D3 DUET DFA INFLUENZA A/RESPIRATORY VIRUS SCREENING KIT

K081746 · Diagnostic Hybrids, Inc. · GNW · Dec 23, 2008 · Microbiology

Device Facts

• Record ID: K081746
• Device Name: D3 DUET DFA INFLUENZA A/RESPIRATORY VIRUS SCREENING KIT
• Applicant: Diagnostic Hybrids, Inc.
• Product Code: GNW · Microbiology
• Decision Date: Dec 23, 2008
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3330
• Device Class: Class 1

Intended Use

The Diagnostic Hybrids, Inc. device, D³ Duet DFA Influenza A/Respiratory Virus Screening Kit, is intended for the qualitative detection and identification of influenza A, while screening for influenza B virus, respiratory syncytial virus, adenovirus, and parainfluenza virus types 1, 2 and 3 viral antigens, in nasal and nasopharyngeal swabs and aspirates or in cell culture. The assay detects viral antigens by immunofluorescence using monoclonal antibodies (MAbs), from patients with signs and symptoms of respiratory infection. It is recommended that specimens found to be negative after examination of the direct specimen result be confirmed by cell culture. Negative results do not preclude influenza virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. Performance characteristics for influenza A virus detection and identification were established when influenza A H3N2 and influenza A H1N1 were the predominant influenza A strains circulating in the United States. When other influenza A viruses are emerging, performance characteristics may vary. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to a state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

Device Story

In vitro diagnostic kit for qualitative detection of respiratory viral antigens; utilizes direct immunofluorescence assay (DFA). Input: nasal/nasopharyngeal swabs, aspirates, or cell culture specimens. Process: specimens fixed on glass slides with acetone; incubated with blend of murine monoclonal antibodies (MAbs). Influenza A MAbs labeled with R-phycoerythrin (R-PE); other viral MAbs labeled with fluorescein isothiocyanate (FITC). Output: fluorescence observed via microscope; golden-yellow fluorescence indicates influenza A; apple-green indicates other screened viruses; red counterstain (Evans Blue) marks uninfected cells. Used in clinical laboratories by trained personnel. Results aid in diagnosis of respiratory infections; negative results require cell culture confirmation. Benefits include rapid identification of influenza A and screening for multiple other respiratory pathogens.

Clinical Evidence

Prospective study of 1184 fresh clinical specimens and 298 cultured specimens across three sites. Compared D³ Duet against predicate DFA assay. For Influenza A, PPA was 99% (94.5-99.8% CI) and NPA was 100% (99.7-100% CI). For the six screened viruses, PPA was 100% (99.0-100% CI) and NPA was 100% (99.5-100% CI). Cultured specimen study showed 100% PPA and NPA for Influenza A and 100% PPA/NPA for the other six viruses. No clinical sensitivity/specificity metrics required as this is an in vitro diagnostic test.

Technological Characteristics

Immunofluorescence assay using murine monoclonal antibodies. Influenza A MAbs labeled with R-phycoerythrin (R-PE); other viral MAbs labeled with fluorescein isothiocyanate (FITC). Includes Evans Blue counterstain, Tween 20/PBS wash solution, and glycerol mounting fluid. Preserved with 0.1% sodium azide. Manual procedure involving slide fixation, incubation, and fluorescence microscopy. No software or electronic components.

Indications for Use

Indicated for qualitative detection/identification of influenza A and screening for influenza B, RSV, adenovirus, and parainfluenza types 1, 2, and 3 in nasal/nasopharyngeal swabs, aspirates, or cell culture from patients with respiratory infection symptoms.

Regulatory Classification

Identification

Influenza virus serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies to influenza in serum. The identification aids in the diagnosis of influenza (flu) and provides epidemiological information on influenza. Influenza is an acute respiratory tract disease, which is often epidemic.

Predicate Devices

• D3 Ultra DFA Respiratory Virus Screening & ID Kit (k081746)

Submission Summary (Full Text)

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K081746

D3 DUET DFA INFLUENZA A/RESPIRATORY VIRUS SCREENING KIT

# SECTION 05, 510(K) SUMMARY

## Applicant:

DIAGNOSTIC HYBRIDS, INC. 1055 East State Street Suite 100 Athens, OHIO 45701

DEC 2 3 2008

#### Contact Information:

Gail R. Goodrum Vice President, Regulatory Affairs E-mail: goodrum(@dhiusa.com Telephone: [phone] Desk Extension: 740-589-3380 FAX: [phone]

#### Date of preparation of 510(k) summary:

June 13, 2008

### Device Name:

Trade name - D3 Duet DFA Influenza A/Respiratory Virus Screening Kit Common name - Fluorescent antibody test for screening Influenza A Classification name - Antisera, Cf, Influenza Virus A, B, C Product Code - GNW Regulation - 21 CFR 866.3330, Class I, Influenza virus serological reagents; Panel Microbiology (83)

### Legally marketed device to which equivalence is claimed:

K061101, D3 Ultra DFA Respiratory Virus Screening & ID Kit

Intended Use: The Diagnostic Hybrids, Inc. device, D3 Duet DFA Influenza A/Respiratory Virus Screening Kit, is intended for the qualitative detection and identification of influenza A, while screening for influenza B virus, respiratory syncytial virus, adenovirus, and parainfluenza virus types 1, 2 and 3 viral antigens. in nasal and nasopharyngeal swabs and aspirates or in cell culture. The assay detects viral antigens by immunofluorescence using monoclonal antibodies (MAbs), from patients with signs and symptoms of respiratory infection.

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It is recommended that specimens found to be negative after examination of the direct specimen result be confirmed by cell culture. Negative results do not preclude influenza virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

Performance characteristics for influenza A virus detection and identification were established when influenza A H3N2 and influenza A H1N1 were the predominant influenza A strains circulating in the United States. When other Influenza A viruses are emerging, performance characteristics may vary. If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to a state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

## Device Description:

The Diagnostic Hybrids, Inc. device, D Duet DFA Influenza A/Respiratory Virus Screening Kit, uses a blend of viral antigen-specific murine MAbs. MAbs for influenza A virus are directly labeled with R-phycoerythrin (R-PE) for the rapid detection and identification of influenza A virus. MAbs for influenza B virus, respiratory syncytial virus, adenovirus, and parainfluenza virus types 1, 2, and 3 are directly labeled with fluorescein isothiocyanate (FITC), for rapid detection of these agents.

### Kit components:

- · D3 Duet DFA Influenza A/Respiratory Virus Screening Reagent Rphycoerythrin-labeled murine MAbs directed against influenza A virus and a mixture of fluorescein-labeled murine MAbs directed against influenza B. respiratory syncytial virus, adenovirus, and parainfluenza virus types 1, 2, and 3. The buffered, stabilized, aqueous solution also contains Evans Blue as a counterstain and 0.1% sodium azide as preservative.
- Normal Mouse Gamma Globulin DFA Reagcnt a mixture of fluorescein labeled murine gamma globulin that has been shown to be non-reactive with any of the listed respiratory viruses. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as preservative.
- · Respiratory Virus Antigen Control Slides five individually packaged control slides containing wells with cell culture-derived positive and negative control cells. Each positive well is identified with the virus infected cells present, i.e., influenza A virus, influenza B virus, respiratory syncvtial virus, adenovirus, and parainfluenza virus types 1, 2 and 3. The negative well contains uninfected cultured cells. Each slide is intended to be stained only one time.
- · Wash Solution Concentrate a 40X concentrate consisting of Tween 20 and 4% sodium azide (0.1% sodium azide after dilution in de-mineralized water) in a 40X phosphate buffered saline solution.

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- Mounting Fluid an aqueous, buffered, stabilized solution of glycerol and . 0.1% sodium azide.
The cells to be tested, derived from a clinical specimen or cell culture, are placed onto a glass slide and allowed to air dry. The cells are fixed in acetone. The D3 Duet DFA Influenza A/Respiratory Virus Screening Reagent is added to the cells which are then incubated for 15 to 30 minutes at 35° to 37°C in a humidified chamber or humidified incubator. The stained cells are then washed with the diluted wash solution, a drop of the supplied Mounting Fluid is added and a coverslip is placed on the prepared cells. The cells are examined using a fluorescence microscope. The influenza A virus infected cells will fluoresce golden-yellow, while cells infected with any of the other six viruses will fluoresce apple-green. Uninfected cells will contain no fluorescence but will be stained rcd by the Evans Blue counter-stain. If only golden-yellow fluorescent cells are present the specimen can be reported as positive for influenza A antigen. If only apple-green fluorescent cells are present, the particular virus may be identified using the individual reagents from the DS Ultra™ DFA Respiratory Virus Screening & ID Kit (D3 Ultra) on new, separate cell preparations. If both golden-yellow and apple-green are present, the additional virus may be identified using the individual reagents from the D3 Ultra on new, separate cell preparations.

## Technological Characteristics:

The DHI device, D3 Duet, has been compared directly to the DHI device, D3 Ultra, as the legally marketed device. The technology used in both devices is based on a standard immunofluorescence assay technique utilizing either R-PF. or FITC-labeled MAbs. A summary is provided in Table 5.1 below:

| TABLE 5.1: Technological Characteristics Comparison | | | |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Characteristic | D3 Duet DFA Influenza A/<br>Respiratory Virus Screening Kit | D3 Ultra DFA Respiratory Virus<br>Screening & ID Kit | |
| Monoclonal antibodies (MAbs) | The Influenza A/Respiratory Virus<br>DFA Screening Reagent<br>contains 12 MAbs to 6 different<br>respiratory viruses (influenza B virus,<br>respiratory syncytial virus, adenovirus,<br>parainfluenza virus type 1,<br>parainfluenza virus type 2,<br>parainfluenza virus type 3), plus 2<br>MAbs to influenza A virus.<br>One of the 2 MAbs to influenza A<br>virus is different from either of those<br>used in the D3 Ultra Reagent; the<br>second is the same. | The Respiratory Virus DFA Screening<br>Reagent<br>contains 12 MAbs to 6 different<br>respiratory viruses (influenza B virus,<br>respiratory syncytial virus, adenovirus,<br>parainfluenza virus type 1,<br>parainfluenza virus type 2,<br>parainfluenza virus type 3), plus 2<br>MAbs to influenza A virus. | |
| TABLE 5.1: Technological Characteristics Comparison | | | |
| Characteristic | | D3 Duet DFA Influenza A/<br>Respiratory Virus Screening Kit | D3 Ultra DFA Respiratory Virus<br>Screening & ID Kit |
| Labeling method | | Direct labeling,<br>- using R-phycoerythrin (R-PE) to<br>label the MAbs to influenza A virus<br>antigens<br>- using fluorescein isothiocyanate<br>(FITC) to label all other MAbs with<br>fluorescein moiety | Direct labeling,<br>- using fluorescein isothiocyanate<br>(FITC) to label all other MAbs with<br>fluorescein moiety |
| Fluorescein-labeled MAbs | | Influenza B virus, respiratory syncytial<br>virus, adenovirus, parainfluenza virus<br>type 1, parainfluenza virus type 2,<br>parainfluenza virus type 3 | Influenza A virus, influenza B virus,<br>respiratory syncytial virus, adenovirus<br>parainfluenza virus type 1,<br>parainfluenza virus type 2,<br>parainfluenza virus type 3 |
| Phycoerythrin-labeled MAbs | | Influenza A virus<br>(Phycoerythrin-labeled influenza A<br>virus MAbs stain with golden-yellow<br>fluorescence) | None<br>(Fluorescein-labeled influenza A virus<br>MAbs stain with apple-green<br>fluorescence) |
| Cell Fixative | | Cell Fixative is the same for both devices:<br>Acetone | |
| Performance characteristics | | | |
| Staining patterns | | Staining patterns are the same for both devices:<br>Influenza A and B: The fluorescence is cytoplasmic, nuclear or both.<br>Cytoplasmic staining is often punctate with large inclusions while nuclear<br>staining is uniformly bright.<br>Respiratory Syncytial Virus: The fluorescence is cytoplasmic and punctate<br>with small inclusions in the syncytia.<br>Parainfluenza 1, 2, 3: The fluorescence is cytoplasmic and punctate with<br>irregular inclusions. Types 2 and 3 cause the formation of syncytia.<br>Adenovirus: The fluorescence is cytoplasmic and punctate or bright nuclear or<br>both. | |
| Analytical sensitivity, according<br>to 96-well cell culture plates<br>infected with Flu A diluted to<br>give a TCID50 of 1 per 0.2-mL<br>inoculum (reported as average<br>of 4 runs) | | There is no significant difference between the two devices for analytical<br>sensitivity.<br><br>34.3 ± 12.0<br>culture positives out of 96 | |
| | | | 34.8 + 9.7<br>culture positives out of 96 |
| Analytical specificity (for<br>influenza A virus strains; MAbs<br>are reactive with all listed<br>strains) | | Mabs to influenza A virus were shown to be reactive with these virus strains: | |
| | | 9 Flu A strains (Aichi, VR-547<br>(H3N2); Mal, VR-98 (H1N1); Hong<br>Kong, VR-544 (I13N2); Denver, VR-<br>546 (H1N1); Port Chalmers, VR-810<br>(H3N2); Victoria, VR-822 (H3N2);<br>New Jersey, VR-897(H1N1); WS, VR-<br>1520 (H1N1); PR, VR-95 (H1N1)) | 9 Flu A strains (Aichi, VR-547<br>(H3N2); Mal, VR-98 (H1N1); Hong<br>Kong, VR-544 (H3N2); Denver, VR-<br>546 (H1N1); Port Chalmers, VR-810<br>(H3N2); Victoria, VR-822 (H3N2);<br>New Jersey, VR-897(H1N1); WS, VR-<br>1520 (H1N1); PR, VR-95 (H1N1)) |
| | | | |
| | | | |
| | | | |
| | | | |
| Analytical<br>specificity (cross<br>reactivity studies;<br>various strains of<br>microorganisms | | Device Screening Reagent is not reactive with these microorganisms: | |
| | Viruses | 32 | 31 |
| | Bacteria | 25 | 18 |
| | Chlamydia<br>spp. | 3 | 1 |
| Characteristic<br>and cell lines) | D3 Duet DFA Influenza A/<br>Respiratory Virus Screening Kit | D3 Ultra DFA Respiratory Virus<br>Screening & ID Kit | |
| Yeast | 1 | 0 | |
| Protozoan | 1 | 0 | |
| Cell lines | 17 | 17 | |

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D³ DUET DFA INFLUENZA A/RESPIRATORY VIRUS SCREFNING KIT Page 5 of 13

## Non-Clinical Performance:

Staining patterns of the phycoerythrin-labeled influenza A virus MAbs on influenza A virus infected cells were similar to those of the Predicate device.

## Precision/Reproducibility:

Assay precision, intra-assay variability and inter assay variability were assessed with a panel of proficiency-level antigen control slides. The panel consisted of slides spotted with cell preparations of the following:

- 1. Low level influenza A (Victoria strain)
- 2. Mid level influenza A (Victoria strain)
- 3. Low level influenza A (Victoria strain) mixed with Mid level RSV (Washington strain)
- 4. Mid level influenza A (Victoria strain) mixed with Low level RSV (Washington strain)
- 5. Low level respiratory virus (either influenza virus B {Taiwan strain}, adenovirus type 1, Parainfluenza virus types 1, 2, or 3 (strains C35, Greer, C243 respectively). This panel member was rotated during the 5-days of testing so that cach virus is tested twice.
- 6. Negative no infected cells present

The low level is estimated to contain between 4 to 10% infected cells per cell spot. The mid level is estimated to contain between 20 to 25% infected cells per cell spot. Both levels were below the level used in quality control slides. Each panel member was re-coded daily to prevent its identification. Each panel was stained twice per day for 5-days by three diffcrent laboratorics.

The following results were recorded for both the control slide and the panel slide:

- 1. Presence or absence of Yellow-gold fluorescence.
- 2. Percent of cells exhibiting Yellow-gold fluorescence
- 3. Presence or absence of Green fluorescence

4. Percent of cells exhibiting Green fluorescence

The combined data for negative specimens -- no infected cells present - from the three sites demonstrates that the R-PF labeled and FITC labeled MAbs reproducibly do not stain uninfected cells. No fluorescent cells were scen in 100% (60/60) of the wells lacking infected cells.

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The combined data from the three sites demonstrates reproducible detection of influenza A virus by the R-PE labeled MAbs. The presence of influenza A virus infected cells was reported in 95.3% (143/150) of the wells in which the infected cells were expected:

| Influenza A virus detection Summary | | | | |
|-------------------------------------|--------------------|--------------------|---------------------------------|---------------------------------|
| Positive<br>Control Slide | Low Level<br>Slide | Mid-Level<br>Slide | Low Level with<br>Mid-Level RSV | Mid-Level with<br>Low Level RSV |
| 100% (30/30) | 100% (30/30) | 100% (30/30) | 83.3% (25/30) | 93.3% (28/30) |

The combined data demonstrates the reproducibility of the detection of respiratory syncytial virus by the FITC labeled MAbs. The presence of respiratory syncytial virus infected cells was reported in 100% (90/90) of the wells in which the infected cells were expected:

Respiratory syncytial virus detection Summary

| Positive Control Slide | Low Level Influenza A<br>with Mid-Level RSV | Mid-Level Influenza A<br>with Low Level RSV |
|------------------------|---------------------------------------------|---------------------------------------------|
| 100% (30/30) | 100% (30/30) | 100% (30/30) |

The combined data demonstrates that the presence of R-PE fluorescent cells reproducibly does not interfere with the detection of respiratory syncytial virus by the FITC labeled MAbs. The presence of respiratory syncytial virus infected cells was reported in 100% (53/53) of the wells in which the R-PE stained infected cells were present:

Respiratory syncytial virus detection in the presence of R-PE positive cells Summary

| Low Level R-PE stained cells with Mid-<br>Level RSV | Mid-Level R-PE stained cells with Low<br>Level RSV |
|-----------------------------------------------------|----------------------------------------------------|
| 100% (25/25) | 100% (28/28) |

The combined data from all three sites demonstrates that the presence of R-PE in the stain reproducibly does not interfere with the FITC staining of other viruses. The presence of influenza B virus infected cells was reported in 100% (36/36) of the wells in which the infected cells were expected. The presence of adenovirus infected cells was reported in 100% (36/36) of the wells in which the infected cells were expected. The presence of parainfluenza virus type 1 virus infected cells was reported in 100% (36/36) of the wells in which the infected cells were expected. The presence of parainfluenza virus type 2 virus infected cells was reported in 100% (36/36) of the wells in which the infected cells were expected. The presence of parainfluenza virus type 3 virus infected cells was reported in 100% (36/36) of the wells in which the infected cells were expected.

| Respiratory virus detection in the presence of R-PE Summary | | | | | |
|-------------------------------------------------------------|--|--|--|--|--|
| | | | | | |

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D³ DUET DFA INFLUENZA A/RESPIRATORY VIRUS SCREENING KIT Page 7 of 13

| Adenovirus<br>Control Slide | Low Level<br>Adenovirus | Influenza B<br>Virus Control<br>Slide | Low Level<br>Influenza B<br>Virus | Parainfluenza<br>type 1<br>Control Slide | Low Level<br>Parainfluenza<br>type 1 |
|------------------------------------------|--------------------------------------|------------------------------------------|--------------------------------------|------------------------------------------|--------------------------------------|
| 100% (30/30) | 100% (6/6) | 100% (30/30) | 100% (6/6) | 100% (30/30) | 100% (6/6) |
| Parainfluenza<br>type 2<br>Control Slide | Low Level<br>Parainfluenza<br>type 2 | Parainfluenza<br>type 3<br>Control Slide | Low Level<br>Parainfluenza<br>type 3 | | |
| 100% (30/30) | 100% (6/6) | 100% (30/30) | 100% (6/6) | | |

The reproducibility study data demonstrates that the presence of R-PE in the stain reproducibly does not interfere with the detection of the 5 respiratory viruses by their respective FITC labeled MAbs.

## Analytical specificity

Results for analytical detection limit for the seven viruses detected by the D3 Duet were reported in numbers of fluorescent cells per cell monolayer. Each master stock virus preparation was diluted in a ten-fold manner. Four wells of a 96-well cell culture plate were inoculated with each dilution. The plates were centrifuged at 700 xg for 60 minutes, and then incubated at 35° to 37°C for 24hours. Four wells from each dilution were stained with the D3 Duet. Each well was then examined at 200x magnification and the number of fluorescent cells counted. The table below lists the virus identity and strain along with the fluorescent cell count.

| Analytical Sensitivity of D3 Duet compared with that of<br>D3 Ultra MAbs | | | |
|--------------------------------------------------------------------------|-----------------------------------------|---------------------------------|------------|
| (values are numbers of fluorescent cells per cell<br>monolayer) | | | |
| Virus strain | Virus<br>Dilutions from<br>master stock | Fluorescent staining cells/well | |
| | | D3 Duet | D3 Ultra |
| Influenza A virus<br>(PR, VR-95 H1N1) | 1x10-5 | 1, 3, 2, 6 | 1, 3, 0, 5 |
| | 1x10-6 | 1, 0, 1, 1 | 0, 0, 1, 0 |
| | 1x10-7 | 0, 0, 0, 0 | 0, 0, 0, 0 |
| Influenza B virus<br>(Hong Kong, VR-823) | 1x10-4 | 4, 1, 6, 2 | 0, 4, 3, 5 |
| | 1x10-5 | 1, 0, 1, 1 | 0, 0, 2, 2 |
| | 1x10-6 | 0, 0, 0, 0 | 0, 0, 0, 0 |
| Adenovirus (Type<br>8, VR-8) | 1x10-6 | 1, 1, 3, 5 | 1, 3, 2, 4 |
| | 1x10-7 | 0, 0, 0, 0 | 0, 0, 0, 0 |
| RSV (Washington, | 1x10-2 | 1, 0, 3, 4 | 2, 3, 2, 0 |

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| Analytical Sensitivity of D3 Duet compared with that of<br>D3 Ultra MAbs | | | |
|--------------------------------------------------------------------------|-----------------------------------------|---------------------------------|------------|
| (values are numbers of fluorescent cells per cell monolayer) | | | |
| Virus strain | Virus<br>Dilutions from<br>master stock | Fluorescent staining cells/well | |
| | | D3 Duet | D3 Ultra |
| VR-1401) | 1x10-3 | 0, 1, 1, 0 | 2, 1, 0, 0 |
| | 1x10-4 | 0, 0, 0, 0 | 0, 0, 0, 0 |
| Parainfluenza 1 (C-<br>35, VR-94) | 1x10-4 | 7, 7, 6, 8 | 9, 8, 4, 6 |
| | 1x10-5 | 2, 2, 3, 0 | 1, 0, 2, 1 |
| | 1x10-6 | 0, 0, 0, 0 | 0, 0, 0, 0 |
| Parainfluenza 2<br>(Greer, VR-92) | 1x10-4 | 4, 0, 3, 1 | 4, 3, 1, 2 |
| | 1x10-5 | 0, 2, 0, 0 | 0, 1, 1, 1 |
| | 1x10-6 | 0, 0, 0, 0 | 0, 0, 0, 0 |
| Parainfluenza 3 (C<br>243, VR-93) | 1x10-6 | 3, 3, 0, 6 | 1, 1, 3, 5 |
| | 1x10-7 | 1, 0, 1, 1 | 1, 1, 1, 0 |
| | 1x10-8 | 0, 0, 0, 0 | 0, 0, 0, 0 |

Analytical reactivity (inclusivity) of the D3 Duet was evaluated using 10 influenza A virus and 4 influenza B virus strains. Four wells of a 96-well cell culture plate were inoculated with each viral strain (diluted to less than 20-TCID50 per 0.2-mL inoculum). The plates were centrifuged at 700xg for 60 minutes, and then incubated at 35° to 37°C for 24-hours. Four wells from cach strain were stained with the D3 Duet, and each well was then examined at 200x magnification and the number of fluorescent cells counted. The table below lists the virus identity and strain along with the fluorescent cell count.

| Analytical Reactivity (inclusivity) of D³ Duet<br>with various influenza A virus and<br>influenza B virus strains<br>(values are numbers of fluorescent cells<br>per cell monolayer) | |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------|
| Influenza<br>strain | Fluorescent staining cells/cell<br>monolayer |
| Influenza A<br>Wisconsin/56/<br>2005 | 3, 2, 1, 0 |
| Influenza A WS,<br>VR-1520 (H1N1) | 6, 6, 6, 4 |
| Influenza A Hong<br>Kong, VR-544<br>(H3N2) | 3, 4, 5, 5 |
| Influenza A New<br>Jersey, VR-897 | 9, 12, 14, 15 |

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| Analytical Reactivity (inclusivity) of D³ Duet<br>with various influenza A virus and<br>influenza B virus strains<br>(values are numbers of fluorescent cells<br>per cell monolayer) | |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------|
| Influenza<br>strain | Fluorescent staining cells/cell<br>monolayer |
| Influenza A Victoria, VR-822 (H3N2) | 3, 3, 3, 5 |
| Influenza A PR, VR-95 (H1N1) | 3, 9, 9, 6 |
| Influenza A Port Chalmers, VR-810 (H3N2) | 6, 6, 9, 10 |
| Influenza A Aichi, VR-547 (H3N2) | 3, 7, 9, 11 |
| Influenza A Denver, VR-546 (H1N1) | 13, 14, 11, 10 |
| Influenza A Mal, VR-98 (H1N1) | 8, 3, 6, 4 |
| Influenza B GL/1739/54, VR-103 | 7, 6, 7, 7 |
| Influenza B Taiwan/2/62, VR-295 | 3, 1, 2, 5 |
| Influenza B Hong Kong/5/72, VR-823 | 3, 2, 0, 1 |
| Influenza B Maryland/1/59, VR-296 | 5, 6, 6, 8 |

Based on the data presented above, the assay can reliably detect influenza A virus and influenza B virus strains exhibiting both temporal and geographical diversity at viral levels near the limit of detection in cell culture. Analytical sensitivity of the phycoerythrin-labeled influenza A MAbs of the D3 Duet was determined, and compared to that of the fluorescein-labeled influenza A MAbs of the D' Ultra. Cell monolayers of R-Mix in 96-well plates were inoculated with prepared virus stock of influenza A virus, Victoria strain, VR-822 (H3N2), diluted to give a TCID50 of 1 per 0.2-mL inoculum. The plates were incubated at 37℃ for 24 hours. Monolayers were stained using the procedures in the D Ultra's labeling or the D Duet's draft labeling. The assay was performed four times. Results indicate that analytical sensitivities of the phycoerythrin-labeled and the fluorcscein-labeled influenza A MAbs are not statistically different, by a paired t-test".

DHI-Duct_FluARespi_Sec05_510k-Summary printed.doc

ª 50% tissue culture infectivity dose

⁶ Microsoft Office Excel, Microsoft Corporation

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### Clinical Performance:

Direct fresh specimens:

A study was performed prospectively at three sites with 1203 fresh specimens that were received for respiratory virus testing. Each specimen was evaluated by the D3 Duet DFA Influenza A/Respiratory Virus Screening Kit and a cleared DSFA device for the presence of influenza A, influenza B, respiratory syncytial virus, adenovirus, parainfluenza 1, parainfluenza 2 and parainfluenza 3 in cells derived from clinical specimens. A total of nineteen specimens were excluded from analysis due to a site deviations, duplicate specimen, insufficient cell numbers, or high background. These exclusions left 1184 specimen results for analysis.

The following tables detail the summary of the comparison of the D3 Duet and the cleared DSFA comparator assay, combined for study sites 1, 2, and 3:

| D3 Duet R-PE identification of influenza A virus positive specimens | | | |
|---------------------------------------------------------------------|-----|------------------------------------------------------|---------------------|
| Direct Specimen (1184 Specimens) | | D3 Ultra Final Identification<br>(influenza A virus) | |
| | | Pos | Neg |
| D3 Duet R-PE<br>(influenza A virus) | Pos | 99 | 0 |
| | Neg | 1 | 1084 |
| Positive Percent Agreement (PPA) | | 99% (99/100) | |
| 95% CI- PPA | | 94.5, 99.8% | |
| Negative Percent Agreement (NPA) | | | 100%<br>(1084/1084) |
| 95% CI- NPA | | | 99.7, 100% |

| D3 Duet FITC detection of influenza B virus, respiratory syncytial virus, adenovirus, and parainfluenza virus types 1, 2, and 3 viruses | | | |
|-----------------------------------------------------------------------------------------------------------------------------------------|-----|-------------------------------|----------------|
| Direct Specimen (1184 Specimens) | | D3 Ultra Final Identification | |
| | | Pos | Neg |
| D3 Duel FITC Screen | Pos | 386 | 0 |
| | Neg | 0 | 798* |
| Positive Percent Agreement (PPA) | | 100% (386/386) | |
| 95% CI- PPA | | 99.0,100% | |
| Negative Percent Agreement (NPA) | | | 100% (798/798) |
| 95% CI- NPA | | | 99.5,100% |

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| Virus Follow-up Identification of 386 D³ Duet FITC Positive Specimens for influenza B<br>virus, respiratory syncytial virus, adenovirus, and parainfluenza virus types 1, 2, and 3<br>viruses, using D³ Ultra Identification Reagents | | | | | | |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------|---------|--------------------|----------------|---------|--------------------|
| | Sensitivity | | | Specificity | | 95% CI |
| Virus | TP /<br>(TP+FN) | percent | for<br>Sensitivity | TN/<br>(TN+FP) | percent | for<br>Specificity |
| Influenza B<br>virus | 11/11 | 100% | 74.12, 100 | 1173/1173 | 100% | 99.7, 100 |
| Adenovirus | 52/52 | 100% | 93.1, 100 | 1132/1132 | 100% | 99.7, 100 |
| Parainfluenza<br>type 1 | 4/4 | 100% | 51.0, 100 | 1180/1180 | 100% | 99.7, 100 |
| Parainfluenza<br>type 2 | 1/1 | 100% | 20.1, 100 | 1183/1183 | 100% | 99.7, 100 |
| Parainfluenza<br>type 3 | 19/19 | 100% | 83.2, 100 | 1165/1165 | 100% | 99.7, 100 |
| Respiratory<br>Syncytial Virus | 299/299 | 100% | 98.7, 100 | 885/885 | 100% | 99.6, 100 |

D³ DUET DFA INFLUENZA A/RESPIRATORY VIRUS SCREENING KIT Page 11 of 13

The D2 Duet's ability to identify influenza A virus using phycoerythrin in direct specimens was compared to the D3 Ultra's ability using fluorescein. The positive percent agreement was 99% (95% CI range of 94.5% to 99.8%). The negative percent agreement was 100% (95% CI range of 99.7% to 100%). When the ability of the D3 Duet to detect the six other respiratory viruses using fluorescein in direct specimens was compared to the D3 Ultra's ability using fluorescein, the positive percent agreement was 100% (95% CI range of 99.0% to 100%). The negative percent agreement was 100% (95% CI range of 99.5% to 100%).

Specimen type distribution:

Tables below show the study results by the claimed specimen type. Results from sites 1, 2, and 3 have been combined.

| Influenza A virus by specimen type | | | | | | |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------|---------|------------------|------------------|---------|-------------------|
| Specimen<br>type | PPA | | NPA | | | |
| | TP /<br>(TP+FN) | percent | 95%CI for<br>PPA | TN/<br>(TN + FP) | percent | 95% CI for<br>NPA |
| NPA | 61/62 | 98.4% | 91.4, 99.7 | 525/525 | 100% | 99.3, 100 |
| NPS | 38/38 | 100% | 90.8, 100 | 501/501 | 100% | 99.2, 100 |
| D³ Duet FITC detection of influenza B virus, respiratory syncytial virus, adenovirus,<br>and parainfluenza virus types 1, 2, and 3 viruses by specimen type | | | | | | |
| Specimen<br>type | PPA | | NPA | | | |
| | TP /<br>(TP+FN) | percent | 95%CI for<br>PPA | TN/<br>(TN+FP) | percent | 95% CI for<br>NPA |

Influenza A virus by specimen type

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D³ DUFT DFA INFLUENZA A/RESPIRATORY VIRUS SCREENING KIT Page 12 of 13

| NPA | 196/196 | 100% | 98.1, 100 | 391/391 | 100% | 99.0, 100 |
|-----|---------|------|-----------|---------|------|-----------|
| NPS | 173/173 | 100% | 97.8, 100 | 366/366 | 100% | 99.0, 100 |

Cultured specimens:

To evaluate the performance of this device using cultured clinical specimens, a fourth study was performed with 298 frozen specimens to compare performance of the D' Duet DFA Influenza A/Respiratory Virus Screening Kit with that of the predicate for the presence of Influenza A, Influenza B, Respiratory Syncytial Virus, Adenovirus, Parainfluenza 1, Parainfluenza 2 and Parainfluenza 3 (Para 3) from cultured clinical specimens. At Study Site 4, 298 frozen specimens were processed for cell culture testing in accordance with the procedure in the Comparator product insert (same procedure for both Subject and Comparator devices) using R-Mix Too™ FreshCells™ in 48/24-fill multi-well plates. All specimens at study site 4 were derived from nasopharyngeal specimens. The results of this study are presented below. The table below shows the age distribution for individuals studied at site 4:

| Site 4 (culture) – Age Distribution | |
|-------------------------------------|-----|
| 0 - 1 month | 5 |
| >1 month - 2 years | 130 |
| >2 - 12 years | 44 |
| >12 - 21 years | 28 |
| 22- 30 years | 19 |
| 31- 40 years | 20 |
| 41- 50 years | 10 |
| 51 - 60 years | 9 |
| 61-70 years | 8 |
| 71 - 80 years | 6 |
| 81-90 years | 8 |
| >90 years | 5 |
| Unknown age | 6 |
| Total | 298 |

The following tables detail the results of the…