WEST NILE VIRUS IGM CAPTURE ELISA

K040854 · Focus Technologies, Inc. · NOP · Jun 30, 2004 · Microbiology

Device Facts

• Record ID: K040854
• Device Name: WEST NILE VIRUS IGM CAPTURE ELISA
• Applicant: Focus Technologies, Inc.
• Product Code: NOP · Microbiology
• Decision Date: Jun 30, 2004
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3940
• Device Class: Class 2

Intended Use

The Focus Technologies West Nile Virus IgM Capture ELISA is intended for qualitatively detecting IgM antibodies to West Nile virus in human serum. In conjunction with the Focus Technologies West Nile Virus ELISA IgG, the test is indicated for testing persons having symptoms of meningoencephalitis, as an aid in the presumptive clinical laboratory diagnosis of West Nile virus infection. Positive results must be tested using the background subtraction method (either on the initial test or on a repeat test). Positive results must be confirmed by neutralization test, or by using the current CDC guidelines for diagnosing West Nile encephalitis. This test is not intended for self-testing, and this test is not FDA cleared or approved for testing blood or plasma donors. Assay performance characteristics have not been established for automated instruments

Device Story

Indirect ELISA for qualitative detection of human serum IgM antibodies to West Nile virus (WNV). Microwells coated with anti-human IgM capture patient IgM; recombinant WNV antigen added; mouse anti-flavivirus conjugated with horseradish peroxidase (HRPO) binds to captured WNV antigen. Colorimetric substrate added; spectrophotometer measures color intensity. Background subtraction procedure required for positive results to mitigate false positives from cross-reacting antibodies (e.g., RF, heterophilic antibodies) by comparing reactivity with and without WNV antigen. Used in clinical laboratories by trained personnel. Output aids clinicians in presumptive diagnosis of WNV infection; positive results require confirmation via neutralization test or CDC guidelines. Benefits include rapid presumptive identification of WNV in symptomatic patients.

Clinical Evidence

Clinical performance evaluated across multiple sites (n=300 encephalitis/meningitis patients, n=476 non-flavivirus samples, n=75 PRNT-positive samples, n=103 IFA-negative samples). Sensitivity for confirmed WNV encephalitis/meningitis was 93.2% (with background subtraction). Negative agreement with presumptive negative samples ranged from 98.0% to 100%. Cross-reactivity assessed against other pathogens (Dengue, SLE, EEE, HSV, EBV, CMV, Borrelia, Polio) and interfering substances (RF, ANA). Reproducibility studies (inter-lot, inter/intra-assay, inter-laboratory) performed.

Technological Characteristics

Polystyrene microwells coated with anti-human IgM (u-chain). Detection via mouse anti-flavivirus conjugated with HRPO. Colorimetric readout via spectrophotometer. Manual assay; performance not established for automated instruments. Requires background subtraction procedure for positive results.

Indications for Use

Indicated for patients with symptoms of meningoencephalitis to aid in presumptive laboratory diagnosis of West Nile virus infection. Not for self-testing or blood/plasma donor screening.

Regulatory Classification

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.

Special Controls

*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.

Predicate Devices

• Focus Technologies West Nile Virus IgM Capture ELISA (K031952)

Submission Summary (Full Text)

{0}------------------------------------------------

# JUN 3 0 2004

# 14085

510(k) Summary of Safety and Effectiveness West Nile Virus IgM Capture ELISA Catalog No. EL0300M Prepared June 28, 2004 Page 1 of 8

| Applicant | Focus Technologies, Inc.<br>10703 Progress Way<br>Cypress, California 90630<br>USA |
|-----------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Establishment Registration<br>No. | 2023365 |
| Contact Person | Michael J. Wagner, Esq.<br>tel [phone]<br>fax [phone]<br>[e-mail] |
| Summary Date | June 28, 2004 |
| Proprietary Name | West Nile Virus IgM Capture ELISA |
| Generic Name | West Nile Virus IgM Capture ELISA |
| Classification | West Nile Virus Serological Reagents<br>21 CFR §866.3940<br>Class II |
| Predicate Device | Focus Technologies Arbovirus IFA IgM (K913618)<br>Focus Technologies HSV-2 ELISA (K993724)<br>Focus West Nile Virus IgM Capture ELISA (K031952)<br>CDC West Nile Virus IgM Capture ELISA<br>West Nile Virus Plaque Reduction Neutralization Test |

# Device Description

Indirect Enzyme-linked immunosorbent assay for qualitatively detecting human serum IgM antibodies to West Nile virus.

# Intended Use

The Focus Technologies West Nile Virus IgM Capture ELISA is intended for qualitatively detecting IgM antibodies to West Nile virus in human serum. In conjunction with the Focus Technologies West Nile Virus ELISA IgG, the test is indicated for testing persons having symptoms of meningioencephalitis, as an aid in the presumptive laboratory diagnosis of West Nile virus infection. Positive results must be tested using the background subtraction method (either on the initial test or on a repeat test). Positive results must be confirmed by neutralization test, or by using the current CDC guidelines for diagnosing West Nile encephalitis. This test is not intended for self-testing, and this test is not FDA cleared nor approved for testing blood or plasma donors. Assay performance characteristics have not been established for automated instruments.

{1}------------------------------------------------

# Test Principle

In the Focus Technologies West Nile Virus IgM Capture ELISA, the polystyrene microwells are coated with anti-human antibody specific for IgM (u-chain). Diluted specimen samples and controls are incubated in the wells, and IgM present in the sample binds to the anti-human antibody (IgM specific reactants are removed by washing. Recombinant WNV antigen is then added to the wells and incubated, and, if anti-WNV IgM is present in the sample, the WNV antigen binds to the anti-WNV in the well.

Unbound WNV antigen is then removed by washing the well Mouse anti-flavivirus comjugated with horseradish peroxidase (HRPO) is then added to the wells and, if WNV antigen has been retained in the well by the anti-flavivirus in the sample, the mouse anti-flavivirus: HRPO binds to the WNV antigen in the wells. Excess conjugate is removed by washing. Enzyme substrate and chromogen are added, and the color is allowed to develop. After adding the Stop Reagent, the resultant color change is read by a spectrophotometer. The color intensity is compared to the Cut-off's to determine if antigen-specific IgM is present in the sample.

# Background Subtract Procedure

All IgM reactive samples must be tested with the background subtract procedure to check for false positives caused by cross-reacting antibodies (e.g., RF and heterophilic antibodies) and other substances. Heterophile antibodies are antibodies that can be present in the patient specimen and can bind to animal antibodies (for example the Capture Wells contain rabbit antibody and the Anti-flavivirus Contains mouse antibody). The background subtract procedure detects false positives by testing initially positive samples with and without West Nile Antigen and comparing the reactivity. If heterophile antibodies are present in the sample, they will cross-link the Capture Well antiflavivirus Conjugate, and both wells will be reactive. If heterophile antibodies are absent, then only the well with Antigen will be reactive. The background subtraction method will not eliminate false positive results due to cross-reactive antibodies to other flaviviruses (e.g. St. Louis encephalitis, dengue etc).

{2}------------------------------------------------

# K040854 510(k) Summary of Safety and Effectiveness West Nile Virus IgM Capture ELISA Catalog No. EL0300M Prepared June 28, 2004 Page 3 of 8

# Expected Values

The prevalence of West Nile antibodies varies depending age, geographic location, testing method used, and other factors. A community based serosurvey for West Nile infection conducted in New York in 2000 found that 0.2% (5/2433) of persons tested overall had antibodies indicating recent West Nile infection, and that 1.1% (2/176) of persons reporting a recent headache and fever had antibodies indicating a recent. West Nile infection. Two serosurveys conducted in New York City (NYC) in 1999 and 2000 showed that approximately 1 in 150 infections (<1%) resulted in meningitis or encephalitis. The NYC results are consistent with a 1996 Romanian serosurvey indicating that 1:140 to 1:320 infections resulted in meningitis or encephalitis.

# Prevalence in Samples Submitted for Non-Flavivirus Testing (n=476)

Focus assessed reactivity with 476 samples prospectively collected from North America during August 2003. The samples had been submitted to a clinical laboratory located in Southern California for non-flavivirus tests (e.g., tests for other infectious diseases). The samples consisted of 64.1% females, and 1.5% from persons of unspecified gender.

# Study Site 4: Focus Reactivity with Non-Flavivirus Test Samples (n = 476)

Focus assessed the device's reactivity with 476 samples prospectively collected from North America during August 2003. The samples had been submitted to a clinical laboratory located in Southern California for infectious diseases. Positive samples were tested with a CDC WNV IgM ELISA and/or the CDC WNV IgG ELISA.

### IgM Results without Background Subtract Prevalence with Samples Submitted for Non-Flavivirus Testing (n=476)

### IgM results with Background Subtract Prevalence with Samples Submitted for Non-Flavivirus Testing (n=476)

| Age | Neg | Eqv | Pos | % Positive | 95%CI |
|----------|-----|-----|-----|--------------|-----------|
| 0 to 9 | 24 | 0 | 0 | 0.0% (0/24) | 0.0-14.2% |
| 10 to 19 | 28 | 0 | 1 | 3.5% (1/29) | 0.1-17.8% |
| 20 to 29 | 70 | 0 | 0 | 0.0% (0/70) | 0.0-5.1% |
| 30 to 39 | 82 | 0 | 0 | 0.0% (0/82) | 0.0-4.4% |
| 40 to 49 | 77 | 0 | 1 | 1.3% (1/78) | 0.0-6.9% |
| 50 to 59 | 48 | 1 | 2 | 3.9% (2/51) | 0.5-13.5% |
| 60 to 69 | 38 | 0 | 1 | 2.6% (1/39) | 0.1-13.5% |
| 70 to 79 | 34 | 0 | 0 | 0.0% (0/34) | 0.0-10.3% |
| 80+ | 17 | 1 | 0 | 0.0% (0/18) | 0.0-18.5% |
| Unknown | 50 | 1 | 0 | 0.0% (0/51) | 0.0-7.0% |
| Overall | 468 | 3 | 5 | 1.1% (5/476) | 0.3-2.4% |

| Age | Neg | Eqv | Pos | % Positive | 95%CI |
|----------|-----|-----|-----|--------------|-----------|
| 0 to 9 | 24 | 0 | 0 | 0.0% (0/24) | 0.0-14.2% |
| 10 to 19 | 29 | 0 | 0 | 0.0% (0/29) | 0.0-11.9% |
| 20 to 29 | 70 | 0 | 0 | 0.0% (0/70) | 0.0-5.1% |
| 30 to 39 | 82 | 0 | 0 | 0.0% (0/82) | 0.0-4.4% |
| 40 to 49 | 78 | 0 | 0 | 0.0% (0/78) | 0.0-4.6% |
| 50 to 59 | 51 | 0 | 0 | 0.0% (0/51) | 0.0-7.7% |
| 60 to 69 | 38 | 0 | 1 | 2.6% (1/39) | 0.1-13.5% |
| 70 to 79 | 34 | 0 | 0 | 0.0% (0/34) | 0.0-10.3% |
| 80+ | 17 | 0 | 1 | 5.6% (1/18) | 0.1-27.3% |
| Unknown | 51 | 0 | 0 | 0.0% (0/51) | 0.0-7.0% |
| Overall | 474 | 0 | 2 | 0.4% (2/476) | 0.1-1.5% |

{3}------------------------------------------------

# Performance Characteristics

Performance characteristics without background subtract are in the beft column, and with background subtract is in the right column.

# Study Site 1: Focus Reactivity with Encephalitis/Meningitis Patients (n = 300)

A state department of health laboratory located in the northeastern U.S. assessed the device's reactivity from encephalitis meningitis patients (n = 300). Patients were suspected of having either viral meningitis. Viral encephalitis criteria included: 1) fever, 2) altered mental status and/or other evidence of cortical involvement; and 3) CSF pleocytosis with predominant lymphocytes and/or elevated protein and a negative gram stain and culture.15 Viral meningitis criteria included: 1) fever, 2) headache, stiff neck and/or other meningeal signs; and 3) CSF pleocytosis with predominant lymphocytes and/or elevated protein and a negative gram stain and culture).12 The sera were sequentially submitted to the laboratory, archived, and masked. The reference methods were the CDC IgM ELISAs, and a plaque reduction neutralization test (PRNT) for West Nile virus. Of 300 encephalitis/meningitis patients, 44 were classified as confirmed positive West Nile encephalitis/neningitis symptoms. CDC IgM ELISA positive and WNV PRNT positive) and 256 had presumptive assay results (CDC WNV IgM ELISA) . 4 of the 256 presumptive assay results showed NS and were excluded.

### Without Background Subtract

The Focus IgM assay was positive with 90.9% (40/44) of the confirmed positive WNV encephalitis patients (including 2 Focus equivocals calculated as negatives). Of the 252 patients with presumptive assay results, 250 were classified as presumed negative patients (CDC WNV IgM ELISA negative), and 2 were classified as presumed positive West Nile encephalitis patients (CDC WNV IgM ELISA positive). The Focus IgM assay was positive with 100% (2/2) of the presumed positive WNV encephalitis patients. The Focus IgM assay was negative with 99.6% (249/250) of the presumed negative patients (including 1 Focus equivocal calculated as positive).

| Study Site 1: Focus Reactivity with |
|------------------------------------------|
| Encephalitis/Meningitis Patients (n=300) |

| Specimens<br>Characterized by<br>Reference Assays | Neg | Eqv | Pos | Total | % |
|-------------------------------------------------------------------------------------------------------------------------|-----|-----|-----|-------|---------------------------------------------------------------------------------------------|
| Clinical sensitivity<br>(encephalitis or<br>meningitis symptoms,<br>CDC IgM ELISA<br>positive and WNV<br>PRNT positive) | 2 | 2 | 40 | 44 | 90.9% (40/44)<br>95%CI 78.3-97.5% |
| Agreement with the<br>presumptive CDC<br>IgM ELISA | 249 | 1 | 0 | 250 | Positive<br>100% (2/2)<br>95%CI 15.8-100%<br>Negative<br>99.6% (249/250)<br>95%CI 97.8-100% |

# With Background Subtract

The Focus IgM assay was positive with 90.9% (40/44) of the confirmed positive WNV encephalitis patients (including 2 Focus equivocals calculated as negatives). Of the 252 patients with presumptive assay results, 250 were classified as presumed negative patients (CDC WNV IgM ELISA negative), and 2 were classified as presumed positive West Nile encephalitis patients (CDC WNV IgM ELISA positive). The Focus IgM assay was positive with 100% (2/2) of the presumed positive WNV encephalitis patients. The Focus IgM assay was negative with 100% (250/250) of the presumed negative patients.

Study Site 1: Focus Reactivity with Encenhalitis/Meningitis Patients (n=300)

| Encephalitis/Meningitis Patients (n=300) | | | | | |
|-------------------------------------------------------------------------------------------------------------------------|-----|-----|-----|-------|------------------------------------------------------------------------------------------------|
| Specimens<br>Characterized by<br>Reference Assays | Neg | Eqv | Pos | Total | % |
| Clinical sensitivity<br>(encephalitis or<br>meningitis symptoms,<br>CDC IgM ELISA<br>positive and WNV<br>PRNT positive) | 2 | 1 | 41 | 44 | 93.2% (41/44)<br>95%CI 78.3-97.5% |
| Agreement with the<br>presumptive CDC<br>IgM ELISA | 250 | 0 | 0 | 250 | Positive<br>100% (2/2)<br>95%CI 15.8-100%<br><br>Negative<br>100% (250/250)<br>95%CI 98.6-100% |

{4}------------------------------------------------

510(k) Summary of Safety and Effectiveness

West Nile Virus IgM Capture ELISA Catalog No. EL0300M Prepared June 28, 2004

Page 5 of 8

Performance Characteristics (continued)

# Study Site 2 & Study Site 4: Focus Reactivity with WNV PRNT Positives (n = 75)

Focus (background subtract) and a clinical laboratory (screening procedure) located in the mid-western U.S. assessed the device's reactivity with 75 retrospective samples with no clinical information that were pre-screened positive (by Focus) with a West Nile virus native antigen ELISA'.0, and confirmed West Nile positive by plaque reduction neutralization test (PRNT). The sera were sequentially submitted to the laboratory, archived, and masked.

# Without Background Subtract

The clinical laboratory located in the mid-western U.S. determined that the Focus IgM ELISA was positive with 100% (75/75) of the WNV PRNT positive samples.

# Study Site 2: Focus Reactivity with WNV PRNT

Positives (n = 75)

| Specimens<br>Characterized by<br>Reference Assays | Focus WNV IgM ELISA Results | | | | |
|---------------------------------------------------|-----------------------------|-----|-----|-------|---------------------------------|
| | Neg | Eqv | Pos | Total | % |
| Serological<br>sensitivity (WNV<br>PRNT positive) | 0 | 0 | 75 | 75 | 100% (75/75)<br>95%CI 95.2-100% |
| Positives (II – 70) | | | | | |
| Specimens<br>Characterized by<br>Reference Assays | Neg | Eqv | Pos | Total | % |
| Serological<br>sensitivity (WNV<br>PRNT positive) | 0 | 0 | 70 | 70 | 100% (70/70)<br>95%CI 94.9-100% |

# With Background Subtract

Focus determined that the Focus IgM ELISA was positive with 100% (70/70) of the WNV PRNT positive samples. Five samples were QNS for the background subtract procedure.

### Study Site 2: Focus Reactivity with WNV PRNT Positives (n = 70)

* Five of the 75 samples were QNS.

# Study Site 3: Focus Reactivity with West Nile IFA Negatives (n=103)

A clinical laboratory located in the southwestern U.S. assessed reactive samples that were West Nile IFA negative. 13

# Without Background Subtract

The Focus IgM ELISA was negative with 96.1% (99/103) of WNV IgM IFA negative samples (including one equivocal calculated as positive).

# Study Site 3: Focus Reactivity with West Nile IFA Negatives (n=103)

| Specimens<br>Characterized by<br>Reference Assays | | Focus WNV IgM ELISA Results | | | |
|---------------------------------------------------|-----------------------------|-----------------------------|-----|-------|-------------------------------------|
| | Neg | Eqv | Pos | Total | % |
| Negative agreement<br>with presumptive<br>WNV IFA | 99 | 1 | 3 | 103 | 96.1% (99/103)<br>95%CI 90.3-98.9% |
| Specimens<br>Characterized by<br>Reference Assays | Focus WNV IgM ELISA Results | | | | |
| | Neg | Eqv | Pos | Total | % |
| Negative agreement<br>with presumptive<br>WNV IFA | 101 | 1 | 1 | 103 | 98.1% (101/103)<br>95%CI 93.2-99.8% |

# With Background Subtract

The Focus IgM ELISA was negative with 98.1% (101/103)) of WNV IgM IFA negative samples (including one equivocal calculated as positive).

# Study Site 3: Focus Reactivity with West Nile IFA Negatives (n=103)

{5}------------------------------------------------

K040854 510(k) Summary of Safety and Effectiveness West Nile Virus IgM Capture ELISA Catalog No. EL0300M Prepared June 28, 2004 Page 6 of 8

Performance Characteristics (continued)

# Study Site 4: Focus Reactivity with Suspected Encephalitis/Meningitis Patients (n= 50)

Focus assessed the device's reactivity with 50 samples from patients mening tis. A U.S. federal government laboratory provided the archived and masked sera. One sample was confirmed positive by WNV PRNT, and the other 49 were presumptively negative (CDC ELISA) for arboviruses present in North America (LAC, EEE, SLE and WNV).

# Without Background Subtract

The Focus IgM ELISA was negative with 98.0% (48/49) of the WNV presumptive negative samples, and positive with the one WNV PRNT confirmed sample.

#### Study Site 4: Reactivity Suspected with Encephalitis/Meningitis Patients (n= 50)

| Specimens<br>Characterized by<br>Reference Assays | Focus WNV IgM ELISA Results | | | | |
|------------------------------------------------------------------------------------|-----------------------------|-----|-----|-------|-----------------------------------|
| | Neg | Eqv | Pos | Total | % |
| Serological<br>sensitivity (CDC<br>IgM ELISA positive<br>and WNV PRNT<br>positive) | 0 | 0 | 1 | 1 | 100% (1/1)<br>95%CI NA |
| Negative agreement<br>with presumptive<br>CDC IgM ELISA | 48 | 0 | 1 | 49 | 98.0% (48/49)<br>95%CI 89.1-99.9% |

# With Background Subtract

The Focus IgM ELISA was negative with 100% (49/49) of the WNV presumptive negative samples, and positive with the one WNV PRNT confirmed sample.

#### Study Site 4:Focus Reactivity with Suspected Encephalitis/Meningitis Patients

| Specimens<br>Characterized by<br>Reference Assays | Focus WNV IgM ELISA Results | | | | % |
|------------------------------------------------------------------------------------|-----------------------------|-----|-----|-------|---------------------------------|
| | Neg | Eqv | Pos | Total | |
| Serological<br>sensitivity (CDC<br>IgM ELISA positive<br>and WNV PRNT<br>positive) | 0 | 0 | 1 | 1 | 100% (1/1)<br>95%CI NA |
| Negative agreement<br>with presumptive<br>CDC IgM ELISA | 49 | 0 | 0 | 49 | 100% (49/49)<br>95%CI 92.7-100% |

# Study Site 4: Focus Reactivity with Non-Flavivirus Test Samples (n = 476)

Focus assessed the device's reactivity with 476 samples prospectively collected from North America during August 2003. The samples had been submitted to a clinical laboratory located in Southern California for infectious diseases. Positive samples were tested with a CDC WNV IgM ELISA.

# Without Background Subtract

The Focus West Nile IgM Capture ELISA was negative with 99.4% (468/471) of the CDC ELISA IgM negative samples (including 3 Focus equivocals included as positive), and positive with 33.3% (1/3) of the CDC ELISA IgM positive samples.. Four CDC ELISA IgM indeterminant samples were excluded from the calculations.

Study Site 4: Focus Reactivity with Non-Flavivirus Test Samples (n = 476)*

| Specimens<br>Characterized by<br>Reference Assays | Focus WNV IgM ELISA Results | | | | |
|---------------------------------------------------------|-----------------------------|-----|-----|-------|-------------------------------------|
| | Neg | Eqv | Pos | Total | % |
| Positive agreement<br>with presumptive<br>CDC IgM ELISA | 0 | 2 | 1 | 3 | 33.3% (1/3)<br>95%CI 0.8-90.6% |
| Negative agreement<br>with presumptive<br>CDC IgM ELISA | 468 | 1 | 0 | 469 | 99.8% (468/469)<br>95% CI 98.8-100% |

* Excludes four samples that were indeterminant with the CDC IgM ELISA.

# With Background Subtract

The Focus West Nile IgM Capture ELISA was negative with 100% (469/469) of the CDC ELISA IgM negative samples, and positive with 66.7% (2/3) of the CDC ELISA IgM positive samples.. Four CDC ELISA IgM samples were excluded from indeterminant the calculations.

| Study Site 4: Focus Reactivity with Non-Flavivirus |
|----------------------------------------------------|
| Test Samples (n = 476)* |

| Specimens<br>Characterized by<br>Reference Assays | Neg | Eqv | Pos | Total | % |
|---------------------------------------------------------|-----|-----|-----|-------|------------------------------------|
| Positive agreement<br>with presumptive<br>CDC IgM ELISA | 1 | 0 | 2 | 3 | 66.7% (2/3)<br>95%CI 9.4-99.2% |
| Negative agreement<br>with presumptive<br>CDC IgM ELISA | 469 | 0 | 0 | 469 | 100% (469/469)<br>95% CI 99.2-100% |

* Excludes four samples that were indeterminant with the CDC IgM ELISA.

{6}------------------------------------------------

# Performance Characteristics (continued)

### Focus Cross-reactivity

Focus (Study Site 4) and a state department of health laboratory located in the northeastern U.S. (DOH) (Study Site 1) assessed the device's cross-reactivity with sera that were sero-positive to other potentially cross-reactive pathogens (n = 75). The DOH tested the SLE positives, and Focus tested the other sera. The sera were retrospective and masked. The results of the studies are summarized in the table below:

| Focus Cross-reactivity without Background Subtract | | | | | | |
|----------------------------------------------------|------|-----------------------------|-----|-----|------------|----------------------------------|
| Specimens<br>characterized by<br>Reference Assays | Site | Focus WNV IgM ELISA Results | | | % Positive | |
| | | Neg | Eqv | Pos | Total | |
| Dengue virus<br>(secondary<br>infections) | 4 | 6 | 1 | 8 | 15 | 40.0% (6/15)<br>95%CI 16.3-67.7% |
| St. Louis<br>encephalitis virus | 1 | 6 | 0 | 7 | 13 | 53.8% (7/13)<br>95%CI 25.1-80.8% |
| Eastern equine<br>encephalitis virus | 4 | 2 | 0 | 0 | 2 | 0.0% (0/2)<br>95%CI 0.0-84.2% |
| Herpes simplex<br>virus | 4 | 18 | 1 | 1 | 20 | 10.0% (2/20)<br>95%CI: 1.2-31.7% |
| Epstein-Barr virus | 4 | 19 | 0 | 0 | 19 | 0.0% (0/19)<br>95%CI 0.0-17.6% |
| Cytomegalovirus | 4 | 13 | 0 | 1 | 14 | 7.1% (1/14)<br>95%CI 0.2-33.9% |
| Borrelia<br>burgdorferi | 4 | 0 | 0 | 1 | 20 | 5.0% (1/20)<br>95%CI 0.1-24.9% |
| Rheumatoid factor | 4 | 0 | 1 | 4 | 20 | 25.0% (5/20)<br>95%CI 3.7-49.1% |
| Anti-nuclear<br>antibodies | 4 | 0 | 0 | 1 | 20 | 5.0% (1/20)<br>95%CI 0.1-24.9% |
| Polio virus | 4 | 10 | 0 | 0 | 10 | 0.0% (0/10)<br>95%CI 0.0-30.8% |

| Focus Cross-reactivity with Background Subtract | | | | | | |
|---------------------------------------------------|------|-----|-----|-----|-------|----------------------------------|
| Specimens<br>characterized by<br>Reference Assays | Site | Neg | Eqv | Pos | Total | % Positive |
| Dengue virus<br>(secondary infections) | 4 | 9 | 3 | 3 | 15 | 40.0% (6/15)<br>95%CI 16.3-67.7% |
| St. Louis<br>encephalitis virus* | NA | NA | NA | NA | NA | Not tested. |
| Eastern equine<br>encephalitis virus | 4 | 2 | 0 | 0 | 2 | 0.0% (0/2)<br>95%CI 0.0-84.2% |
| Herpes simplex<br>virus | 4 | 20 | 0 | 0 | 20 | 0.0% (0/20)<br>95%CI: 0.0-16.8% |
| Epstein-Barr virus | 4 | 19 | 0 | 0 | 19 | 0.0% (0/19)<br>95%CI 0.0-17.6% |
| Cytomegalovirus | 4 | 13 | 0 | 1 | 14 | 0.0% (0/14)<br>95%CI 0.0-23.2% |
| Borrelia<br>burgdorferi | 4 | 20 | 0 | 0 | 20 | 0.0% (0/20)<br>95%CI: 0.0-16.8% |
| Rheumatoid factor | 4 | 20 | 0 | 0 | 20 | 0.0% (0/20)<br>95%CI: 0.0-16.8% |
| Anti-nuclear<br>antibodies | 4 | 20 | 0 | 0 | 20 | 0.0% (0/20)<br>95%CI: 0.0-16.8% |
| Polio virus | 4 | 10 | 0 | 0 | 10 | 0.0% (0/10)<br>95%CI 0.0-30.8% |

* Positive and equivocal SLE samples were not tested with the background subtract procedure.

#### Focus Reproducibility

Focus (Study Site 4), a clinical laboratory located in the mid-west United States (Study Site 5), and a university laboratory located in northern California (Study Site 6) assessed the reproducibility of the assay with and without the background subtract procedure. Each laboratory tested seven samples in three runs per day for three days. Of the seven samples, three samples were negative (BS1, BS2 and BS6), two samples were positive in the assay and with background subtract (BS22 and BS3), and two samples were positive in the assay but negative in background subtract (BS21 and BS23, these samples were masked replicates). The studies are summarized in the tables below:

| Focus Reproducibility without Background Subtract | | | | | |
|---------------------------------------------------|--|--|--|--|--|
|---------------------------------------------------|--|--|--|--|--|

| ID | Mean<br>Index | Inter-Lab<br>%CV | Inter-<br>assay<br>%CV | Intra-<br>assay<br>%CV |
|-------|---------------|------------------|------------------------|------------------------|
| BS1 | 0.06 | 36.9 | 42.5 | 15.4 |
| BS6 | 0.07 | 22.5 | 31.2 | 13.2 |
| BS2 | 0.09 | 15.1 | 27.6 | 14.7 |
| BS22 | 1.49 | 1.5 | 5.2 | 3.0 |
| BS3 | 2.49 | 3.6 | 6.2 | 3.7 |
| BS21* | 2.72 | 24.4 | 23.3 | 4.3 |
| BS23* | 2.75 | 25.3 | 24.0 | 2.6 |

* These samples were masked replicates

#### Focus Reproducibility with Background Subtract

| ID | Mean<br>Index | Inter-Lab<br>%CV | Inter-<br>assay<br>%CV | Intra-<br>assay<br>%CV |
|-------|---------------|------------------|------------------------|------------------------|
| BS1 | NA | NA | NA | NA |
| BS6 | NA | NA | NA | NA |
| BS2 | NA | NA | NA | NA |
| BS22 | 1.46 | 2.1 | 7.6 | 3.4 |
| BS3 | 2.47 | 1.2 | 8.6 | 3.6 |
| BS21* | -0.08 | -92.0 | -198.3 | -351.7 |
| BS23* | -0.06 | -41.8 | -194.2 | -127.6 |

* These samples were masked replicates

{7}------------------------------------------------

### Performance Characteristics (continued)

### Specificity of the Focus WNV IgM Assay

Focus (Study Site 4) assessed specificity of the WNV IgM Assay by selecting fifteen different sera that were positive for both WNV IgM and IgG. The sera were treated with 5 uL of 1.43 M (10% v/v) 2-mercaptoethanol (2-ME). Treating with 2-ME caused 100% (15/15) of the samples to become IgM negative.

#### Sera Freeze-Thaw Study

Focus (Study Site 4) assessed the impact on the WNV IgM assay's reactivity by selecting 8 sera (5 positive and 3 negative), subjecting them to up to 5 repeated freeze-thaw cycles, and testing them in parallel with aliquots that had not been frozen. There were no changes in interpretation in any of the sera. Positive samples trended slightly towards increasing indices, while negative sera did not appear to change.

#### Reproducibility

Reproducibility studies included Inter-lot Reproducibility, Inter/Intra-assay Reproducibility, and Inter-laboratory Reproducibility. In each study, two sets of samples were masked duplicates. Focus (Study Site 4) assessed the device's Inter-lot Reproducibility by testing five samples on three separate lots. For one lot. For one lot. the samples were run in triplicate, and run in duplicate with the other two lots. Each of the three lots had a different lot of Antigen and Capture Wells. Focus (Study Site 4) assessed the device's Inter/Intra-assay Reproducibility by testing seven samples in triplicate, once a day, for three days, for a total of 63 data points. A state department of health laboratory located in the northeastern U.S. (Study Site 1), a clinical laboratory located in the mid-western U.S. (Study Site 2), and Focus (Study Site 4), assessed the device's Inter-laboratory Reproducibility. Each of the three laboratories in triplicate on three different days.

| Sample | Inter- & Intra-assay | | | Inter-lot | | Inter-Lab | |
|--------|----------------------|--------------------|--------------------|---------------|--------------|---------------|--------------|
| | Index<br>Mean | Intra-assay<br>%CV | Inter-assay<br>%CV | Index<br>Mean | Index<br>%CV | Index<br>Mean | Index<br>%CV |
| M2* | 0.21 | 2.9 | 10.3 | 0.22 | 1.2 | 0.23 | 9.7 |
| M6* | 0.23 | 3.4 | 20.0 | 0.23 | 0.4 | 0.24 | 13.2 |
| M5 | 0.69 | 1.6 | 5.7 | 0.70 | 0.7 | 0.71 | 6.4 |
| M1* | 1.43 | 1.5 | 2.9 | 1.41 | 2.6 | 1.45 | 4.0 |
| M7* | 1.53 | 1.8 | 4.0 | 1.54 | 2.1 | 1.49 | 12.8 |
| M3 | 2.37 | 2.7 | 1.7 | 2.33 | 3.6 | 2.23 | 2.5 |
| M4 | 2.99 | 1.9 | 0.3 | 2.98 | 1.9 | 2.78 | 2.3 |

# re du ~iktlitz

* There were two sets of masked pairs (same sample, different labeled identity): M2 & M6 were one masked pair, and M1 & M7 were the second masked pair.

{8}------------------------------------------------

| 510(k) Number (if known): | K040854 |
|---------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Device Name: | West Nile Virus IgM Capture ELISA |
| Indications for Use: | The Focus Technologies West Nile Virus IgM Capture ELISA is<br>intended for qualitatively detecting IgM antibodies to West Nile<br>virus in human serum. In conjunction with the Focus Technologies<br>West Nile Virus ELISA IgG, the test is indicated for testing<br>persons having symptoms of meningioencephalitis, as an aid in the<br>presumptive laboratory diagnosis of West Nile virus infection.<br>Positive results must be tested using the background subtraction<br>method (either on the initial test or on a repeat test). Positive<br>results must be confirmed by neutralization test, or by using the<br>current CDC guidelines for diagnosing West Nile encephalitis.<br>This test is not intended for self-testing, and this test is not FDA<br>cleared nor approved for testing blood or plasma donors. Assay<br>performance characteristics have not been established for<br>automated instruments. |

Prescription Use _ X (Part 21 CFR 801 Subpart D)

AND/OR

Over-the-Counter Use (21 CFR 807 Subpart C)

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

Concurrence of CDRH, Office of In Vitro Diagnostic Devices (OIVD)

{9}------------------------------------------------

# DEPARTMENT OF HEALTH & HUMAN SERVICES

Public Health Service

Image /page/9/Picture/2 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES U.S.A." arranged around the perimeter. Inside the circle is an abstract symbol that resembles a stylized caduceus or a bird in flight, composed of three curved lines.

JUN 3 0 2004

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Michael J. Wagner, Esq. Senior Regulatory Affairs Specialist Focus Technologies, Inc. 10703 Progress Way Cypress, CA 90630

k040854 Re:

> Trade/Device Name: Focus West Nile Virus IgM Capture ELISA Regulation Number: 21 CFR 866.3940 Regulation Name: West Nile Virus Serological Reagents Regulatory Class: Class II Product Code: NOP Dated: June 6, 2004 Received: June 8, 2004

Dear Mr. Wagner:

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.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. 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); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820).

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

# Page 2

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

If you desire specific information about the application of labeling requirements to your device, or questions on the promotion and advertising of your device, please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at [phone]. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21CFR Part 807.97). You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number [phone] or [phone] or at its Internet address http://www.fda.gov/cdrh/dsma/dsmamain.html.

Sincerely yours,

Salazar

Sally A. Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

{11}------------------------------------------------

# Indications for Use

510(k) Number (if known):K040854

Device Name: Focus West Nile Virus IgM Capture ELISA

Indications For Use: The Focus Technologies West Nile Virus IgM Capture ELISA is intended for qualitatively detecting IgM antibodies to West Nile virus in human serum. In conjunction with the Focus Technologies West Nile Virus ELISA IgG, the test is indicated for testing persons having symptoms of meningioencephalitis, as an aid in the presumptive laboratory diagnosis of West Nile virus infection. Positive results must be tested using the background subtraction method (either on the initial test or on a repeat test). Positive results must be confirmed by neutralization test, or by using the current CDC quidelines for diagnosing West Nile encephalitis. This test is not intended for selftesting, and this test is not FDA cleared nor approved for testing blood or plasma donors. Assay performance characteristics have not been established for automated instruments.

Prescription Use
(Part 21 CFR 801 Subpart D)

AND/OR

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

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

Concurrence of CDRH, Office of Device Evaluation (ODE)

Saga Atto 6/23/04
Division Sign-Off

Office of In Vitro Diagnostic Device Evaluation and Safety

KO40854 510(k) _

Page 1 of