Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on traditional immunofluorescence techniques and manual microscopic interpretation, with no mention of automated image analysis, algorithms, or machine learning for interpretation.

Therapeutic

No.
The device is intended for the qualitative detection and identification of viruses, making it a diagnostic tool, not a therapeutic device.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the kit is "intended for the qualitative detection and identification" of various viruses and notes that "Negative results do not preclude respiratory virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions," implying it contributes to diagnosis. The "Device Description" also refers to it as a "rapid detection and identification" kit.

SaMD (Software as a Medical Device)

No

The device is a kit containing reagents (monoclonal antibodies) and requires a fluorescence microscope for interpretation, indicating it is a hardware and reagent-based medical device, not software-only.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The intended use explicitly states the device is for the "qualitative detection and identification of... virus in respiratory specimens... by immunofluorescence". This is a diagnostic purpose performed on a biological sample (respiratory specimens) in vitro (outside the body).
Device Description: The description details a laboratory test using reagents (monoclonal antibodies) to detect viral antigens in fixed cells from clinical specimens or cell cultures. This is a typical process for an in vitro diagnostic test.
Performance Studies: The performance studies involve testing clinical specimens and isolates to evaluate the device's ability to detect and identify viruses, which is a requirement for demonstrating the effectiveness of an IVD.
Predicate Device: The mention of a "Predicate Device" with a K number (K061101) indicates that this device is being compared to a previously cleared medical device, which is a common regulatory pathway for IVDs.

All these elements align with the definition and characteristics of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Diagnostic Hybrids, Inc. D3 Ultra DFA (direct fluorescent antibody) RESPIRATORY VIRUS SCREENING & ID KIT is intended for the qualitative detection and identification of the Influenza A. Influenza B. Respiratory Syncytial Virus (RSV), Adenovirus, Parainfluenza 1, Parainfluenza 2 and Parainfluenza 3 virus in respiratory specimens, by either direct detection or cell culture method, by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs). 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 respiratory virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. Performance characteristics for influenza A were established when influenza A/H3 and A/H1 were the predominant influenza A viruses in circulation. 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 state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL3+ facility is available to receive and culture specimens.

Product codes

GNW

Device Description

The Diagnostic Hybrids, Inc. D3 Ultra DFA RESPIRATORY VIRUS SCREENING & ID KIT uses viral antigen-specific murine monoclonal antibodies that are directly labeled with fluorescein for the rapid detection and identification of respiratory viruses. The kit includes a DFA Screening Reagent that contains a blend of murine monoclonal antibodies (MAbs) directed against seven respiratory viruses (Influenza A, Influenza B, Respiratory Syncytial Virus, Adenovirus, Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3) plus seven separate DFA Reagents, each consisting of MAb blends directed against a single respiratory virus. The kit can be used for direct specimen or cell culture screening and final virus identification. The cells to be tested, either derived from a clinical specimen or cell culture, are fixed in acetone. The DFA Screening Reagent is added to the cells to determine the presence of viral antigens. After incubating at 35℃ to 37℃, the stained cells are rinsed 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. Virus infected cells will be stained with viral specific apple-green fluorescence when stained with the DFA Screening Reagent while uninfected cells will contain no fluorescence but will be stained red by the Evan's Blue counter-stain. If the specimen contains fluorescent cells, the particular virus is identified using the separate DFA Reagents on new, separate cell preparations. If on examination of a direct stained specimen, no fluorescent-stained cells are found and all the cells stain red from the Evan's Blue, it is recommended that the specimen be cultured and stained using the DFA Screening Reagent. If fluorescent cells are seen, the identification of the virus is determined as described above. Cell preparations are fixed in acetone. The individual DFA reagents are added to the cell preparations. After incubating at 35° to 37°C, the stained cells are rinsed with the diluted Wash Solution. A drop of the supplied Mounting Fluid is added and a coverslip is placed on the stained cells. The cells are examined using a fluorescence microscope for the presence of viral specific apple-green fluorescence. The unknown respiratory virus is then identified and reported.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Respiratory specimens

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Study 1-DS - Direct Specimen Method:

Sample Size: 329 fresh specimens (326 for evaluation)
Data Source: Specimens submitted February through May, 2006, to the laboratory for respiratory virus testing. These were prospectively collected.
Annotation Protocol: Slides were prepared from PBS-washed cells from the fresh specimens and fixed according to the prescribed protocol. The slides were stored at -70°C until testing was performed. The slides were brought to room temperature and stained in accordance with the procedure in the Predicate product insert (same procedure for both Subject and Predicate devices).

Study 2-DS - Direct Specimen Method:

Sample Size: 192 specimens
Data Source: Specimens submitted to the laboratory for respiratory virus testing during December 2005 through February 2006, with residual specimen material stored at -70°C from a few days to 2 months. These were prospectively collected.
Annotation Protocol: The frozen specimens were thawed and processed between 13 February to 17 February 2006 according to the procedure in the Predicate product insert (same procedure for both Subject and Predicate devices). Slides were prepared from the specimens according to instructions detailed in the Predicate device's product insert. These slides were stained with both the Predicate and Subject devices and interpreted according to the Predicate device's product insert procedure (same procedure for both Subject and Predicate devices).

Study 2-CC - Cell Culture Method:

Sample Size: 192 specimens
Data Source: The same 192 specimens that were evaluated by DS testing (prospectively collected, December 2005 through February 2006).
Annotation Protocol: Processed according to the Predicate device's product insert procedure for cell culture (same procedure for both Subject and Predicate devices). Briefly, 200-uL from the specimens were inoculated onto each of 4 monolayers of R-Mix™ Too FreshCells™ contained in shell vials which were centrifuged for 60 minutes at 700xg and incubated for 24-hours at 35° to 37°C. The shell vials were processed according to instructions detailed in the Predicate device's product insert.

Study 3-DS - Direct Specimen Method:

Sample Size: 298 specimens (282 for evaluation)
Data Source: Specimens originally received by a hospital laboratory in the eastern US for respiratory virus testing during January through March 2006, with residual specimen material stored at -70°C from 3 to 6 months. These were prospectively collected.
Annotation Protocol: The frozen specimens were sent to DHI, where they were thawed and processed between 30 May and 1 June 2006, according to the predicate device's product insert. All specimens used in the studies were tested by both the DS and CC procedures as detailed in the Predicate device's product insert.

Study 3-CC - Cell Culture Method:

Sample Size: 298 specimens
Data Source: The same 298 specimens that were evaluated by DS testing (prospectively collected, January through March 2006).
Annotation Protocol: Processed for CC testing according to the Predicate device's product insert for cell culture using R-Mix™ Too FreshCellsTM in 48/24-fill cluster plates.

Study 3a-DS - Direct Specimen Method:

Sample Size: 30 specimens (26 for evaluation)
Data Source: Archived specimens originally received by a hospital laboratory in Italy for respiratory virus testing during the period from October 2005 through April 2006, with residual specimen material stored at -70℃ from 2 to 6 months. These were identified as containing Parainfluenza (types 1, 2, or 3).
Annotation Protocol: The frozen specimens were sent to DHI, where they were thawed and processed between June 7 and 8, 2006, according to the prescribed protocol. All specimens used in the studies were tested by both the DS and CC procedures as detailed in the Predicate device's product insert. Original results reported by the laboratory were unknown to the study investigator.

Study 3a-CC - Cell Culture Method:

Sample Size: 30 frozen specimens (29 for evaluation)
Data Source: The same 30 frozen specimens that were evaluated by DS testing (archived from Italy, October 2005 through April 2006).
Annotation Protocol: Processed for CC testing according to the Predicate device's product insert for cell culture using R-Mix™ FreshCells™ in 48/24-fill cluster plates.

Study 3b-CC - Cell Culture Method:

Sample Size: 81 clinical isolates
Data Source: A collection of banked clinical isolates known to contain respiratory viruses that had been frozen from the 2005/2006 respiratory season. These were selected because they were previously shown to contain at least one of the seven virus analytes detected by the Subject Test.
Annotation Protocol: Processed according to the Predicate device's product insert for cell culture using R-Mix™ FreshCells™ cultures in shell vials.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Study 1-DS - Direct Specimen Method:

Study Type: Clinical study (comparison against Predicate device)
Sample Size: 326 fresh specimens
Key Results:
- Positive Percent Agreement (PPA): 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 3, and Respiratory Syncytial Virus (no Parainfluenza 2 detected).
- Negative Percent Agreement (NPA): 98.3% for Screen +, 100% for Adenovirus, Influenza A, Parainfluenza 1, Parainfluenza 2, Respiratory Syncytial Virus, and 98.7% for Influenza B, 96.7% for Parainfluenza 3.
- Concordance: With the exception of 4 specimens, the DS test results were concordant for both the screen and the identification of the individual viruses.

Study 2-DS - Direct Specimen Method:

Study Type: Clinical study (comparison against Predicate device)
Sample Size: 192 specimens (frozen/thawed)
Key Results:
- PPA: 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2, and Respiratory Syncytial Virus (no Parainfluenza 3 detected).
- NPA: 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and Respiratory Syncytial Virus.
- Concordance: The DS test results were concordant for both the Screen and the ID reagents.

Study 2-CC - Cell Culture Method:

Study Type: Clinical study (comparison against Predicate device)
Sample Size: 192 specimens
Key Results:
- PPA: 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2, and Respiratory Syncytial Virus (no Parainfluenza 3 detected).
- NPA: 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and Respiratory Syncytial Virus.
- Concordance: The CC test results were concordant for both the Screen and the ID of the specific viruses.

Study 3-DS - Direct Specimen Method:

Study Type: Clinical study (comparison against Predicate device)
Sample Size: 282 specimens (frozen/thawed)
Key Results:
- PPA: 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and Respiratory Syncytial Virus.
- NPA: 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and Respiratory Syncytial Virus.
- Concordance: The DS test results were concordant for both the Screen and the ID reagents.
- Co-infections: 10 co-infections identified.

Study 3-CC - Cell Culture Method:

Study Type: Clinical study (comparison against Predicate device)
Sample Size: 298 specimens (frozen/thawed)
Key Results:
- PPA: 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and Respiratory Syncytial Virus.
- NPA: 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and Respiratory Syncytial Virus.
- Concordance: The CC test results were concordant for both the Screen and the ID reagents.
- Co-infections: 16 co-infections identified.

Study 3a-DS - Direct Specimen Method (Archival Parainfluenza specimens):

Study Type: Clinical study (comparison against Predicate device)
Sample Size: 26 specimens (frozen/thawed)
Key Results:
- PPA: 100% for Screen +, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2. 78.4% for Para 3. (Other viruses not found).
- NPA: 88.9% for Screen +. 100% for Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2. 78.4% for Para 3. (Other viruses not found).
- Concordance: With the exception of one specimen, the DS test results were concordant for both the Screen and the ID of individual viruses.

Study 3a-CC - Cell Culture Method (Archival Parainfluenza specimens):

Study Type: Clinical study (comparison against Predicate device)
Sample Size: 29 specimens (frozen/thawed)
Key Results:
- PPA: 100% for Screen +, Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3. (Other viruses not found).
- NPA: 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and Respiratory Syncytial Virus.
- Concordance: The CC test results were concordant for both the Screen and the ID reagents.

Study 3b-CC - Cell Culture Method (Archival clinical isolates):

Study Type: Clinical study (comparison against Predicate device)
Sample Size: 81 clinical isolates
Key Results:
- PPA: 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and Respiratory Syncytial Virus.
- NPA: 100% for Screen +, Adenovirus, Influenza A, Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and Respiratory Syncytial Virus.
- Concordance: The CC test results were concordant for both the Screen Reagent and the specific virus ID Reagents.
- Co-infections: One co-infection (Para 1 + Para 3) identified.

Cross-reactivity Testing:

Study Type: Analytical study
Sample Size: 64 virus strains, 18 host culture cell types, 18 bacterial cultures.
Key Results: No cross-reactivity observed for 64 virus strains or 18 host culture cell types. Staining of S. aureus appeared as small points of fluorescence, while all other bacterial cultures were negative.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Key metrics are reported as Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA).

Study 1-DS - Direct Specimen Method (326 specimens):

Screen+: PPA 100% (95% CI: 95.1% - 100%), NPA 98.3% (95% CI: 95.4% - 99.5%)
Adenovirus: PPA 100% (95% CI: 79.3% - 100%), NPA 100% (95% CI: 94.2% - 100%)
Influenza A: PPA 100% (95% CI: 87.3% - 100%), NPA 100% (95% CI: 93.0% - 100%)
Influenza B: PPA 100% (95% CI: 79.3% - 100%), NPA 98.7% (95% CI: 92.2% - >99.9%)
Parainfluenza 1: PPA 100% (95% CI: 29.0% - 100%), NPA 100% (95% CI: 95.2% - 100%)
Parainfluenza 2: PPA ---, NPA 100% (95% CI: 95.3% - 100%) (No positives)
Parainfluenza 3: PPA 100% (95% CI: 29.0% - 100%), NPA 96.7% (95% CI: 90.5% - 99.3%)
Respiratory Syncytial Virus: PPA 100% (95% CI: 79.3% - 100%), NPA 100% (95% CI: 94.2% - 100%)

Study 2-DS - Direct Specimen Method (192 specimens):

Screen+: PPA 100% (95% CI: 91.5% - 100%), NPA 100% (95% CI: 96.8% - 99.5%)
Adenovirus: PPA 100% (95% CI: 29.0% - 100%), NPA 100% (95% CI: 92.6% - 100%)
Influenza A: PPA 100% (95% CI: 84.8% - 100%), NPA 100% (95% CI: 96.2% - 100%)
Influenza B: PPA 100% (95% CI: 38.3% - 100%), NPA 100% (95% CI: 96.8% - >99.9%)
Parainfluenza 1: PPA 100% (95% CI: 16.8% - 100%), NPA 100% (95% CI: 96.8% - 100%)
Parainfluenza 2: PPA 100% (95% CI: 16.8% - 100%), NPA 100% (95% CI: 96.8% - 100%)
Parainfluenza 3: PPA ---, NPA 100% (95% CI: 96.8% - 100%) (No positives)
Respiratory Syncytial Virus: PPA 100% (95% CI: 80.5% - 100%), NPA 100% (95% CI: 89.4% - 100%)

Study 2-CC - Cell Culture Method (192 specimens):

Screen+: PPA 100% (95% CI: 91.5% - 100%), NPA 100% (95% CI: 96.8% - 99.5%)
Adenovirus: PPA 100% (95% CI: 38.3% - 100%), NPA 100% (95% CI: 96.8% - 100%)
Influenza A: PPA 100% (95% CI: 84.8% - 100%), NPA 100% (95% CI: 96.2% - 100%)
Influenza B: PPA 100% (95% CI: 38.3% - 100%), NPA 100% (95% CI: 96.8% - >99.9%)
Parainfluenza 1: PPA 100% (95% CI: 16.8% - 100%), NPA 100% (95% CI: 96.8% - 100%)
Parainfluenza 2: PPA 100% (95% CI: 16.8% - 100%), NPA 100% (95% CI: 96.8% - 100%)
Parainfluenza 3: PPA ---, NPA 100% (95% CI: 96.8% - 100%) (No positives)
Respiratory Syncytial Virus: PPA 100% (95% CI: 77.3% - 100%), NPA 100% (95% CI: 96.4% - 100%)

Study 3-DS - Direct Specimen Method (282 specimens):

Screen+: PPA 100% (95% CI: 96.2% - 100%), NPA 100% (95% CI: 97.3% - 100%)
Adenovirus: PPA 100% (95% CI: 55.7% - 100%), NPA 100% (95% CI: 96.0% - 100%)
Influenza A: PPA 100% (95% CI: 92.7% - 100%), NPA 100% (95% CI: 92.7% - 100%)
Influenza B: PPA 100% (95% CI: 77.3% - 100%), NPA 100% (95% CI: 95.6% - >99.9%)
Parainfluenza 1: PPA 100% (95% CI: 29.0% - 100%), NPA 100% (95% CI: 96.2% - 100%)
Parainfluenza 2: PPA 100% (95% CI: 29.0% - 100%), NPA 100% (95% CI: 96.2% - 100%)
Parainfluenza 3: PPA 100% (95% CI: 65.5% - 100%), NPA 100% (95% CI: 95.9% - 100%)
Respiratory Syncytial Virus: PPA 100% (95% CI: 87.9% - 100%), NPA 100% (95% CI: 91.3% - 100%)

Study 3-CC - Cell Culture Method (298 specimens):

Screen+: PPA 100% (95% CI: 96.6% - 100%), NPA 100% (95% CI: 97.3% - 100%)
Adenovirus: PPA 100% (95% CI: 67.9% - 100%), NPA 100% (95% CI: 96.3% - 100%)
Influenza A: PPA 100% (95% CI: 93.5% - 100%), NPA 100% (95% CI: 93.2% - 100%)
Influenza B: PPA 100% (95% CI: 81.0% - 100%), NPA 100% (95% CI: 96.0% - >99.9%)
Parainfluenza 1: PPA 100% (95% CI: 51.1% - 100%), NPA 100% (95% CI: 96.4% - 100%)
Parainfluenza 2: PPA 100% (95% CI: 38.3% - 100%), NPA 100% (95% CI: 96.5% - 100%)
Parainfluenza 3: PPA 100% (95% CI: 65.5% - 100%), NPA 100% (95% CI: 96.3% - 100%)
Respiratory Syncytial Virus: PPA 100% (95% CI: 87.6% - 100%), NPA 100% (95% CI: 95.5% - 100%)

Study 3a-DS - Direct Specimen Method (26 specimens):

Screen+: PPA 100% (95% CI: 85.7% - 100%), NPA 88.9% (95% CI: 54.3% - >99.9%)
Adenovirus: PPA ---, NPA 100% (95% CI: 79.3% - 100%) (No positives)
Influenza A: PPA 100% (95% CI: 16.8% - 100%), NPA 100% (95% CI: 79.3% - 100%)
Influenza B: PPA 100% (95% CI: 51.1% - 100%), NPA 100% (95% CI: 79.3% - 100%)
Parainfluenza 1: PPA 100% (95% CI: 16.8% - 100%), NPA 100% (95% CI: 79.3% - 100%)
Parainfluenza 2: PPA 100% (95% CI: 70.0% - 100%), NPA 100% (95% CI: 78.4% - 100%)
Parainfluenza 3: PPA 78.4% (95% CI: 46.7% - 95.2%), NPA 100% (95% CI: 73.4% - 100%)
Respiratory Syncytial Virus: PPA ---, NPA 100% (95% CI: 79.3% - 100%) (No positives)

Study 3a-CC - Cell Culture Method (29 specimens):

Screen+: PPA 100% (95% CI: 81.8% - 100%), NPA 100% (95% CI: 62.8% - 100%)
Adenovirus: PPA ---, NPA 100% (95% CI: 81.8% - 100%) (No positives)
Influenza A: PPA ---, NPA 100% (95% CI: 81.8% - 100%) (No positives)
Influenza B: PPA ---, NPA 100% (95% CI: 81.8% - 100%) (No positives)
Parainfluenza 1: PPA 100% (95% CI: 38.3% - 100%), NPA 100% (95% CI: 79.3% - 100%)
Parainfluenza 2: PPA 100% (95% CI: 51.1% - 100%), NPA 100% (95% CI: 77.3% - 100%)
Parainfluenza 3: PPA 100% (95% CI: 73.4% - 100%), NPA 100% (95% CI: 62.8% - 100%)
Respiratory Syncytial Virus: PPA ---, NPA 100% (95% CI: 81.8% - 100%) (No positives)

Study 3b-CC - Cell Culture Method (81 specimens):

Screen+: PPA 100% (95% CI: 94.6% - 100%), NPA 100% (95% CI: 97.3% - 100%) (No negatives reported)
Adenovirus: PPA 100% (95% CI: 70.0% - 100%), NPA 100% (95% CI: 93.8% - 100%)
Influenza A: PPA 100% (95% CI: 79.3% - 100%), NPA 100% (95% CI: 93.1% - 100%)
Influenza B: PPA 100% (95% CI: 78.4% - 100%), NPA 100% (95% CI: 93.2% - 100%)
Parainfluenza 1: PPA 100% (95% CI: 45.4% - 100%), NPA 100% (95% CI: 94.3% - 100%)
Parainfluenza 2: PPA 100% (95% CI: 16.8% - 100%), NPA 100% (95% CI: 94.5% - 100%)
Parainfluenza 3: PPA 100% (95% CI: 84.8% - 100%), NPA 100% (95% CI: 92.2% - 100%)
Respiratory Syncytial Virus: PPA 100% (95% CI: 51.1% - 100%), NPA 100% (95% CI: 94.2% - 100%)

Predicate Device(s)

K061101

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.3330 Influenza virus serological reagents.

(a)
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.(b)
Classification. Class I (general controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 866.9.

Summary

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Special 510(k): Device Modification De Ultra DFA Respiratory Virus Screening & ID Kit

Image /page/0/Picture/1 description: The image shows a logo for Diagnostic Hybrids. The logo includes the company name in bold, black letters, with the tagline "Integrating Science and Humanity" underneath. To the right of the text is a stylized graphic that resembles a DNA strand intertwined with a human figure. The number K092300 is written below the logo.

DATE OF PREPARATION OF 510(k) SUMMARY

July 23, 2009

APPLICANT

AUG 2 8 2009

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

CONTACT INFORMATION

Ronald H. Lollar Senior Director, Product Realization, Management, and Marketing E-mail: lollar@dhiusa.com Telephone: 740-589-3300 Desk Extension: 740-589-3373 FAX: 740-593-8437

DEVICE NAME

५:

Trade name: D 3 Ultra DFA Respiratory Virus Screening & ID Kit Common name: Respiratory Virus DFA Assay Classification name: Antisera, Cf, Influenza A, B, C Product Code: GNW Regulation: 21 CFR § 866.3330, Class I, Influenza virus serological reagents, Panel Microbiology (83)

LEGALLY MARKETED DEVICE

Do Ultra DFA Respiratory Virus Screening & ID Kit, K061101

DESCRIPTION of DEVICE MODIFICATION

The product insert has been modified. The following has been added (see below):

Table 15 in the product insert has been updated to include reactivity data on influenza A virus Mexico/4108/2009 and California/07/2009 strains. The following language was included with the data:

"Although this test has been shown to detect the 2009 H1N1 influenza virus in two cultured isolates, the performance characteristics of this device with clinical

1

specimens that are positive for the 2009 H1N1 influenza virus have not been established. The D Ultra DFA Respiratory Virus Screening & ID Kit can distinguish between influenza A and B viruses, but it cannot differentiate influenza subtypes."

INTENDED USE

The Diagnostic Hybrids, Inc. D3 Ultra DFA (direct fluorescent antibody) RESPIRATORY VIRUS SCREENING & ID KIT is intended for the qualitative detection and identification of the Influenza A. Influenza B. Respiratory Syncytial Virus (RSV), Adenovirus, Parainfluenza 1, Parainfluenza 2 and Parainfluenza 3 virus in respiratory specimens, by either direct detection or cell culture method, by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs). 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 respiratory virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

Performance characteristics for influenza A were established when influenza A/H3 and A/H1 were the predominant influenza A viruses in circulation. 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 state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL3+ facility is available to receive and culture specimens.

ASSESSMENT OF NON-CLINICAL PERFORMANCE DATA FOR EQUIVALENCE

Not Applicable

ASSESSMENT OF NON-CLINICAL PERFORMANCE DATA FOR EQUIVALENCE

The risk analysis method used to assess the impact of the modification was a Failure Modes and Effects Analysis (FMEA). The modification to device labeling poses no additional risk.

BIOCOMPATABILITY

www.cdc.gov

2 FDA Guidance Document: In Vitro Diagnostic Devices to Detect Influenza A Viruses: Labeling and Regulatory Path; Issued 4/10/2006

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Not applicable

STERILIZATION

Not applicable

:

3

D3 Ultra DFA 1 Respiratory Virus Screening & ID 2 Kit 3 4 For In Vitro Diagnostic Use I. INTENDED USE 5 б The Diagnostic Hybrids, Inc. Do Ultra DFA (direct fluorescent antibody) RESPIRATORY 7 8 VIRUS SCREENING & ID KIT is intended for the qualitative detection and identification 9 of the Influenza A. Influenza B. Respiratory Syncytial Virus (RSV), Adenovirus, Parainfluenza 1, Parainfluenza 2 and Parainfluenza 3 virus in respiratory specimens, by 10 either direct detection or cell culture method, by immunofluorescence using 11 fluoresceinated monoclonal antibodies (MAbs). It is recommended that specimens found 12 13 to be negative after examination of the direct specimen result be confirmed by cell culture. Negative results do not preclude respiratory virus infection and should not be used as the 14 ાં ર sole basis for diagnosis, treatment or other management decisions. 16 Performance characteristics for influenza A were established when influenza A/H3 and 17 . A/H1 were the predominant influenza A viruses in circulation. When other influenza 18 A viruses are emerging, performance characteristics may vary. 19 If infection with a novel influenza A virus is suspected based on current clinical and 20 . epidemiological screening criteria recommended by public health authorities, 21 specimens should be collected with appropriate infection control precautions for novel 22 virulent influenza viruses and sent to state or local health departments for testing. 23 Viral culture should not be attempted in these cases unless a BSL3+ facility' is 24 available to receive and culture specimens.2 ટર્ટ II. SUMMARY AND EXPLANATION OF THE TEST ટેર 27 With the addition of new antiviral drugs for the treatment of Influenza', more rapid and 28 sensitive tests for respiratory virus detection 45 and the increasing need to be more 29 discriminating in the use of antibiotics , early detection and identification of the infecting 30 viral agent has grown substantially in importance. Viral identification is becoming 31 increasingly important in ruling out bacteria as the cause of respiratory infections. Virus 32 33 identification by either direct antigen detection or cell culture using fluorescent monoclonal antibodies continues to be the standard method in virology laboratories. 34 રે રે Influenza A and B Influenza viruses (family Orthomyxoviridae) contain a single-stranded RNA genome 36 which is present in 8 separate segments of ribonucleoprotein. This segmentation of the 37 38 genome is rare among viruses and probably contributes to the rapid development of new influenza strains through interchange of gene segments if two different viruses infect the 39 same cell. There are 3 types of influenza, A, B and C. Type A has counterparts in birds 40 and pigs as well as humans, while types B and C are known only in man. Due to the 41 possibility of another pandemic caused by Influenza A, as occurred in 1918 when 25-35 42 million people worldwide died, the Centers for Disease Control (CDC) and the World 43

4

Health Organization (WHO) maintain surveillance of influenza strains and make 44

predictions of suitable strains for vaccine production. વર્સ

Influenza infects an estimated 120 million people in the US, Europe and Japan each year 46

and it is estimated that in the US there are 75,000 deaths annually from pneumonia caused 47
by influenza. Primary viral pneumonia or pneumonia from secondary bacterial infections 48
are the primary causes of morbidity of the viral infection. Pandemics of influenza A 49
occur about every 10 to 30 years and epidemics of either influenza A or B occur annually. ર૦
Infections are seasonal, typically extending from November to April in the northern રી I
hemisphere. Complications tend to occur in the young, elderly and persons with chronic 52 રે 3 cardio-pulmonary diseases.
રવે Incubation time is 1-3 days with rapid spread by inhalation via aerial droplets and fomites. ર ર It is characterized by fever, myalgia, headache and pharyngitis. .
Influenza A and B are most commonly isolated in A549/Mv1Lu mixtures (R-Mix™1), ર્ડ
A549/MDCK mixtures (R-Mix Too™1), Rhesus MK, MDCK, MRC-5 and A549 cells . 57

ર 8 Adenovirus

Adenoviruses (family Adenoviridae) are non-enveloped, double stranded DNA viruses. રતે
There are 49 serotypes, further divided into 6 groups, A to F, with most associated with રેજી
respiratory and ocular infections. Generally, adenovirus infections in adults have a low ୧।
morbidity with the exceptions of immunocompromised patients and individuals living in 62
cramped quarters where infections can cause atypical pneumonia. Virus spread is 63
commonly via aerial droplets and fomites where they infect the mucous membranes of the 64 ર્ણ રહ્યું eye, respiratory tract and gut'.
Adenovirus can be isolated in A549/Mv1Lu mixtures (R-Mix™), A549/MDCK mixtures 66
(R-Mix Too™), HEp2, HEK, A549 and MRC-5 cells.8 67

Parainfluenza Viruses 1, 2 and 3 68

Parainfluenza viruses (family Paramyxoviridae) are enveloped viruses with a single, રેતે
negative strand RNA genome. The 4 different types, 1 to 4, cause croup and viral 70
pneumonia in children under the age of 5 years and cause upper respiratory illness in 71
adults. Parainfluenza is the number 2 leading cause of lower respiratory illness in children 72
(after RSV). Outbreaks caused by parainfluenza viruses occur during alternate years in the 73
fall (P1 and P2) or throughout the year, with increased activity in the spring (P3) 10 74
75 Parainfluenza viruses can be isolated in A549/Mv1Lu mixtures (R-Mix™), A549/MDCK
mixtures (R-Mix Too™), Rhesus MK, MRC-5 and LLC-MK2 cells. Trypsin is helpful in 76
the medium for recovery of types 1 and 2 but not type 3 8. 77
78 Respiratory Syncytial Virus (RSV)
RSV (family Paramyxoviridae) is an enveloped virus with a single, negative strand RNA 79
genome. RSV infections cause viral bronchiolitis and pneumonia in infants and the 80
common cold in adults". RSV is usually a seasonal (winter and early spring) infection 81
82 with epidemics lasting up to 5 months. Peak mortality due to RSV occurs in 3-4 month
old infants. There are two major subtypes. A and B; Subtype B is characterized as the 83
asymptomatic strain that the majority of the population experiences. The more severe 84

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The use of R-Mix™ and R-Mix Too™ cells is covered by U.S. Patent Number 6,168,915 with additional patents pending.

5

clinical illnesses involve Subtype A strains which tend to predominate in most outbreaks 12. 8 રે

૪૯ RSV is the primary viral cause of lower respiratory disease in infants and young children.

Re-infections do occur but tend to be limited to minor upper respiratory infections13. RSV 87

is also now recognized as a significant problem in certain adult populations. These include 88 the elderly, persons with cardiopulmonary diseases, and immunocompromised hosts 4. 89
90

RSV is commonly detected directly in cells from the nasopharyngeal epithelium by ਰੇ I

staining with immunofluorescent reagents' although it can be isolated in cell cultures of 92
A549/Mv1Lu mixtures (R-Mix™), A549/MDCK mixtures (R-Mix Too™), HEp2, Vero, ਰੇਤੇ

े पै LLC-MK2 and MRC-5 cells®.

તેરે

III. PRINCIPLE OF THE PROCEDURE તેરિ

97
The Diagnostic Hybrids, Inc. D3 Ultra DFA RESPIRATORY VIRUS SCREENING & ID 98

ਰੇਰੇ KIT uses viral antigen-specific murine monoclonal antibodies that are directly labeled

with fluorescein for the rapid detection and identification of respiratory viruses. 100
The kit includes a DFA Screening Reagent that contains a blend of murine monoclonal 101
antibodies (MAbs) directed against seven respiratory viruses (Influenza A, Influenza B, 102
103 Respiratory Syncytial Virus, Adenovirus, Parainfluenza 1, Parainfluenza 2, and
Parainfluenza 3) plus seven separate DFA Reagents, each consisting of MAb blends 104
directed against a single respiratory virus. The kit can be used for direct specimen or cell 105
106 culture screening and final virus identification.
The cells to be tested, either derived from a clinical specimen or cell culture, are fixed in 107
acetone. The DFA Screening Reagent is added to the cells to determine the presence of 108
viral antigens. After incubating at 35℃ to 37℃, the stained cells are rinsed with the 109
diluted Wash Solution. A drop of the supplied Mounting Fluid is added and a coverslip is 110
placed on the prepared cells. The cells are examined using a fluorescence microscope. 111
Virus infected cells will be stained with viral specific apple-green fluorescence when 112
stained with the DFA Screening Reagent while uninfected cells will contain no 113
fluorescence but will be stained red by the Evan's Blue counter-stain. If the specimen 114
contains fluorescent cells, the particular virus is identified using the separate DFA । । ર
Reagents on new, separate cell preparations. 116

If on examination of a direct stained specimen, no fluorescent-stained cells are found and 117

all the cells stain red from the Evan's Blue, it is recommended that the specimen be 118
cultured and stained using the DFA Screening Reagent. If fluorescent cells are seen, the 119 120 identification of the virus is determined as described above.
Cell preparations are fixed in acetone. The individual DFA reagents are added to the cell 121
preparations. After incubating at 35° to 37°C, the stained cells are rinsed with the diluted 122
Wash Solution. A drop of the supplied Mounting Fluid is added and a coverslip is placed 123
on the stained cells. The cells are examined using a fluorescence microscope for the 124
presence of viral specific apple-green fluorescence. The unknown respiratory virus is then 125 identified and reported. 126
127

IV. REAGENTS 128

129
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350 West State Street Athens, OH 45701

6

A. Kit Components 130

Respiratory Virus DFA Screening Reagent - 10-mL. One dropper bottle containing a 131 132 mixture of fluorescein labeled murine monoclonal antibodies directed against respiratory

133 viral antigens of Influenza A, Influenza B, Respiratory Syncytial Virus (RSV),

Adenovirus, Parainfluenza 1, Parainfluenza 2 and Parainfluenza 3. The buffered, 134
stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium ા ૩૨
azide as preservative. I 36

Influenza A DFA Reagent - 2-mL. One dropper bottle containing fluorescein labeled 137 murine monoclonal antibodies directed against antigens produced by Influenza A virus 138 I ਤੇਰੇ (strain Texas 1/77, H3N2) infected cells. The buffered, stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. 140
Influenza B DFA Reagent - 2-mL. One dropper bottle containing fluorescein labeled 141 murine monoclonal antibodies directed against antigens produced by Influenza B virus 142 (Hong Kong 5/72) infected cells. The buffered, stabilized, aqueous solution contains 143 Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. 144
RSV DFA Reagent - 2-mL. One dropper bottle containing fluorescein labeled murine 145 monoclonal antibodies directed against antigens produced by RSV (Long strain) infected 146 147 cells. The buffered, stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. 148
Adenovirus DFA Reagent - 2-mL. One dropper bottle containing fluorescein labeled 149 murine monoclonal antibodies directed against antigens produced by Adenovirus (Type 3-1 20 GB strain and Type 6-tonsil 99 strain) infected cells. The buffered, stabilized, aqueous 151 solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. 152
Parainfluenza 1 DFA Reagent - 2-mL. One dropper bottle containing fluorescein 153 labeled murine monoclonal antibodies directed against antigens produced by Parainfluenza 154 ાં રેર 1 (VP-1 strain) infected cells. The buffered, stabilized, aqueous solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative. । રહ
Parainfluenza 2 DFA Reagent - 2-mL. One dropper bottle containing fluorescein 157 labeled murine monoclonal antibodies directed against antigens produced by Parainfluenza I રાજ 2 (Greer strain) infected cells. The buffered, stabilized, aqueous solution contains Evan's । રેતે Blue as a counter-stain and 0.1% sodium azide as preservative. 160
Parainfluenza 3 DFA Reagent - 2-mL. One dropper bottle containing fluorescein 161 labeled murine monoclonal antibodies directed against antigens produced by Parainfluenza 162 3 (C243 strain) infected cells. The buffered, stabilized, aqueous solution contains Evan's 163

164 Blue as a counter-stain and 0.1% sodium azide as preservative.
। ୧୯ 9. Respiratory Virus Antigen Control Slides - 5-slides. Five individually packaged

control slides containing wells with cell culture derived positive and negative control cells. 166

Each positive well is identified as to the virus infected cells present, i.e., Influenza A, 167
Influenza B. Respiratory Syncytial Virus (RSV), Adenovirus, Parainfluenza 1, 1 68
Parainfluenza 2 and Parainfluenza 3. The Negative well contains uninfected cells. Each । ୧୯୯
slide is intended to be stained only one time. 170

7

171 10. Normal Mouse Gamma Globulin DFA Reagent - 10-mL. One dropper bottle

172 containing a mixture of fluorescein labeled murine gamma globulin that has been shown to

173 be un-reactive with any of the listed respiratory viruses. The buffered, stabilized, aqueous

174 solution contains Evan's Blue as a counter-stain and 0.1% sodium azide as preservative.

Wash Solution Concentrate - 25-mL. One bottle containing a 40X concentrate 175

176 consisting of Tween 20 and 4% sodium azide (after dilution to 1X in water, the

177 concentration of sodium azide in the solution is 0.1%) in Phosphate Buffered Saline.

178 12. Mounting Fluid - 15-mL. One dropper bottle containing an aqueous, buffered, stabilized solution of glycerol and 0.1% sodium azide. 179

B. Warnings and Precautions 180

181 1. For in vitro diagnostic use.
1. Cells may have some potential to be hazardous. Personnel working with these cultures 182 must be properly trained in safe handling techniques1516/7, and have experience with 183 tissue culture before attempting this procedure. 184
I 85 3. All procedures must be conducted in accordance with the CDC 4" edition Biosafety in Microbiological and Biomedical Laboratories, 1999, and CLSI Approved Guideline 186 M29-A, Protection of Laboratory Workers from Instrument Biohazards and Infectious 187 Disease Transmitted by Blood, Body Fluids, and Tissue. 188
189 4. Acetone, a reagent that is required for the test but not provided in the kit, is a flammable, volatile organic solvent. Use it in a well-ventilated area and keep away 190 from flames and other sources of ignition. । ਰੇ।
192 5. Sodium azide is included in the Wash Solution Concentrate at 4%, and in the other
solutions in this kit at 0.1%. A MSDS for sodium azide or for Diagnostic Hybrids, Inc 193 (DHI) reagents containing sodium azide is available by contacting a Diagnostic 194
I તેર Hybrids' Technical Service Representative.
196 a. Reagents containing sodium azide should be considered a poison. If products 197 containing sodium azide are swallowed, seek medical advice immediately and show product container or label. [Refer to NIOSH, National Institute for 198 Occupational Safety and Health; CAS#: 2628-22-8; and also to GHS, The । ਉਹੇ 200 Globally Harmonized System of Classification and Labeling of Chemicals.]
- b. Aqueous solutions of sodium azide, when mixed with acids, may liberate toxic gas (sodium azide in water exists is ionic equilibrium with hydrazoic acid. which when mixed with acid may liberate a toxic gas).
c. Any reagents containing sodium azide should be evaluated for proper disposal. 204 205 Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. If products containing sodium azide are discarded into 206 207 a drain, flush with a large volume of water to prevent azide build-up. Check 208 with regulatory agencies to determine at what concentration sodium azide may 209 cause a product to be regulated as hazardous waste.

210 6. Evan's Blue counter-stain is a potential carcinogen. If skin contact occurs. flush with 211 water immediately.

The DFA Reagents are supplied at working strength. Any dilution of the DFA 212

213 Reagents will decrease sensitivity.

201

202 203

8

1. Reagents should be used prior to their expiration date. 214 215 9. Each Respiratory Virus Antigen Control Slide should be used only once. Do not re-216 use a Control Slide. 217 10. Microbial contamination of DFA Reagents may cause a decrease in sensitivity. 218 11. Store 1X Wash Solution and PBS in a clean container to prevent contamination. 12. All specimens and materials used to process them should be considered potentially 219 infectious and handled in a manner which prevents infection of laboratory personnel. 220 221 Decontamination is most effectively accomplished using a 0.05% solution of sodium 222 hypochlorite (1:100 dilution of household bleach). 223 13. Although Antigen Control Slides have been shown to be non-infectious, the same precautions taken in handling and disposing of other infectious materials should be 224 225 employed in their use. 226 14. Never pipette reagents or clinical samples by mouth; avoid all contact of clinical 227 samples with broken skin. 228 15. Avoid splashing and the generation of aerosols with clinical samples. 229 16. Use aseptic technique and sterile equipment and materials for all tissue culture 230 procedures. 231 17. Reusable glassware must be washed and thoroughly rinsed free of all detergents. 232 18. Do not expose DFA Reagents to bright light during staining or storage. 19. Use of other reagents than those specified with the components of this kit may lead to 233 234 erroneous results. 235 236 C. Preparation of 1X Wash Solution 237 1. After storage at 2° to 8°C, some salts in the Wash Solution Concentrate may have 238 crystallized. Warm the solution to room temperature to re-dissolve the crystals and 239 mix. 240 2. Add contents of the fully dissolved 25-mL Wash Solution Concentrate to 975-mL of 241 de-mineralized water. 3. · Label the 1X Wash Solution with a sixty (60) day expiration date after reconstitution 242 243 and store at room temperature (20° to 25°C).
244

245 D. Storage Instructions

TABLE 1
1.	Respiratory Virus DFA Screening Reagent	Store at 2° to 8°C in the dark.
2.	Influenza A DFA Reagent
3.	Influenza B DFA Reagent
4.	RSV DFA Reagent
5.	Adenovirus DFA Reagent
6.	Parainfluenza 1 DFA Reagent
7.	Parainfluenza 2 DFA Reagent
8.	Parainfluenza 3 DFA Reagent
9.	Mounting Fluid
10.	Normal Mouse Gamma Globulin DFA Reagent

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9

11. Respiratory Virus Antigen Control Slides	Store at 2° to 8°C.
12. Wash Solution Concentrate
NOTE: The Concentrate may crystallize when
stored at 2° to 8°C. The crystals will
dissolve when the Concentrate is warmed
to room temperature.	Store liquid at 2° to 8°C
prior to dilution.
13. 1X Wash Solution	Store at room temperature
(20° to 25°C).

246

247 E. Stability

248 Reagents and components will retain their full potency through the expiration date shown 249 on the label of each bottle when stored at recommended temperatures. Light exposure of 250. the DFA Reagents should be kept to a minimum.

Discard 1X Wash Solution if it becomes cloudy. 251

252

V. SPECIMEN COLLECTION AND PREPARATION 253

254

255 Proper collection and handling of the patient specimen are the most important factors in successful respiratory virus detection. Specimen collection, specimen processing, and cell 256 culture of viruses should be attempted only by personnel that have been trained in such 257 procedures. Care should be taken during all specimen collection and handling to avoid 258 259 generation of aerosols.

260

A. Specimen Collection18 261

Aspirates and Washes containing secretions from the nasopharyngeal epithelium provide 262

the best specimens for direct specimen testing since they will contain large numbers of 263 264 epithelial cells.

265 Aspirates can be collected using a sterile, soft polyethylene #8 infant feeding tube attached 266 to a disposable aspiration trap connected to a suction device.

267 Washes can be collected by instilling and aspirating 1- to 2-mL of saline in the patient's 268 nostril while the patient is in a supine position.

Aspirates and washes should be diluted with equal volumes of transport medium contained 269 in a centrifuge tube with several sterile glass beads. 270

Swabs from nasal, throat and nasopharyngeal areas often do not contain sufficient 271
272 numbers of columnar epithelial cells to allow for direct specimen detection of respiratory 273 viruses.
274

275 B. Specimen Transport and Storage

276 All potentially infectious agents should be transported according to International Air

Transport Association (IATA), International Civil Aviation Organization, (ICAO), Titles 277
42 and 49 of the US Code of Federal Regulations, or other regulatory requirements, as 278
279 may be applicable.
280 Specimens should be transported on wet ice to the laboratory and processed and tested as
281 soon as possible and then stored at 2° to 8°C.

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10

Specimens should be stored at 2° to 8°C for no longer than 48 hours before being tested. If 282

longer storage is required, the specimens should be frozen at -70℃ or lower. 283

284 Freezing and thawing of specimens should be avoided since this will result in a loss of

285 viability of viruses, leading to decreased sensitivity of the test.

286

VI. PROCEDURE 287

288

A. Materials Provided 289

290 1. Respiratory Virus DFA Screening Reagent
291 2. Influenza A DFA Reagent
1. Influenza B DFA Reagent 292
1. RSV DFA Reagent 293
294 5. Adenovirus DFA Reagent
જીતેર 6. Parainfluenza 1 DFA Reagent
296 7. Parainfluenza 2 DFA Reagent
297 8. Parainfluenza 3 DFA Reagent
298 9. Normal Mouse Gamma Globulin DFA Reagent
299 10. Respiratory Virus Antigen Control Slides
300 11. Mounting Fluid
301 12. Wash Solution Concentrate
302

B. Materials Required But Not Provided 303

1. Fluorescence microscope with the correct filter combination for FITC (excitation peak 304 305 = 490 nm, emission peak = 520nm).
1. Cell culture for respiratory virus isolation. Suggested cell lines that are susceptible to 306 respiratory viruses include LLC-MK2, HEp-2, A549 cells, R-Mix™ and R-Mix TooTM 307 Mixed Cells, and primary Rhesus monkev kidnev cells, all available from DHI. 308
300 3. Cover slips (22 x 50mm) for Antigen Control Slides and for specimen slides.
310 4. Universal Transport Medium (available from DHI).
1. R-Mix Refeed medium (for use with R-Mix™ and R-Mix Too™ Mixed Cells) or 311 other standard Refeed medium. Available from DHI. 312
313 6. Reagent grade acetone (>99% pure) chilled at 29 to 8°C for fixation of direct specimen slides and shell vials. 314
NOTE 1: Keep the reagent grade acetone container tightly sealed to avoid hygroscopic 315 absorption of water, which may cause a hazy, nonspecific, background fluorescence. 316
NOTE 2: A mixture of 80% acetone/20% de-mineralized water is used for fixing cells 317
318 in plastic multi-well plates. Store at ambient temperature.
319 7. Sterile graduated pipettes: 10-mL, 5-mL, and 1-mL.
320 8. Sterile Pasteur pipettes or other "transfer"-type pipettes.
Fine-tipped forceps. 321 9.
200-mL wash bottle. 322 10.
1. Bent-tip teasing needle (for removal of coverslip from a shell vial for the typing 323
324 portion of the procedure); fashion the teasing needle by bending the tip of a syringe

11

325		needle or similar object (i.e. mycology teasing needle) against a benchtop or with a pair of forceps taking care to avoid injury.
326		pair of forceps taking care to avoid injury.
327	12.	Sodium hypochlorite solution, 0.05% (1:100 dilution of household bleach).
328	13.	Humid chamber (e.g. covered Petri dish with a damp paper towel placed in the bottom).
329		bottom).
330	14.	Glass microscope slides.
331	15.	Acetone-cleaned multi-well glass microscope slides (2-well and 8-well masked slides).
332		slides).
333	16.	Blotters for multi-well glass microscope slides: Two- and 8-well absorbent blotters, used to blot excess liquid from the mask to prevent spread of liquid or stained cells from one well to the other.
334		used to blot excess liquid from the mask to prevent spread of liquid or stained cells
335		from one well to the other.
336	17.	Sterile nylon flock swab or polyester swab, non-inhibitory to respiratory viruses and tissue culture.
337		tissue culture.
338	18.	Incubator, 35° to 37°C (CO2 or non-CO2 , depending on the cell culture format used).
339	19.	Centrifuge with free swinging bucket rotor.
340	20.	De-mineralized water for dilution of Wash Concentrate Solution and for dilution of the reagent grade acetone for use in polystyrene multi-well plates (see item VI.B.5).
341		the reagent grade acetone for use in polystyrene multi-well plates (see item VI.B.5).
342	21.	PBS (Phosphate Buffered Saline), sterile, for use in rinsing and suspending cells.
343	22.	Control viruses: Known strains of the 7 respiratory viruses for use in monitoring the cell culture and staining procedures. Such control virus strains can be obtained from Diagnostic Hybrids, Inc.
344		cell culture and staining procedures. Such control virus strains can be obtained from
345		Diagnostic Hybrids, Inc.
346	23.	Aspirator Set-up: Vacuum aspirator with disinfectant trap containing sufficient household bleach (5%) that the concentration is not decreased by more than 100 fold as it is diluted with discarded fluids.
347		household bleach (5%) that the concentration is not decreased by more than 100 fold
348		as it is diluted with discarded fluids.
349	24.	Wash Container: Beaker, wash bottle or Coplin jar for washing slides.
350	25.	Fixing Container: Coplin jar, slide dish or polyethylene holder for slides for use in fixing the cells on the slides.
351		fixing the cells on the slides.
352	26.	Inverted Light Microscope: Used for examining the monolayers of cells prior to inoculation and examination for toxicity and CPE. It should have between 100X to 400X magnification capability.
353		inoculation and examination for toxicity and CPE. It should have between 100X to
354		400X magnification capability.
355
356		C. Preliminary Comments and Precautions
357		1. Adhere to the recommended volumes and times in the following procedure to ensure that accurate results are obtained.
358		2. For specimen swabs received in transport medium with glass beads, vortex vigorously for about 15 seconds to dissociate adhered cells. For swabs not received in transport medium, transfer them to a tube of transfer medium containing glass beads and vortex vigorously for about 15 seconds to dissociate adhered cells.
359		For specimen swabs received in transport medium with glass beads, vortex
360		vigorously for about 15 seconds to dissociate adhered cells. For swabs not received
361		in transport medium, transfer them to a tube of transfer medium containing glass
362		beads and vortex vigorously for about 15 seconds to dissociate adhered cells.
363		3. When staining with fluorescent reagents and examining cells microscopically for fluorescence, it is very important to include controls, both positive and negative, to monitor the procedure and performance of the reagents. It is recommended that such controls be run with each batch of patient specimens.
364		fluorescence, it is very important to include controls, both positive and negative, to
365		monitor the procedure and performance of the reagents. It is recommended that
366		such controls be run with each batch of patient specimens.
367		4. The closed, humidified container for holding the slides during incubation should be kept in the incubator so it is at incubator temperature when the slides are placed in
368		kept in the incubator so it is at incubator temperature when the slides are placed in
369		it. By doing this, the cells and antibody solution will come up to temperature more
370		rapidly, yielding more intense stains in shorter periods of time.
371		REGARDING CELL CULTURE TESTING:
372		5. Good Laboratory Practice dictates that positive and negative virus controls be run
373		with each new batch of cells to confirm their performance in culturing specific
374		viruses.
375		6. It is good practice to retain the medium removed from the positive monolayers
376		until after staining results have been obtained. If there is any question concerning
377		the specimen results, the medium can be passed to another monolayer for repeat
378		testing.
379		7. If using cell cultures in polystyrene multi-well plates, the acetone fixative must be
380		diluted with water to 80% by adding 20 mL of water to 80 mL of acetone.
381		8. Do not allow the monolayers to dry before fixing; this can lead to high background
382		staining and decreased sensitivity.
383		9. Do not allow the antibody reagents to dry on the monolayers; this can lead to high
384		background.
385		REGARDING IMMUNOFLUORESCENCE MICROSCOPY:
386		10. It is good practice to examine the positive and negative controls before examining
387		the test specimens. If one of these fails to perform as expected, review the steps
388		and conditions under which the test was performed to determine the cause(s). Do
389		not report results until controls perform properly.
390		11. There are three aspects of the fluorescence microscope that must be functioning
391		properly and optimally in order to achieve maximum brightness of fluorescence:
392		i. The activation light source has a finite life and as it ages, its output decreases,
393		resulting in lower fluorescence intensity from the DFAs.
394		ii. The light source is focused by a number of lenses and mirror(s). For
395		maximum intensity, these must be properly aligned.
396		iii. The filters used in the light path must be appropriate for the particular fluor,
397		in this case, fluorescein.
398		12. There are several fluorescent artifacts that may be observed in the cell monolayers
399		being examined:
400		i. Cell debris, lint, etc. can nonspecifically adsorb DFAs, resulting in highly
401		intense fluorescence. These can be identified by their morphology, i.e., they
402		don't have the appearance of a complete cell and typically do not appear to
403		be a part of the monolayer like the other cells.
404		ii. A low grade, yellow-green fluorescence may sometimes be seen, particularly
405		in areas that have piled cells or are near holes in the cell monolayer. In both
406		cases, the diffusion of the entrapped DFAs is retarded during the wash step,
407		resulting in the nonspecific fluorescence.
408		iii. Intense fluorescence around the periphery of slide wells is indicative of
409		drying of the DFA Reagent during incubation, suggesting that it was
410		incubated too long or the humidity was not controlled.
411		iv. Inadequate removal of the mucus from direct specimens can lead to
412		nonspecific adsorption of DFAs.
413	v. Inadequate washing can lead to a general low grade fluorescence due to residual DFAs remaining on the monolayer of cells.
414
415	vi. On direct specimens, beware of trapping of fluorescence by leukocytes and monocytes. Also, the presence of RBCs in the specimen may leave a green haze on the sample.
416
417
418	13. Quenching or fading of the fluorescence of the stained cells may occur on exposure to light, particularly light of high intensity. Slides should be protected as much as possible during the assay.
419
420
421
422	D. Specimen Preparation
423	1. Vortex the specimen vigorously for 10 to 15 seconds.
424	2. Centrifuge at 400 to 600xg for 5 to 10 minutes.
425	3. Collect and set aside the supernatant for viral isolation. (See Step VI.G.9 below.)
426	4. Add 5 mL of PBS and vortex vigorously for 10 to 15 seconds.
427	5. Centrifuge at 400 to 600xg for 5 to 10 minutes.
428	6. Remove the supernatant and the mucus layer above the cell pellet taking care not to disturb the cell pellet.
429
430	7. Repeat steps 4 through 6 until the mucus layer has been completely removed.
431	NOTE: It is important to remove all the mucus since it can cause nonspecific fluorescence.
432
433	8. Add 0.5 to 1-mL of PBS.
434	9. Mix the suspension by pipetting up and down to re-suspend the cell pellet, forming a slightly cloudy suspension. This cell suspension will be used for Direct Specimen Testing (See Section VI.E., below).
435
436
437	NOTE: The quality of the slide preparation is dependent on the concentration of cells in the suspension; too many cells make it difficult to read the result and too few decrease the sensitivity of the procedure. Specimens may also be cytofuged if a monolayer is preferred.
438
439
440
441	10. For use in Cell Culture Testing (See Section VI. G., F., and H.), add the supernatant that was reserved in Step VI.D.3. above, to the cell suspension that remains after Direct Specimen Testing.
442
443
444	Add a few sterile glass beads to the tube and vortex for about 15 seconds to break up the cells and release any virus. Repeat this step for each specimen.
445
446
447	E. Direct Specimen Testing
448	1. Spot 25 $ \mu $ L of the suspension on each well of a 2-well and an 8-well slide. Repeat this step for each specimen.
449
450	2. Air dry the wells completely.
451	3. Fix the cells to the slides using fresh, chilled acetone for 5 to 10 minutes.
452	4. Remove the slides from the fixative and allow to air dry.
453	5. Add one drop of the DFA Screening Reagent to completely cover the dried, fixed cells on one well of each of the 2-well slides.
454
455	6. Also, to each of the wells of a fresh Respiratory Virus Antigen Control Slide add
457	stained only once, as it contains individual wells of viral infected cells and non-
458	infected cells.
459	7. Add one drop of the Normal Mouse Gamma Globulin DFA Reagent to completely
460	cover the dried, fixed cells on the other well of each of the 2-well slides.
461	8. Place the slides in a covered chamber at 35° to 37°C for 15 to 30 minutes.
462	9. Rinse the stained cells using the 1X Wash Solution. For only a few slides, this can
463	be done using a beaker of the 1X Wash Solution. For many slides, a slide carrier
464	that holds 10 to 20 slides can be placed in its container of 1X Wash Solution. For
465	effective rinsing, dip the slide(s) up and down a minimum of four times.
466	10. Discard the used wash and repeat the washing step using new 1X Wash Solution.
467	11. Rinse the stained cells using de-mineralized water. For only a few slides, this can
468	be done using a beaker of the de-mineralized water. For many slides, a slide
469	carrier that holds 10 to 20 slides can be placed in its container with de-mineralized
470	water. For effective rinsing, dip the slide(s) up and down a minimum of four
471	times.
472	12. Blot the excess 1X Wash Solution, add a small drop of Mounting Fluid to each
473	cell-containing well and cover the wells with a coverslip.
474	13. Examine the stained, mounted cells using a fluorescence microscope with
475	magnifications between 100X to 400X. (See Section VI. C. 10-12, 'Regarding
476	Immunofluorescence Microscopy')
477	14. Refer to Section VII., 'Interpretation of Results'.
478	15. If the result is positive for respiratory virus, the staining procedure may be repeated
479	using the reserved 8-well specimen slides in order to identify which respiratory
480	virus may be present.
481	i. Add one drop of each individual virus DFA Reagent to its corresponding well
482	on the 8-well specimen slide. Leave one well as a negative.
483	ii. For the Respiratory Virus Antigen Control Slide, add one drop of each
484	individual virus DFA Reagent to its corresponding labeled well. An
485	Antigen Control Slide should be stained only once, as it contains individual
486	wells of viral infected cells and non-infected cells.
487	iii. Continue with steps 8 through 14, above.
488
489	F. Cell Culture Testing - Tube Culture
490	1. Examine the monolayers for proper morphology prior to inoculation.
491	2. Aspirate maintenance medium from the monolayers and add 0.2 to 0.5-mL of each
492	prepared specimen (Step VI.D., above) to each of the cell lines used for respiratory
493	virus culture.
494	3. Place the tubes at an angle sufficient for the monolayers to be covered by the
495	inoculum and allow virus adsorption to occur for 1 hour at 35° to 37°C.
496	4. After adsorption, add 2-mL of appropriate refeed medium.
497	5. Incubate the tubes at 35° to 37°C in a roller drum at 1 to 3 rpm. Examine the
498	monolayers daily for evidence of toxicity or viral CPE or test for hemadsorption.
499	6. When the monolayers are ready to be stained, remove the medium by aspiration
500	and gently rinse the monolayer two times with 1 to 2-mL PBS.
501
502
503	7.	Add 0.5-mL of PBS to the tube and prepare a suspension of the cells by scraping the monolayer using a pipette and breaking the cell aggregates up by pipetting up and down several times.
504
505	8.	Prepare cell spots using about 25-µL of the suspension on each well of a 2-well and an 8-well slide. Repeat this step for each specimen.
506	9.	Air dry the wells completely.
507
508	10.	Fix the cells to the slides using fresh, chilled acetone. Let stand for 5 to 10 minutes, at 20° to 25°C.
509	11.	Remove the slides from the fixative and allow to air dry.
510
511	12.	Add one drop of the DFA Screening Reagent to completely cover the dried, fixed cells on one well of each of the 2-well slides.
512
513
514
515	13.	Also, to each of the wells of a fresh Respiratory Virus Antigen Control Slide, add one drop of the DFA Screening Reagent. An Antigen Control Slide should be stained only once, as it contains individual wells of viral infected cells and non-infected cells.
516	14.	Place the slides in a covered chamber at 35°C to 37°C for 15 to 30 minutes.
517
518
519
520	15.	Rinse the stained cells using the 1X Wash Solution. For only a few slides, this can be done using a beaker of the 1X Wash Solution. For many slides, a slide carrier that holds 10 to 20 slides can be placed in its container with 1X Wash Solution. For effective rinsing, dip the slide(s) up and down a minimum of four times.
521	16.	Discard the used wash and repeat the washing step using new 1X Wash Solution.
522
523
524
525	17.	Rinse the stained cells using de-mineralized water. For only a few slides, this can be done using a beaker of the de-mineralized water. For man slides, a slide carrier that holds 10 to 20 slides can be placed in its container with de-mineralized water. For effective rinsing, dip the slide(s) up and down a minimum of four times.
526	18.	Remove the de-mineralized water by aspiration.
527
528	19.	Blot the excess liquid, add a small drop of Mounting Fluid to each cell-containing well and cover the wells with a coverslip.
529
530
531	20.	Examine the stained, mounted cells using a fluorescence microscope with magnifications between 100X to 400X (See Section VI.C. 10-12, 'Regarding Immunofluorescence Microscopy', page 10).
532	21.	Refer to Section VII., 'Interpretation of Results'.
533
534
535	22.	If the result is positive for respiratory virus, the staining procedure may be repeate using the reserved 8-well specimen slides in order to identify which respiratory virus may be present.
536
537		i. Add one drop of each individual virus DFA Reagent to its corresponding well on the 8-well specimen slide. Leave one well as a negative.
538
539
540
541
542		ii. For the Respiratory Virus Antigen Control Slide, add one drop of each individual virus DFA Reagent to its corresponding labeled well. An Antigen Control Slide should be stained only once, as it contains individual wells of viral infected cells and non-infected cells.
iii. Continue with steps 14 through 21 above.
544		G. Cell Culture Testing - Shell Vial

12

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350 West State Street
Athens, OH 45701

14

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15

16

Calculate the number of vials needed based on the staining protocol to be used (this રવર 1. staining protocol requires 3-vials): ર્સ્વર્ i. One vial is required for each day the culture will be screened with the DFA 547 Screening Reagent (i.e. staining at 16- to 24-hours, and then at 48- to 72-ર્સ્વેઠ રવેતે hours, requires 2 vials). One additional vial is required for preparing 8-well slides used to identify ર રા ii. રુદ્ય the viruses from positive screens. Examine the monolavers for proper morphology prior to inoculation. રુટર 2. 3. Aspirate maintenance medium from the monolayers and add 1-mL of appropriate 233 રર્સ refeed medium to each shell vial. રરુર 4. Add 0.2 to 0.4-mL of prepared specimen to each shell vial. Centrifuge the shell vials at 700xg for 1-hour at 20° to 25°C. న. રર્સ્ Place stoppered shell vials in an incubator at 35° to 37°C. રું રહ્યું રહ્યું રહ્યું રહ્યું રહ્યું રહ્યું રહ્યું રહ્યું રહ્યું રહ્માન કરત 6. When a monolayer is ready to be stained using the DFA Screening Reagent, ર રજ 7. remove the medium and add 1-mL of PBS. ર રેતે 8. Swirl to mix and then aspirate. ર્સ્વ Repeat this wash with another 1-mL of PBS and then aspirate. 9. 261 ર્સ્ટ 10. Add 1-mL of chilled 100% acetone and allow to stand for 5 to 10 minutes at 18° to ર્સ્ડ 26°C. Remove the fixative by aspiration. 564 11. ર્સ્ટ 12. Add 0.5-mL of PBS to wet the monolayer. 13. Swirl and then aspirate completely. ર્ રહ્યુ Add 4 drops of the DFA Screening Reagent to the fixed monolayers of patient and 567 14. control samples, and rock to ensure complete coverage of the monolayer by the રૂર્શ્વ ર્સ્વેત Reagent. Place stoppered shell vials in a 35° to 37°C incubator for 15 to 30 minutes. 570 15. Aspirate the DFA Screening Reagent from the monolayers. 571 16. 572 17. Add 1-mL of the 1X Wash Solution. Remove the 1X Wash Solution by aspiration, repeat the wash step and again 573 18. 574 remove by aspiration. 575 19. Add 1-mL of de-mineralized water. Remove the de-mineralized water by aspiration. ર 76 20. Lift the coverslip from the bottom of the shell vial using a bent-tip needle on a 577 21. syringe barrel, and, grasping it with the fine tipped forceps, transfer it, monolayer-578 side down, to a small drop of Mounting Fluid on a standard microscope slide. 579 Examine the stained monolayers using a fluorescence microscope with રજૂરી 22. magnifications between 100X to 400X. (See Section VI. C. 10-12, 'Regarding રજા ર્ રજૂટ Immunofluorescence Microscopy') Refer to Section VII., 'Interpretation of Results'. ર્સ્કર્ 23. If the result is positive for respiratory virus, process a reserved replicate culture as 584 24. a cell suspension and spot onto an 8-well specimen slide in order to identify which ર્ ૪૨ respiratory virus may be present (see Section VI.F. steps 6 through 11, for રક્ષર ર્સ્ડ procedure to prepare a specimen slide), then: i. Add one drop of each individual virus DFA Reagent to its corresponding ર 88 well on the 8-well specimen slide. Leave one well as a negative. ર 80

17

| 590 | ii. | For the Respiratory Virus Antigen Control Slide, add one drop of each
individual virus DFA Reagent to its corresponding labeled well. An
Antigen Control Slide should be stained only once, as it contains individual |
|-----|------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 591 | | |
| 592 | | |
| 593 | | wells of viral infected cells and non-infected cells. |
| 594 | iii. | Continue with VI. F. steps 14 through 15. |
| 595 | | |
| 596 | | |
| 597 | | H. Cell Culture Testing – Multi-well Plate |
| 598 | 1. | Calculate the number of wells needed for the staining protocol to be used (this
staining protocol requires 3-wells): |
| 599 | | i. One well is required for each day the culture will be screened with the DFA |
| 600 | | Screening Reagent (i.e. staining at 16- to 24-hours, and again at 48- to 72- |
| 601 | | hours, requires 2-wells). |
| 602 | | ii. One additional well is required for preparing 8-well slides used to identify the |
| 603 | | viruses from positive screens. |
| 604 | | iii. It is recommended that each replicate well be on a different multi-well plate. |
| 605 | | This allows each plate to be processed on the appropriate day. |
| 606 | 2. | Examine the monolayers for proper morphology prior to inoculation. |
| 607 | 3. | Aspirate maintenance medium from the monolayers and add 1-mL of appropriate |
| 608 | | refeed medium to each 24-well multi-well plate monolayer; add 0.8-mL to each |
| 609 | | 48-well plate monolayer. |
| 610 | 4. | Add 0.2 to 0.4-mL of prepared specimen to the appropriate well of a multi-well |
| 611 | | plate. |
| 612 | 5. | Centrifuge the multi-well plates at 700xg for 1-hour at 20° to 25°C. |
| 613 | 6. | Place the covered multi-well plates in a 35° to 37°C incubator with a humidified, |
| 614 | | 5% CO2 atmosphere. |
| 615 | 7. | When a monolayer is ready to be stained using the DFA Screening Reagent, |
| 616 | | remove the medium and add 1-mL of PBS. |
| 617 | 8. | Swirl to mix and then aspirate. |
| 618 | 9. | Repeat this wash with another 1-mL of PBS and then aspirate. |
| 619 | 10. | Add 1-mL of 80% aqueous acetone and let stand 5 to 10 minutes. |
| 620 | | NOTE: Do not allow the 80% acetone fixative to remain in the polystyrene wells |
| 621 | | longer than 10 minutes since it may craze and cloud the plastic, making it difficult |
| 622 | | to examine the monolayers. |
| 623 | 11. | Remove the fixative by aspiration. |
| 624 | 12. | Add 0.5-mL of the PBS to wet the monolayer. |
| 625 | 13. | Swirl and then aspirate completely. |
| 626 | 14. | Add 4 drops of the DFA Screening Reagent to the fixed monolayers of patient and |
| 627 | | control samples in each 24-well multi-well plate monolayer; add 3 drops of the |
| 628 | | DFA Screening Reagent to the fixed monolayers of patient and control samples in |
| 629 | | each 48-well plate monolayer. Rock to ensure complete coverage of the |
| 630 | | monolayer by the Reagent. |
| 631 | 15. | Place the covered multi-well plate in a 35° to 37°C, humidified incubator for 15 to |
| 632 | | 30 minutes. |
| 633 | 16. | Aspirate the DFA Screening Reagent from the monolayers. |
| 634 | 17. | Add 1-mL of the 1X Wash Solution. |

18

Remove the 1X Wash Solution by aspiration, repeat the wash step and again

ર્ણ કર્ remove by aspiration. 19. 637 Add 1-mL of de-mineralized water. 20. 638 Remove the de-mineralized water by aspiration. 639 21. Add 2 to 3 drops of Mounting Fluid to each monolayer, then cover the plate. 640 22. Examine the stained monolayers using a fluorescence microscope with magnifications between 100X to 400X. (See Section VI.C. 10-12, 'Regarding 641 642 Immunofluorescence Microscopy') 643 23. Refer to Section VII. 'Interpretation of Results'. If the result is positive for respiratory virus, process a reserved replicate culture as 644 24. 645 a cell suspension and spot onto an 8-well specimen slide in order to identify which 646 respiratory virus may be present (see Section VI.F. steps 6 through 11, for 647 procedure to prepare a specimen slide), then: i. Add one drop of each individual virus DFA Reagent to its corresponding well 648 on the 8-well specimen slide. Leave one well as a negative. 649 650 ii. For the Respiratory Virus Antigen Control Slide, add one drop of each individual virus DFA Reagent to its corresponding labeled well. An Antigen 651 Control Slide should be stained only once, as it contains individual wells of 652 viral infected cells and non-infected cells. 653 654 iii. Continue with VI.F. steps 14 through 21. 655

656 I. Quality Control

657 A fresh Respiratory Virus Antigen Control Slide should be stained each time the staining procedure is performed to ensure proper test performance. The positive wells will show 658 multiple infected cells of bright apple-green fluorescence with negative cells staining a ર્ણવત્ત્વના વિદ્યારત તાલુકામાં આવેલું એક ગામનાં મુખ્યત્વે આવેલું એક ગામનાં મુખ્યત્વે આવેલું એક ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં મુખ્યત્ ୧୧୦ dull red due to the included Evan's Blue counter-stain. The negative well will show only 661 negative cells staining a dull red. Positive and negative controls must demonstrate appropriate fluorescence for specimen results to have validity. Antigen Control Slides 662 663 may also aid in the interpretation of patient specimens.

The use of the Normal Mouse Gamma Globulin DFA Reagent in the direct specimens is to 664 665 rule out those rare instances where cells are present that bind the Fe portion of the mouse ર્ભર્ gamma globulin which could lead to a false positive result.

667 668
ર્ણ્ડ રે

VII. INTERPRETATION OF RESULTS ୧୧ରୁ

It is recommended that controls be examined first to ensure proper test performance before 670 examination of the specimens. A positive reaction is one in which bright apple-green 671 672 fluorescence is observed in the infected cells. Uninfected cells will stain dull red due to the Evan's Blue counter-stain included in the DFA Reagent. Technologists should not 673 confuse cell clumps which may fluoresce due to entrapment of antibody with virus-674 specific staining. Occasionally, dead, rounded cells due to specimen toxicity or improper 675 cell storage may nonspecifically stain a dull olive green due to trapped antibody. 676 677 Adequate washing between steps will help to eliminate this type of nonspecific staining,

19

FLUORESCENT STAINING PATTERN OF RESPIRATORY VIRUS INFECTED 678

679 CELLS
The "typical" apple-green fluorescence staining pattern for each virus is as follows: 680
Influenza A and B: The fluorescence is cytoplasmic, nuclear or both. Cytoplasmic 681 682 staining is often punctate with large inclusions while nuclear staining is uniformly
683 bright.
Respiratory Syncytial Virus: The fluorescence is cytoplasmic and punctate with 684 small inclusions in the syncytia. 685
Adenovirus: The fluorescence is cytoplasmic and punctate or bright nuclear or both. 686
687 Parainfluenza 1, 2, 3: The fluorescence is cytoplasmic and punctate with irregular
inclusions. Types 2 and 3 cause the formation of syncytia. 688

689 Co-infection with more than one infecting virus present in the specimen has been reported in a number of studies. The presence of multiple viruses is indicated when more than one 690 રતા રાજ્યના ઉત્તર પાકની ખેતી કરવામાં આવે છે. આ ગામમાં પ્રાથમિક શાળા, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ well of the 8-well slide has fluorescent cells. The identification of the viruses is based on 692 the individual virus DFA Reagents showing fluorescence. In such a case, it should be reported as "... and ... detected by direct specimen testing." or "... and ... isolated by ર્ભ્તે રેતા રાજ્યના સ્વિડે 694 cell culture."

રતેર A. Results from Direct Specimen Testing

ર્સ્ત્રેર્ણ The quality of the specimen with respect to the number of epithelial cells in the sample 697 can be assessed by examining the different fields at a magnification of 200X. A satisfactory specimen should have at least 2 columnar epithelial cells per field. A ર્સ્વેજ ર્જિતે negative result is indicated by the absence of fluorescence in a minimal sampling of 20 700 columnar evithelial cells. An inadequate sample is indicated by fewer than 20 columnar 701 epithelial cells present in the sample, in which case the test is considered invalid. A new specimen should be obtained and tested or cell culture of the remaining specimen should 702 703 be initiated.

704 A satisfactory specimen with no fluorescent cells found should be reported as

"Presumptively negative, no Influenza A. Influenza B. Adenovirus. Respiratory Syncytial 705 706 Virus. Parainfluenza 1, Parainfluenza 2, or Parainfluenza 3 detected by direct specimen

testing". However, such negative results should be confirmed using cell culture. 707

708 Specimens negative by direct specimen testing but vielding positive culture results should be reported as " ... isolated by cell culture", where ' ... ' is the appropriate virus, e.g. 709 710 Influenza A. Influenza B. Adenovirus, Respiratory Syncytial Virus, Parainfluenza 1,

711 Parainfluenza 2, or Parainfluenza 3 (see section VII.B, 'Results from Culture Isolation /
Confirmation', below). 712

713 If fluorescent cells are found, continue with the Testing Procedure, staining with the 714 individual virus DFA Reagents (according to section VI.E.). The individual virus DFA 715 Reagent that yields fluorescent cells represents the identification of the respiratory virus. 716 In such a case, it should be reported as " ... detected by direct specimen testing", where 717 · .. ' is the appropriate virus, e.g. Influenza A, Influenza B, Adenovirus, Respiratory 718 Syncytial Virus, Parainfluenza 1, Parainfluenza 2, or Parainfluenza 3.

719

720 B. Results from Culture Isolation / Confirmation

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Page 13-17 of 13-31 pages

20

The entire cell spot or monolayer of cells must be examined for virus-infected, fluorescent 721 722 cells. If no fluorescent cells are found, the results of testing of the specimen should be 723 reported as, "No Influenza A, Influenza B, Adenovirus, Respiratory Syncytial Virus, Parainfluenza 1, Parainfluenza 2, or Parainfluenza 3 isolated by cell culture." 724 725 If fluorescent cells are found, continue with the Testing Procedure, staining with the 726 individual virus DFA Reagents (according to the appropriate sections VI. F., G., and H.). 727 The individual virus DFA Reagent that yields fluorescent cells represents the identification 728 of the respiratory virus. In such a case, it should be reported as " ... isolated by cell 729 culture", where " ... ' is the appropriate virus, e.g. Influenza B. Respiratory 730 Syncytial Virus, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, or Adenovirus. 731 732 VIII. LIMITATIONS OF PROCEDURE 733 734 1. Inappropriate specimen collection, storage, and transport may lead to false negative 735 culture results 19. 736 2. Assay performance characteristics have not been established for direct specimen 737 staining on specimens other than respiratory specimens. It is the user's responsibility to establish assay performance for specimens other than respiratory 738 739 specimens. 740 3. Incubation times or temperatures other than those cited in the test instructions may 741 give erroneous results. 742 Detection of viruses will vary greatly depending upon the specimen quality and 4. 743 subsequent handling. A negative result does not exclude the possibility of virus 744 infection. Results of the test should be interpreted in conjunction with information 745 available from epidemiological studies, clinical evaluation of the patient and other 746 diagnostic procedures. 747 5. The effects of antiviral therapy on the performance of this kit have not been 748 established. The monoclonal antibodies used in this kit are from hybridomas created using viral 749 6. 750 infected cells as the immunogen. The specific viral antigens detected by the 751 antibodies are undetermined. Since the monoclonal antibodies have been prepared using defined virus strains, 752 7. 753 they may not detect all antigenic variants or new strains of the viruses, should they arise. Monoclonal antibodies may fail to detect strains of viruses which have 754 755 undergone minor amino acid changes in the target epitope region. 756 The monoclonal antibodies used in this kit are not group-specific and therefore 8. 757 cannot be used to differentiate among the different types of Adenovirus and RSV. The viral antigens detected in some direct specimens may be from non-viable virus 758 9. 759 and cannot be isolated by culture. This is particularly true of RSV which is known 760 for its instability and loss of viability. 761 10. A negative direct specimen should be inoculated into an appropriate cell culture 762 and incubated to isolate and identify any respiratory virus that may be present in 763 the specimen. A negative result on a direct or cultured specimen does not rule out the presence of 764 11. 765 virus. Tel 866-344-3477 Diagnostic Hybrids, Inc. 350 West State Street FAX 740-593-0980 www.dhiusa.com Athens, OH 45701

Page 13-18 of 13-31 pages

21

766 12. Performance of the kit can only be assured when components used in the assay are 767 those supplied by Diagnostic Hybrids.
1. Prolonged storage of the DFA Reagents under bright light will decrease the 768 769 staining intensity.
770 14. Light background staining may occur with specimens contaminated with
Staphylococcus aureus strains containing large amounts of protein A. Protein A 771 772 will nonspecifically bind to the Fc portions of conjugated antibodies. Such binding
773 can be distinguished from viral antigen binding on the basis of morphology, i.e., S.
774 aureus-bound fluorescence appears as small (~1 micron diameter), bright dots.
Results from cell cultures with bacterial contamination must, therefore, be 775 776 interpreted with caution.
777

778

IX. EXPECTED VALUES 779

780

Respiratory virus infections are often seasonal, with Influenza typically extending from 781 782 November to April in the northern hemisphere, and Adenovirus infections occurring more 783 often during late winter to early summer. RSV is usually a seasonal (winter and early 784 spring) infection as well, with epidemics lasting up to 5 months, while outbreaks caused 785 by parainfluenza viruses may occur throughout a year.

786
787 The clinical studies described in Section X ("Specific Performance Characteristics') were 788 comprised of respiratory specimens collected during the winter to early spring months of 789 2005/2006. Prevalence of the respiratory viruses within the population of specimens that 790 were prospectively collected and tested fresh are noted in Table 2 below (also, see Study 791 1-DS in Section X).
792

| Expected

Values	Adenovirus	Influenza A	Influenza B	Parainfluenza 1	Parainfluenza 2	Parainfluenza 3	Respiratory Syncytial Virus
Fresh
Specimens
(n = 326)	18	32	19	2	0	5	18
Prevalence	5.5%	9.8%	5.8%	0.6%	0	1.5%	5.5%

793

794

X. SPECIFIC PERFORMANCE CHARACTERISTICS 795

796 This study included eight hundred and forty nine (849) original specimens evaluated by this product ("Subject" test) and a currently marketed DFA Screening & Identification Kit 797 798 ("Predicate" test). All 849 specimens were studied by Direct Specimen (DS) testing with 799 22 of these specimens having insufficient cell numbers to be evaluated, and one other 800 which could not be evaluated because it exhibited non-specific staining from the Normal 801 Mouse Gamma Globulin DFA Reagent: 520 of the specimens also were studied by Cell 802 Culture (CC) method with one specimen not evaluated because it produced a toxic cell

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Page 13-19 of 13-31 pages

22

803 culture monolayer. All but 30 of the specimens were prospectively collected during the 804 2005-2006 season: those 30 specimens had been archived as Parainfluenza-positive. In addition, a set of 81 clinical isolates were tested by CC methods only. The evaluations 805 806 were conducted at three laboratory sites: (1) A reference laboratory in northeast United 807 States; (2) A hospital laboratory in northeast United States; and (3) An internal reference 808 laboratory using specimens collected from an external hospital laboratory. 809 Percent Agreement between the Subject and Predicate tests was calculated for prospectively collected specimens. For the DFA Screening Reagent: 810 811 - By DS method using fresh specimens, positive percent agreement is 95.5% and 812 negative percent agreement is 98.3% (see Table 3). By DS method using frozen specimens, both positive percent agreement and negative percent agreement are 813 814 100% (see Table 4).

815 - By CC method using frozen specimens, both positive percent agreement and negative percent agreement are 100% (see Table 5). 81 Q

817 [See individual study results, in this section, parts A through C, below.] -

818

TABLE 3
DS - fresh
326 specimens	Negativ
e	Screen +	Adenovirus	Influenza A	Influenza B	Parainfluenza 1	Parainfluenza 2	Parainfluenza 3	Respiratory
Syncytial
Virus
Predicate Results:	236	90	18	32	18	2	0	2	18
Subject Results:	232	94	18	32	19	2	0	5	18
Positive Percent
Agreement 2 (PPA)		95.5%	100.0%	100.0%	100.0%	100.0%	---	100.0%	100.0%
95% CI 3- PPA		89.0-
98.2%	82.4-
100%	89.3-100%	82.4-100%	34.2-
100%	---	34.2-
100%	82.4-
100%
Negative Percent
Agreement 4 (NPA)	98.3%		100.0%	100.0%	98.7%	100.0%	100.0%	96.7%	100.0%
95% CI - NPA	95.7-
99.3%		95.2-
100%	94.2-100%	92.9-
99.8%	96.0-
100%	96.1-
100%	90.8-
98.9%	95.2-
100%

819

2 "Positive Percent Agreement", or "PPA", values were calculated according to {[Total Number of Positive Results in Agreement by both Subject and Predicate Tests) divided by [(Total Number of Positive Results in Agreement by both Subject and Predicate Tests) plus (Number of Results Positive by Predicate but Negative by Subject)]} multiplied by 100%.

3 "95% CI" refers to 95% Confidence Intervals, which were calculated according to Exact method (Clopper, C. and S. Pearson. Biometrika 26:404-413. 1934).

4 "Negative Percent Agreement", or "NPA", values were calculated according to {[Total Number of Negative Results in Agreement by both Subject and Predicate Tests) divided by [(Total Number of Negative Results in Agreement by both Subject and Predicate Tests) plus (Number of Results Negative by Predicate but Positive by Subject)]} multiplied by 100%.

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23

TABLE 4
DS - frozen
474 specimens	Negativ
e	Screen +	Adenovirus	Influenza
A	Influenza
B	Parainfluenza 1	Parainfluenza 2	Parainfluenza 3	Respiratory
Syncytial
Virus
Predicate Results:	306	168	8	85	19	3	3	9	51
Subject Results:	306	168	8	85	19	3	3	9	51
PPA		100%	100%	100%	100%	100%	100%	100%	100%
95% CI – PPA		97.8-100%	63.1-
100%	95.7-100%	82.3-100%	38.3-
100%	38.3-
100%	70.1-
100%	93.0-100%
NPA	100%		100%	100%	100%	100%	100%	100%	100%
95% CI- NPA	98.8-
100%		97.7-
100%	95.6-100%	97.6-100%	97.8-
100%	97.8-
100%	97.6-
100%	96.7-100%

820

TABLE 5
CC - frozen
490 specimens	Negative	Screen +	Adenovirus	Influenza A	Influenza B	Parainfluenza 1	Parainfluenza 2	Parainfluenza 3	Respiratory
Syncytial Virus
Predicate Results:	309	181	13	93	23	6	4	9	49
Subject Results:	309	181	13	93	23	6	4	9	49
PPA		100%	100%	100%	100%	100%	100%	100%	100%
95% CI - PPA		98.0-
100%	73.4-
100%	95.2-
100%	83.1-
100%	55.7-
100%	45.4-
100%	65.5-
100%	91.3-100%
NPA	100%		100%	100%	100%	100%	100%	100%	100%
95% CI - NPA	98.5 -
100%		97.3-
100%	95.0-
100%	97.1-
100%	97.4-
100%	97.4-
100%	96.6-
100%	96.6-100%

821

822 Specimens and culture isolates used in these studies came from nasopharyngeal (NP)

823 aspirates, washes, swabs, bronchial alveolar lavages (BAL) and/or tracheal aspirates.

824
825 Table 6 below summarizes the participant age demographics according to test results for a 826 population of 326 fresh specimens, prospectively collected and evaluated for performance

using the predicate assay (see 'Study 1-DS - Direct Specimen Method', below).

827

TABLE 6
	Virus:	Adenovirus	Influenza A	Influenza B	Parainfluenza 1	Parainfluenza 2	Parainfluenza 3	Respiratory Syncytial Virus	Negative
	Age
	Totals	18	32	18	2	0	2	18	236
	21y	0	12	1	0	0	0	1	78
Not reported	1	3	1	1	0	0	6	25

Age: m = months, and y = years

828

829

830 A. Prospectively collected specimens:

Clinical Study Sites 1, 2, and 3 generated data for Direct Specimen (DS) Testing 831 according to the study design briefly summarized for each site. 832

Clinical Study Sites 2 and 3 generated data for Cell Culture (CC) Testing according to 833 study design as summarized for each site. 834

835

Study 1-DS - Direct Specimen Method: The study consisted of a total of 329 836 fresh specimens submitted February through May, 2006, to the laboratory for 837 respiratory virus testing. Slides were prepared from PBS-washed cells from the 838 fresh specimens and fixed according to the prescribed protocol. The slides were 839 stored at -70°C until testing was performed. The slides were brought to room 840 temperature and stained in accordance with the procedure in the Predicate product 841 insert (same procedure for both Subject and Predicate devices). Three (3) 842 specimens were found to contain insufficient numbers of cells for interpretation of 843 DS stain results, leaving 326 specimens for evaluation. The results of this testing 844 are summarized in Table 7 below: 845 846

326 specimens	Negative	Screen +	Adenovirus	Influenza A	Influenza B	Parainfluenza 1	Parainfluenza 2	Parainfluenza 3	Respiratory Syncytial Virus
Predicate Results:	236	90	18	32	18	2	0	2	18
Subject Results:	232	94	18	32	19	2	0	5	18
PPA		100%	100%	100%	100%	100%	--	100%	100%
95% CI - PPA		95.1% -
100%	79.3% -
100%	87.3% -
100%	79.3% -
100%	29.0% -
100%	--	29.0% -
100%	79.3% -
100%
NPA	98.3%		100%	100%	98.7%	100%	100%	96.7%	100%
95% CI - NPA	95.4% -
99.5%		94.2% -
100%	93.0% -
100%	92.2% -

99.9% | 95.2% -
100% | 95.3% -
100% | 90.5% -
99.3% | 94.2% -
100% |

Study 1-DS - Direct Specimen Results TABLE 7

847

く

848
849

With the exception of 4 specimens, the DS test results were concordant for both the screen and the identification of the individual viruses; the Predicate device

25

identified 4 specimens as being negative while the Subject device identified one as 850 Flu B and three as Para 3 infections. All but one of the Para 3 specimens were 851 confirmed by culture: the one Para 3, although strongly positive by the Subject 852 assay, could not be cultured to confirm it as a Para 3. The culture method was not 853 performed on the rest of the specimens from this site. 854

Study 2-DS - Direct Specimen Method: The study consisted of 192 specimens submitted to the laboratory for respiratory virus testing during December 2005 through February 2006, with residual specimen material stored at -70°C from a few days to 2 months. The frozen specimens were thawed and processed between 13 February to 17 February 2006 according to the procedure in the Predicate product insert (same procedure for both Subject and Predicate devices).

Slides were prepared from the specimens according to instructions detailed in the 862 Predicate device's product insert. These slides were stained with both the 863 864 Predicate and Subject devices and interpreted according to the Predicate device's product insert procedure (same procedure for both Subject and Predicate devices). 865 All of the frozen/thawed specimens had sufficient intact cells for interpretation. 866 867

The results of this testing are summarized in Table 8 below:

TABLE 8	Study 2-DS - Direct Specimen Results
192 specimens	Negative	Screen +	Adenovirus	Influenza A	Influenza B	Parainfluenz
a 1	Parainfluenz
a 2	Parainfluenz
a 3	Respiratory
Syncytia
Virus
Predicate
Results:	142	50	2	26	3	1	1	0	17
Subject Results:	142	50	2	26	3	1	1	0	17
PPA		100%	100%	100%	100%	100%	100%	--	100%
95% CI - PPA		91.5% -
100%	29.0
% -
100%	84.8
% -
100%	38.3
% -
100%	16.8
% -
100%	16.8
% -
100%	--	80.5% -
100%
NPA	100%		100%	100%	100%	100%	100%	100%	100%
95% CI - NPA	96.8% -
99.5%		92.6
% -
100%	96.2
% -
100%	96.8
% -

99.9% | 96.8
% -
100% | 96.8
% -
100% | 96.8
% -
100% | 89.4% -
100% |

869 870

871

872

873

874

875

876

877

878

879 880

૪૨૨

856

857 858

8 રેતે

860

861

868

The DS test results were concordant for both the Screen and the ID reagents.

Study 2-CC - Cell Culture Method: The same 192 specimens that were evaluated by DS testing were also processed according to the Predicate device's product insert procedure for cell culture (same procedure for both Subject and Predicate devices). Briefly, 200-uL from the specimens were inoculated onto each of 4 monolayers of R-Mix™ Too FreshCells™ contained in shell vials which were centrifuged for 60 minutes at 700xg and incubated for 24-hours at 35° to 37°C. The shell vials were processed according to instructions detailed in the Predicate device's product insert. The results of this testing are summarized in Table 9 below:

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26

| 192 specimens | Negative | Screen + | Adeno
virus | Influen
za A | Influen
za B | Parain
fluenz
a 1 | Parain
fluenz
a 2 | Parain
fluenz
a 3 | Respirato
ry Syncytia
Virus |
|-----------------------|------------------|-----------------|---------------------|---------------------|---------------------------|-------------------------|-------------------------|-------------------------|-----------------------------------|
| Predicate
Results: | 142 | 50 | 3 | 26 | 3 | 1 | 1 | 0 | 16 |
| Subject Results: | 142 | 50 | 3 | 26 | 3 | 1 | 1 | 0 | 16 |
| PPA | | 100% | 100% | 100% | 100% | 100% | 100% | -- | 100% |
| 95% CI - PPA | | 91.5% -
100% | 38.3
% -
100% | 84.8
% -
100% | 38.3
% -
100% | 16.8
% -
100% | 16.8
% -
100% | -- | 77.3% -
100% |
| NPA | 100% | | 100% | 100% | 100% | 100% | 100% | 100% | 100% |
| 95% CI - NPA | 96.8% -
99.5% | | 96.8
% -
100% | 96.2
% -
100% | 96.8
% -

99.9
% | 96.8
% -
100% | 96.8
% -
100% | 96.8
% -
100% | 96.4% -
100% |

Study 2-CC - Cell Culture Results TABLE 9

881 882

883 884

896 897

898

The CC test results were concordant for both the Screen and the ID of the specific viruses.

Study 3-DS - Direct Specimen Method: The study consisted of 298 specimens 885 886 originally received by a hospital laboratory in the eastern US for respiratory virus testing during January through March 2006, with residual specimen material stored 887 at -70°C from 3 to 6 months. The frozen specimens were sent to DHI, where they 888 889 were thawed and processed between 30 May and 1 June 2006, according to the predicate device's product insert. All specimens used in the studies were tested by 890 both the DS and CC procedures as detailed in the Predicate device's product insert; 891 however, a total of 16 specimens were inadequate for interpretation of DS stain 892 893 results (15 were found to contain insufficient numbers of cells, and one other specimen exhibited non-specific staining with the Mouse Gamma Globulin DFA 894 Reagent), leaving 282 specimens for evaluation. 8તેર

The DS results for these specimens tested using the Predicate and Subject devices are summarized in Table 10 below:

| 282 specimens | Negative | Screen + | Adenovirus | Influenza A | Influenza B | Parainfluenz
a 1 | Parainfluenz
a 2 | Parainfluenz
a 3 | Respiratory Syncytia
Virus |
|-----------------------|-----------------|-----------------|---------------------|---------------------|---------------------------|---------------------|---------------------|---------------------|-------------------------------|
| Predicate
Results: | 164 | 118 | 6 | 59 | 16 | 2 | 2 | 9 | 34 |
| Subject Results: | 164 | 118 | 6 | 59 | 16 | 2 | 2 | 9 | 34 |
| PPA | | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% |
| 95% CI - PPA | | 96.2% -
100% | 55.7
% -
100% | 92.7
% -
100% | 77.3
% -
100% | 29.0
% -
100% | 29.0
% -
100% | 65.5
% -
100% | 87.9% -
100% |
| NPA | 100% | | 100% | 100% | 100% | 100% | 100% | 100% | 100% |
| 95% CI - NPA | 97.3% -
100% | | 96.0
% -
100% | 92.7
% -
100% | 95.6
% -

99.9
% | 96.2
% -
100% | 96.2
% -
100% | 95.9
% -
100% | 91.3% -
100% |

TABLE 10 Study 3-DS - Direct Specimen Results

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Page 13-24 of 13-31 pages

27

8 ਰੇਖੇ The DS test results were concordant for both the Screen and the ID reagents. 900 There were ten (10) specimens identified with co-infections as follows: three (3) 901 Flu A+Para 3, one (1) Flu B+Para 2, one (1) Flu B+Para 3, one (1) RSV+Para 1, 902 three (3) RSV+Para 3 and one (1) Adeno+Para 3. Because of the ten (10) co-903 904 infections, the Negatives and Positives add up to 292 ID results.

Study 3-CC - Cell Culture Method: The same 298 specimens that were evaluated by DS testing were also processed for CC testing according to the Predicate device's product insert for cell culture using R-Mix™ Too FreshCellsTM in 48/24-fill cluster plates. The results of this testing are summarized in Table 11 below:

| 298 specimens | Negative | Screen + | Adeno
virus | Influen
za A | Influen
za B | Parain
fluenz
a 1 | Parain
fluenz
a 2 | Parain
fluenz
a 3 | Respirator
y Syncytia
Virus |
|-----------------------|-----------------|-----------------|---------------------|---------------------|---------------------------|-------------------------|-------------------------|-------------------------|-----------------------------------|
| Predicate
Results: | 167 | 131 | 10 | 67 | 20 | 5 | 3 | 9 | 33 |
| Subject Results: | 167 | 131 | 10 | 67 | 20 | 5 | 3 | 9 | 33 |
| PPA | | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% |
| 95% CI – PPA | | 96.6% -
100% | 67.9
% -
100% | 93.5
% -
100% | 81.0
% -
100% | 51.1
% -
100% | 38.3
% -
100% | 65.5
% -
100% | 87.6% -
100% |
| NPA | 100% | | 100% | 100% | 100% | 100% | 100% | 100% | 100% |
| 95% CI - NPA | 97.3% -
100% | | 96.3
% -
100% | 93.2
% -
100% | 96.0
% -

99.9
% | 96.4
% -
100% | 96.5
% -
100 | 96.3
% -
100% | 95.5% -
100% |

Study 3-CC - Celi Culture Results TABLE 11

911 912

913 914

જેવર దిరి రెడ్

907

908

909 910

The CC test results were concordant for both the Screen and the ID reagents.

There were sixteen (16) co-infections as follows: three (3) Flu A+Para 3, one (1) Flu A+Para 1,

તે । one (1) Flu A+Para 2, two (2) Flu A+RSV, one (1) Flu A+Adeno, one (1) Flu B+Para 2, one (1) Flu B+Para 3, one (1) Flu B+RSV, one (1) RSV+Para 1, two (2) ਰੇ। Q RSV+Para 3, one (1) Adeno+Para 1 and one (1) Adeno+Para 3. Because of the 917 sixteen (16) co-infections, the Negatives and Positives in the table add up to 314 918 919 ID results.

920 921

922 B. Non-prospective archival specimens:

Due to relative low prevalence of Parainfluenza infections in populations of respiratory 923 specimens, few specimens in the studies detailed above were reactive with the 924 925 Parainfluenza DFA Reagents. In order to better demonstrate performance ૭૮૯ characteristics of the Parainfluenza DFA Reagents, frozen original specimens 927 previously determined to contain Parainfluenza (types 1, 2, or 3) during the 2006 928 "respiratory season" were obtained from an additional laboratory, and were tested in 929 an internal reference laboratory using the Subject and Predicate Tests by Direct Specimen method (Study 3a-DS; see Table 12, below). The same specimens were 930

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28

tested by Cell Culture method (see Table 13). Original results reported by the 931 laboratory were unknown to the study investigator. Although the study design has a 932 selection bias, this study offers further analytical information on the assay's ability to 933 934 detect Parainfluenza viruses.

Study 3a-DS - Direct Specimen Method: The study consisted of 30 specimens તે તે રેણ 937 originally received by a hospital laboratory in Italy for respiratory virus testing તે તે જેવી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં પ્રાથમિક શાળા, આંગણવાડી તેમ જ દૂધની during the period from October 2005 through April 2006, with residual specimen ਹੇਤੇ ਹੋ ਹੈ ਹੈ . ਹੋ ਹੋ . . ਹੇ 3 . . material stored at -70℃ from 2 to 6 months. The frozen specimens were sent to 94() DHI, where they were thawed and processed between June 7 and 8, 2006, 941 according to the prescribed protocol. All specimens used in the studies were tested 942 by both the DS and CC procedures as detailed in the Predicate device's product 943 insert; however, a total of four specimens were found to contain insufficient 944 numbers of cells for interpretation of DS stain results, leaving 26 specimens.

945 The DS results for these specimens tested using the Predicate and Subject devices 946 are summarized in Table 12 below:

Respirat Parain Parain Parain Adeno Influen Influen огу 26 specimens Negative Screen + fluenz fluenz fluenz Syncytia za B virus za A a 1 a 2 ਬ ਤ Virus Predicate 9 17 0 0 0 1 5 11 0 Results: Subject 0 0 5 12 ಕಿ 18 0 1 O Results: PPA 100% 100% 100% 100% -------------16.8 51.1 70.0 78.4% 95% CI - PPA % -% -% -ーーー -----------100% 100% 100% 100% 85.7 100% 100% 100% 100% 100% 100% NPA 88.9% 0/0 46.7 79.3 79.3 79.3 78.4 73.4 79.3 % -54.3% -% -95% CI - NPA % -0/6 -0/0 -0/0 -0/0 ->99.9% 99.5 100% 100% 100% 100% 100% 100 %
TABLE 12 Study 3a-DS - Direct Specimen Results

948

તે તે રેણે તે જે રેણવાડી તેમ જ દૂધની ડેરી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં પ્રાથમિક શાળા, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં પ્રાથમિક શાળા,

947

049 તેરી

તે રે I 952

વેરે 3

With the exception of one specimen, the DS test results were concordant for both the Screen and the ID of individual viruses; the Subject device identified one specimen as positive for Para 3 while the Predicate device was negative for this specimen.

Study 3a-CC - Cell Culture Method: The same 30 frozen specimens that were 954 evaluated by DS testing were also processed for CC testing according to the તેરેર તેરૂરિ Predicate device's product insert for cell culture using R-Mix™ FreshCells™ in 957 48/24-fill cluster plates. One specimen was found to be unsuitable for CC testing because it was toxic to the monolayer of cells. The results of this testing are 958 તે રેતે તે રેતે તે તે તે તે તે તે તે આ રાજીની તે આ રાજીની આવે છે રેતા પાસની વસે છે તે છે. આ ગામનાં પ્રાથમિક શાળા, આંગણવાડી તેમ જ દૂધની ડેરી જેવી જેવી સવલતો પ્રાપ્ય થયેલી છે. summarized in Table 13 below:

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Page 13-26 of 13-31 pages

29

TABLE 13 Study 3a-CC – Cell Culture Results
29 specimens	Negative	Screen +	Adenovirus	Influenza A	Influenza B	Parainfluenz
a 1	Parainfluenz
a 2	Parainfluenz
a 3	Respiratory
Syncytial
Virus
Predicate
Results:	8	21	0	0	0	3	5	13	0
Subject Results:	8	21	0	0	0	3	5	13	0
PPA		100%	---	---	---	100%	100%	100%	---
95% CI – PPA		81.8% -
100%	---	---	---	38.3 % -
100%	51.1 % -
100%	73.4 %-
100%	---
NPA	100%		100%	100%	100%	100%	100%	100%	100%
95% CI – NPA	62.8% -
100%		81.8 % -
100%	81.8 % -
100%	81.8 %-
100%	79.3 %-
100%	77.3 % -
100%	62.8 %-
100%	81.8% -
100%

TARI E 13 Study 3a-CC Cell Culture Results

તેરી તેમને તે જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામમાં મુખ્યત્વે ખેત-ઉત્પાદની તેમ જ દૂધની ડેરી જેવી સવલતો પ્રાપ્ય ರ್ಕಾರ

663

તેરૂર

તેરણ

967

તે 668

ਰੇਉਰੇ

970

971 972

973

974

The CC test results were concordant for both the Screen and the ID reagents.

તેરવ C. Non-prospective archival clinical isolates:

To further demonstrate the proficiency of the Screening and Typing Reagents in the Subject Test, a study was conducted using a collection of banked clinical isolates known to contain respiratory viruses that had been frozen from the 2005/2006 respiratory season. These specimens were selected because they were previously shown to contain at least one of the seven virus analytes detected by the Subject Test.

Study 3b-CC- Cell Culture Method: A total of 81 clinical isolates from a frozen archival repository were processed according to the Predicate device's product insert for cell culture using R-Mix™ FreshCells™ cultures in shell vials. The results of this testing are summarized in Table 14 below:

975 976

TABLE 14	Study 3b-CC – Cell Culture Results
81 specimens	Negative	Screen +	Adenovirus	Influenza A	Influenza B	Parainfluenza 1	Parainfluenza 2	Parainfluenza 3	Respiratory Syncytial Virus
Predicate Results:	0	81	11	18	17	4	1	26	5
Subject Results:	0	81	11	18	17	4	1	26	5
PPA		100%	100%	100%	100%	100%	100%	100%	100%
95% CI – PPA		94.6% – 100%	70.0% – 100%	79.3% – 100%	78.4% – 100%	45.4% – 100%	16.8% – 100%	84.8% – 100%	51.1% – 100%
NPA		100%	100%	100%	100%	100%	100%	100%	100%
95% CI – NPA		97.3% – 100%	93.8% – 100%	93.1% – 100%	93.2% – 100%	94.3% – 100%	94.5% – 100%	92.2% – 100%	94.2% – 100%

Study 3b-CC – Cell Culture Results TARI C 14

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Page 13-27 of 13-31 pages

30

977

978 The CC test results were concordant for both the Screen Reagent and the specific 979 virus ID Reagents. Because of the one co-infection, Para 1+ Para 3, the positive ID

980 results added up to 82.

981

982 D. Cross-reactivity Testing

Diagnostic Hybrids, Inc. D3 Ultra DFA RESPIRATORY VIRUS SCREENING & ID KIT 983 984 DFA Reagents were tested for cross-reactivity against a wide variety of cells and તે જેવી જેવી તેમ જ દૂધની ડેરી જેવી સવલતો પ્રતિષ્ઠા તેમ જ દૂધની ડેરી જેવી સવલતો પ્રતિષ્ઠા જિલ્લામાં આવેલું એક ગામનાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સ microorganisms. No cross-reactivity was observed for 64 virus strains (cultured and તે જણ્વ processed for staining) or for 18 host culture cell types. Eighteen (18) bacterial cultures 987 were stained and examined for cross-reactivity, including Staphylococcus aureus, a 988 protein-A-producing bacterium. Staining of S. aureus appeared as small points of 989 fluorescence (see Limitations of Procedure, Section 12.) while all other bacterial cultures 990 were negative. [See Table 15 for cross-reactivity study results. The table indicates which ਹੇਰੇ I organisms were reactive with which DFA Reagent.]

992
993 Stringent conditions for cross-reactivity testing were achieved by using high concentration ਰੇਰੇਖ DFAs and high titers of microorganisms. The DFAs (i.e. directly fluoresceinated તેત્વે રે monoclonal antibodies) were prepared at 1.5X the concentration that is provided in the તેત્વે રે kit. Each of the tested viruses was prepared as infected cell monolayers (250 infected cells 997 inoculated into a shell vial culture and incubated for 24 to 48 hours, to yield a 3+ to 4+ 998 infection), and processed and stained with the 1.5X DFAs according to the procedure ਰੇਰੇ ਹੋ detailed in this product insert. Bacterial strains were cultured, processed as suspensions, 1000 then spotted on microscope slides (yielding > 150 bacteria per 400X microscope field), then stained with the 1.5X DFAs according to the procedure in this product insert. Cell 1001 1002 cultures were stained as confluent monolayers.

1003

TABLE 15		DFA Reagent (Results are Positive (+) or Negative (-) for Reactivity)
Organism	Strain	Adeno	Flu A	Flu B	Para 1	Para 2	Para 3	RSV
Adenovirus	Type 1	+	-	-	-	-	-	-
	Type 3	+	-	-	-	-	-	-
	Type 5	+	-	-	-	-	-	-
	Type 6	+	-	-	-	-	-	-
	Type 7	+	-	-	-	-	-	-
	Type 10	+	-	-	-	-	-	-
	Type 13	+	-	-	-	-	-	-
	Type 14	+	-	-	-	-	-	-
	Type 18	+	-	-	-	-	-	-
	Type 31	+	-	-	-	-	-	-
	Type 40	+	-	-	-	-	-	-
	Type 41	+	-	-	-	-	-	-
Influenza A	Mexico/4108/2009
(H1N1) from CDC*	-	+	-	-	-	-	-
	California/07/2009
(H1N1) from CDC*	-	+	-	-	-	-	-
	Aichi (H3N2)	-	+	-	-	-	-	-
	Mal (H1N1)	-	+	-	-	-	-	-
	Hong Kong (H3N2)	-	+	-	-	-	-	-
	Denver (H1N1)	-	+	-	-	-	-	-
	Port Chalmers (H3N2)	-	+	-	-	-	-	-
	Victoria (H3N2)	-	+	-	-	-	-	-

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Page 13-28 of 13-31 pages

31

TABLE 15		DFA Reagent (Results are Positive (+) or Negative (-) for Reactivity)
Organism	Strain	Adeno	Flu A	Flu B	Para 1	Para 2	Para 3	RSV
Influenza B	New Jersey (HswN1)	-	+	-	-	-	-	-
	WS (H1N1)	-	+	-	-	-	-	-
	PR (H1N1)	-	+	-	-	-	-	-
	Hong Kong	-	+	+	-	-	-	-
	Maryland	-	-	+	-	-	-	-
	Mass	-	-	+	-	-	-	-
	Taiwan	-	-	+	-	-	-	-
	GL	-	-	+	-	-	-	-
	Russia	-	-	+	-	-	-	-
RSV	Long	-	-	-	-	-	-	+
	Wash	-	-	-	-	-	-	+
	9320	-	-	-	-	-	-	+
Rhinovirus 39	209 Picornavirus	-	-	-	-	-	-	-
Parainfluenza 1	C-35	-	-	-	+	-	-	-
Parainfluenza 2	Greer	-	-	-	-	+	-	-
Parainfluenza 3	C 243	-	-	-	-	-	+	-
Parainfluenza 4a	M-25	-	-	-	-	-	-	-
Parainfluenza 4b	CH19503	-	-	-	-	-	-	-
Metapneumovirus	A1	-	-	-	-	-	-	-
	A2	-	-	-	-	-	-	-
	B3	-	-	-	-	-	-	-
	B4	-	-	-	-	-	-	-
Coronavirus	OC43	-	-	-	-	-	-	-
	229E	-	-	-	-	-	-	-
Herpes simplex virus Type 1	1F	-	-	-	-	-	-	-
	MacIntyre	-	-	-	-	-	-	-
Herpes simplex virus Type 2	MS	-	-	-	-	-	-	-
	Strain G	-	-	-	-	-	-	-
Cytomegalovirus	Towne	-	-	-	-	-	-	-
	Davis	-	-	-	-	-	-	-
	AD169	-	-	-	-	-	-	-
Varicella-zoster	Webster	-	-	-	-	-	-	-
	Ellen	+	-	-	-	-	-	-
Echovirus	9	-	-	-	-	-	-	-
	11	-	-	-	-	-	-	-
	30	-	-	-	-	-	-	-
	34	-	-	-	-	-	-	-
Coxsackievirus	B1	-	-	-	-	-	-	-
	B2	-	-	-	-	-	-	-
	B3	-	-	-	-	-	-	-
	B4	-	-	-	-	-	-	-
	B5	-	-	-	-	-	-	-
	B6	-	-	-	-	-	-	-
Mumps		-	-	-	-	-	-	-
Rubeola		-	-	-	-	-	-	-
Rhinovirus	209 Picornavirus	-	-	-	-	-	-	-
Acholeplasma laidlawii		-	-	-	-	-	-	-
Bordetella bronchiseptica		-	-	-	-	-	-	-
Bordetella pertussis		-	-	-	-	-	-	-
Chlamydia pneumoniae		-	-	-	-	-	-	-
Clostridium diphtheriae		-	-	-	-	-	-	-
Haemophillus influenzae type A		-	-	-	-	-	-	-
Klebsiella pneumoniae		-	-	-	-	-	-	-
Listeria pneumophila		-	-	-	-	-	-	-
Moraxella cartarrhalis		-	-	-	-	-	-	-
Mycobacterium avium		-	-	-	-	-	-	-
Mycobacterium intracellulare		Mycoplasma hominis type 1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Mycoplasma orale	-	-	-	-	-	-	•	-

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32

TABLE 15		DFA Reagent (Results are Positive (+) or Negative (-) for Reactivity)
Organism	Strain	Adeno	Flu A	Flu B	Para 1	Para 2	Para 3	RSV
Mycoplasma pneumoniae		-	-	-	-	-	-	-
Mycoplasma salivarium		-	-	-	-	-	-	-
Pseudomonas aeruginosa		-	-	-	-	-	-	-
Streptococcus pneumoniae		-	-	-	-	-	-	-
Streptococcus pyogenes		-	-	-	-	-	-	-
Ureaplasma urealyticum		-	-	-	-	-	-	-
Cell cultures:
A549		-	-	-	-	-	-	-
BGMK		-	-	-	-	-	-	-
HEp-2		-	-	-	-	-	-	-
LLC-MK2		-	-	-	-	-	-	-
MDCK		-	-	-	-	-	-	-
MRC-5		-	-	-	-	-	-	-
MRHF		-	-	-	-	-	-	-
Mv1Lu		-	-	-	-	-	-	-
NCI-H292		-	-	-	-	-	-	-
pCMK		-	-	-	-	-	-	-
pRhMK		-	-	-	-	-	-	-
pRK		-	-	-	-	-	-	-
RD		-	-	-	-	-	-	-
RhMK II		-	-	-	-	-	-	-
R-Mix™		-	-	-	-	-	-	-
R-Mix™ Too		-	-	-	-	-	-	-
Vero		-	-	-	-	-	-	-
WI-38		-	-	-	-	-	-	-

1004

1005 1006
1007

Although this test has been shown to detect the 2009 H1 N1 influenza virus in two cultured isolates, the performance characteristics of this device with clinical specimens that are positive for the 2009 H1N1 influenza virus have not been established. The D3 Ultra DFA Respiratory Virus Screening & ID Kit can distinguish between influenza A and B viruses, but it cannot differentiate influenza subtypes.

1008 1009

XI. BIBLIOGRAPHY 1010

1 www.cdc.gov

2 FDA Guidance Document: In Vitro Diagnostic Devices to Detect Influenza A Viruses: Labeling and Regulatory Path; Issued 4/10/2006

5 Englund, J.A., (2002). Antiviral therapy of influenza. Semin. Pediatr. Infect. Dis., 13(2):120-128.

4 Patel, N., Hartwig, R., Kauffmann, L. and Evans, M. (2000). Rapid influenza A and B culture 20-hour detection using R-Mix: A new gold standard. Presented at The Sixteenth Annual Clinical Virology Symposium, April 30-May 3, Clearwater Beach, FL.

S Rodriguez, W.J., Schwartz, R.H. and Thorne, M.M. (2002). Evaluation of diagnostic tests for influenza in a pediatric practice. Pediatr. Inf. Dis. J., 3:193-6

6 Gould, I.M. (2002). Antibiotic Policies and control of resistance. Curr. Opin. Infect. Dis., 15(4):395-400.

7 Bischofberger, N., Webster, R.G. and Laver, G. (1999). Disarming Flu Viruses. Scientific American, January.

8 Wiedbrauk, D.L. and Johnston, S.L.G. (1993). Chapter 17, Influenza Virus. In: Manual of Clinical Virology. New York, Raven Press, 127-140.

33

9 Foy, H.M. (1997). Adenoviruses. In: Evans, A., Kaslow, R., eds. Viral Infections in Humans: Epidemiology and Control. 4th ed., New York, Plenum, 119-138.

10 Easton, A.J., Eglin, R.P. (1989). Epidemiology of Parainfluenza virus type 3 in England and Wales over a 10 year period. Epidemiol. Infect., 102:531-535.

11 Fete, T.J., Noyes, B. (1996). Common (but not always considered) viral infections of the lower respiratory tract. Pediatr. Ann., 25:10), 577-584.

12 Hall, C.B. (1981). Respiratory Syncytial Virus. In: Feigin, R. D., Cherry, J.D., eds. Textbook of Pediatric Infectious Diseases, Phila., W.B. Saunders, 1247-1267.

13 Hall, C.B., Hall, W.J., Gala, C.L., MaGill, F.B., Leddy, J.P. (1984). Longterm prospective study in children after Respiratory Syncytial Virus infection. J. Pediatr., 105:358-364.

14 Falsey, Ann R. and Walsh, E.E. (2000). Respiratory Syncytial Virus Infection in Adults. Clinical Microbiology Reviews 13(3):371-384.

15 Biosafety in Microbiological and Biomedical Laboratories (BMBL), 4th edition, 1999, CDC-NIH manual. [http://www.cdc.gov/od/ohs/biosfty/bmb14/bmb14toc.html

16 Biosafety Manual, 3th edition, 2004. World Health Organization [Manual may be available in additional languages: refer to WHO web page

[http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_1 1/en/]

17 Laboratory Biosafety Guidelines, 3th edition, 2004. Published by authority of the Minister of Health, Population and Public Health Branch, Centre for Emergency Preparedness and Response [Guideline is available in French or English; refer to web page [http://www.phac-aspc.gc.ca/publicat/lbg-ldmbl-04/index.html]

18 Eisenberg, Henry D. (1992). Clinical Microbiology Procedures Handbook, published by American Society for Microbiology, Washington DC, pg. 8.2.3.

19 Leland, Diane S. (1996). Clinical Virology, published by W.B. Saunders, Philadelphia, PA.

34

Special 510(k): Device Modification D3 Ultra DFA Respiratory Virus Screening & ID Kit

Image /page/34/Picture/1 description: The image shows the logo for Diagnostic Hybrids. The logo features the company name in bold, stacked text, with the tagline "Integrating Science and Humanity" underneath. To the right of the text is a stylized graphic of a human figure intertwined with a DNA double helix.

DATE OF PREPARATION OF 510(k) SUMMARY

July 23, 2009

APPLICANT

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

CONTACT INFORMATION

Ronald H. Lollar Senior Director, Product Realization, Management, and Marketing E-mail: lollar@dhiusa.com Telephone: 740-589-3300 Desk Extension: 740-589-3373 FAX: 740-593-8437

DEVICE NAME

Trade name: D Ultra DFA Respiratory Virus Screening & ID Kit Common name: Respiratory Virus DFA Assay Classification name: Antisera, Cf, Influenza A, B, C Product Code: GNW Regulation: 21 CFR § 866.3330, Class I, Influenza virus serological reagents, Panel Microbiology (83)

LEGALLY MARKETED DEVICE

Do Ultra DFA Respiratory Virus Screening & ID Kit, K061101

DESCRIPTION of DEVICE MODIFICATION

The product insert has been modified. The following has been added (see below):

Table 15 in the product insert has been updated to include reactivity data on influenza A virus Mexico/4108/2009 and California/07/2009 strains. The following language was included with the data:

"Although this test has been shown to detect the 2009 H1N1 influenza virus in two cultured isolates, the performance characteristics of this device with clinical

35

specimens that are positive for the 2009 H1N1 influenza virus have not been established. The D Ultra DFA Respiratory Virus Screening & ID Kit can distinguish between influenza A and B viruses, but it cannot differentiate influenza subtypes."

INTENDED USE

The Diagnostic Hybrids, Inc. D Ultra DFA (direct fluorescent antibody) RESPIRATORY VIRUS SCREENING & ID KIT is intended for the qualitative detection and identification of the Influenza A. Influenza B. Respiratory Syncytial Virus (RSV), Adenovirus, Parainfluenza 1, Parainfluenza 2 and Parainfluenza 3 virus in respiratory specimens, by either direct detection or cell culture method, by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs). 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 respiratory virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

Performance characteristics for influenza A were established when influenza A/H3 and A/H1 were the predominant influenza A viruses in circulation. 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 1 precautions for novel virulent influenza viruses and sent to state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL3+ facility is available to receive and culture specimens.

ASSESSMENT OF NON-CLINICAL PERFORMANCE DATA FOR EQUIVALENCE

·Not Applicable

ASSESSMENT OF NON-CLINICAL PERFORMANCE DATA FOR EQUIVALENCE

The risk analysis method used to assess the impact of the modification was a Failure Modes and Effects Analysis (FMEA). The modification to device labeling poses no additional risk.

BIOCOMPATABILITY

08272009 FDA modified_510(k) Summary_09AUG_26_Final (2),doc

www.cdc.gov

2 FDA Guidance Document: In Vitro Diagnostic Devices to Detect Influenza A Viruses: Labeling and Regulatory Path; Issued 4/10/2006

36

.

:

Not applicable

STERILIZATION

Not applicable

ﺮ ﺍﻟﻤﺮﺍﺟﻊ ﺍﻟﻤﺮﺍﺟﻊ

37

Image /page/37/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo is a circular seal with the words "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" arranged around the perimeter. Inside the circle is a stylized image of an eagle with its wings spread, symbolizing the department's mission to protect the health of all Americans.

AUG 2 8 2009

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

Ron Lollar Senior Director, Product Realization, Management, and Marketing DIAGNOSTIC HYBRIDS, INC. 1055 East State Street, Suite 100 Athens, Ohio 45701

Re: K092300

Trade/Device Name: D3 Ultra DFA Respiratory Virus Screening & ID Kit Regulation Number: 21 CFR 866.3330 Regulation Name: Influenza virus serological reagents Regulatory Class: Class I Product Code: GNW Dated: July 23, 2009 Received: July 29, 2009

Dear Mr. Lollar:

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. Iisting of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into class II (Special Controls), 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); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter will allow you to begin marketing your device as described in your Section 510(k) premarket

38

Page 2 -

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 advice for your device on our labeling regulation (21 CFR Parts 801 and 809), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 796-5450. 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 Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

Sempabym

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

Enclosure

39

Indications for Use

510(k) Number (if known): K092300

Device Name: D3 Ultra DFA Respiratory Virus Screening & ID Kit

Indication For Use:

The Diagnostic Hybrids. Inc. D Ultra DFA (direct fluorescent antibody) Respiratory Virus Screening & ID Kit is intended for the qualitative detection and identification of the Influenza A. Influenza B, Respiratory Syncytial Virus (RSV), Adenovirus, Parainfluenza 1, Parainfluenza 2 and Parainfluenza 3 virus in respiratory specimens, by either direct detection or cell culture method, by immunofluorescence using fluoresceinated monoclonal antibodies (MAbs). 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 respiratory virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

Performance characteristics for influenza A were established when influenza A/H3 and A/H1 were the predominant influenza A viruses in circulation. 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 state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL3+ facility is available to receive and culture specimens.2

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)

of CDRH, Office of In Vitro Diagnostic Devices (OIVD) Concurrence Division Sign-Off

Office of In Vitro Diagnostic Device Evaluation and Safety

Page 1 of 1

$\frac{5300(x)}{3300(x)} = 0.92300$

www.coc.gov

FDA Guidance Document: In Vitro Diagnostic Devices to Detect Influenza A Viruses: Labeling and Regulatory Path; Issued 4/10/2006