See § 866.1(e) for the availability of this guidance document.