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SUBJECT: INFLUENZA A & B FIA (Sofia®)

1. PRINCIPLE:

The Sofia® Influenza A+B FIA employs immunofluorescence technology that is used with the Sofia® Analyzer to detect influenza virus nucleoproteins. This test allows for the differential detection of influenza A and influenza B antigens.

The Sofia® Influenza A+B FIA involves the disruption of influenza A and B viral antigens. The patient specimen is placed in the Reagent Tube, during which time the virus particles in the specimen are disrupted, exposing internal viral nucleoproteins. After disruption, the specimen is dispensed into the Cassette sample well. From the sample well, the specimen migrates through a test strip containing various unique chemical environments. If influenza viral antigen is present, they will be trapped in a specific location.

Note: Depending upon the user’s choice, the cassette is either placed inside of the Sofia® Analyzer for automatically timed development (Walk Away Mode) or placed on the counter or bench top for a manually timed development and then placed into the Sofia® Analyzer to be scanned (Read Now Mode).

The Sofia® Analyzer will scan the test strip and measure the fluorescent signal by processing the results using method-specific algorithms. The Sofia® Analyzer will display the test results (Positive, Negative, or Invalid) on the screen. The results can also be automatically printed on an integrated printer if this option is selected.

2. CLINICAL SIGNIFICANCE:

Influenza is a highly contagious, acute respiratory illness characterized by the abrupt onset of fever, myalgia, headache, malaise, nonproductive cough, sore throat, and rhinitis. Worldwide, the occurrence of influenza follows seasonal patterns unique to the geographical area, but with international travel the disease may become more of a year-round phenomenon. Influenza type A and B viruses are responsible for the epidemics of disease that occur almost every winter. In the United States, these winter influenza epidemics can cause illness in 5-20% of the population and are associated with an average of 36,000 deaths and 200,000 hospitalizations per year. Influenza type C is usually associated with only mild or asymptomatic disease. Influenza typically resolves in one or two weeks, but some people, such as adults 65 years of age or older, young children and individuals with certain health conditions, are at high risk for serious influenza complications. Influenza can exacerbate underlying medical conditions (e.g., cardiopulmonary conditions), lead to secondary bacterial pneumonia or primary influenza viral pneumonia, or occur as part of a co-infection with other viral or bacterial pathogens. The appropriate treatment of patients with influenza-like illness depends on accurate and timely diagnosis, which can help reduce the inappropriate use of antibiotics and provide the option of using antiviral therapy. Diagnosis of influenza based on symptoms alone is difficult because the initial symptoms can be similar to those caused by other infectious agents. A variety of laboratory methods are available for the detection of influenza virus, including conventional cell culture, shell vial culture, immunofluorescent staining, serologic tests, amplified nucleic acid detection assays, and rapid immunoassays. If diagnosed within 48 hours of the onset of symptoms, several antiviral medications are available which may shorten the duration and lessen the severity of symptoms associated with uncomplicated influenza illness.
3. SPECIMEN: A. COLLECTION AND PROCESSING:

Nasal Swab Sample: Use the foam nasal swab supplied in the kit.

Follow standard PPO policies when collecting and handling patient samples.

To collect a nasal swab sample, carefully insert the swab (provided in the kit) into the nostril that presents the most secretion under visual inspection. Using gentle rotation, push the swab until resistance is met at the level of the turbinates (less than one inch into the nostril). Rotate the swab several times against the nasal wall, then remove it from the nostril.

Return swab to paper sleeve, label appropriately. Place in biohazard transport bag and process as soon as possible.

B. REJECTION:

Mislabeled or improperly labeled swabs / containers will not be accepted Specimens received in unacceptable transport media will be rejected.

C. STORAGE AND PRESERVATION:

Specimens should be tested as soon as possible after collection.

4. REAGENTS, STANDARDS, AND CONTROLS: A. PREPARATION:

Store the kit at room temperature, 59–86°F (15–30°C), out of direct sunlight.

Kit contents are stable until the expiration date printed on the outer box.

Do not freeze.

B. CONTROL PROCEDURE:
QUALITY CONTROL The external positive and negative controls should be assayed once upon receipt of each new lot and kit shipment. Thereafter, the procedural control (internal control) performed with each test, is sufficient to verify reagent integrity and assay performance. Record the lot / shipment results in the Microbiology QC book and the LIS system accordingly. The internal (procedural) control (valid) is recorded with each patient result. There are three types of Quality Control for the Sofia® Analyzer and Cassette: Sofia® Analyzer Calibration Check procedure, built-in procedural control features, and External Controls. Sofia® Analyzer Calibration Check Procedure The Calibration Check Procedure should be performed every thirty (30) days. The Sofia® Analyzer is set to remind the user to complete the Calibration Check Procedure. The Calibration Check is a required function that checks the Sofia® Analyzer optics and calculation systems using a specific Calibration Cassette. This Calibration Cassette is shipped with the Sofia® Analyzer. Refer to the Sofia® Analyzer User Manual for details regarding the Calibration Check Procedure. Important: Ensure that the Calibration Cassette is stored in the provided storage pouch between uses to protect from exposure to light. Do not use past the expiration date. a. To check the calibration of the Sofia® Analyzer, select “Calibration” from the Main Menu. b. Following the prompts, insert the Calibration Cassette into the Sofia® Analyzer and close the drawer. The Analyzer performs the Calibration Check automatically with no user input required. c. The Sofia® Analyzer indicates when the Calibration Check is completed. Select OK to return to the Main Menu. NOTE: If calibration cannot be completed successfully, notify the on-site Supervisor or contact Quidel Technical Support for assistance from 7:00 a.m.–5:00 p.m. PST at (800) 874-1517 (within the USA); (858) 552-1100 (outside the USA); Fax: (858) 455-4960; custserv@quidel.com (Customer Service); technicalsupport@quidel.com (Technical Support) or contact your local distributor. Built-in Procedural Control The Sofia® Influenza A+B FIA contains a built-in procedural control feature. Each time a test is run in the Sofia® Analyzer, the procedural control zone is scanned by the Sofia® Analyzer and the result is displayed on the Analyzer screen. The manufacturer’s recommendation for daily control is to document the results of these built-in procedural controls for the first sample tested each day. This documentation is automatically logged into the Analyzer with each test result, and is recorded in the LIS system with each patient result. A valid result obtained from the procedural control Demonstrates that the test flowed correctly and the functional integrity of the Cassette was maintained. The procedural control is interpreted by the Sofia® Analyzer after the Cassette has developed for fifteen (15) minutes. If the test does not flow correctly, the Sofia® Analyzer will indicate that the result is invalid. Should this occur, review the procedure and repeat the test with a new patient sample and a new test Cassette. External Quality Control External Controls are used to demonstrate that the reagents and assay procedure perform properly. External Positive and Negative Control swabs are supplied in the kit and should be tested using the Swab Test Procedure. The external positive and negative controls should be assayed once upon receipt of each new lot and kit shipment. Thereafter, the procedural control (internal control) performed with each test, is sufficient to verify reagent integrity and assay performance. The user must first select Run QC on the main Menu of the Sofia® Analyzer and then, when prompted, scan the QC Card (located on kit box). This card provides information specific to the kit lot, including lot number and expiration date. The Analyzer will then prompt the user to run the External Control swabs. Run the Positive Control Swab first before the Negative Control swab. Note: the Influenza Positive Control Swab should give a positive result for both influenza A and influenza B. Do not perform patient tests or report patient test results if the control tests do not produce the expected results. Repeat the test or contact Quidel Technical Support before testing patient specimens. Additional External Control swabs may be obtained separately by contacting Quidel Customer Support Services at (800) 874.1517 (toll-free in the U.S.A.) or (858) 552.1100.

5. EQUIPMENT: A. INSTRUMENTS:

Sofia® Analyzer.

B. MICROSCOPIC EXAMINATIONS (AND REJECTIONS):

Not applicable

6. PROCEDURE: A. PERFORMANCE:

NOTE: THE EXTRACTION PORTION OF INFLUENZAE TESTING SHOULD BE PERFORMED BEHIND A PROTECTIVE SHIELD OR UNDER A BIOSAFETY HOOD. (Steps 1 thru 5.) All clinical specimens must be at room temperature before beginning the assay. Check expiration dates on each individual test package before using. Verify that the Sofia® Analyzer is set to the desired mode: Walk Away or Read Now. Swab Test Procedure (Nasal Swab) a.)Add the Reagent Solution to the Reagent Tube. b.)Gently swirl the Reagent Tube to dissolve its contents. a.) Immediately place the patient swab sample into the Reagent Tube. Roll the swab a minimum of three (3) times while pressing the head against the bottom and side of the Reagent Tube. b.) Leave the swab in the Reagent Tube for one (1) minute. a.) Express all liquid from the swab head by rolling it against the inside of the Reagent Tube as the swab is being removed. b.) Discard the swab in a biohazard waste container. Fill the provided small, clear fixed volume pipette (120 μl) or a micropipettor with 120 μl of the patient sample from the Reagent Tube. To fill the fixed volume pipette with the patient sample: a. FIRMLY a. a.) Firmly squeeze the top bulb. b.) Still squeezing, place the pipette tip into the liquid sample. c. ) With the pipette tip still in the liquid sample, release pressure on bulb to fill the pipette. Dispense the patient sample into the Cassette sample well. Firmly squeeze the top bulb to empty the contents of the fixed volume pipette into the Cassette sample well (extra liquid in the overflow bulb is OK). NOTE: The fixed volume pipette is designed to collect and dispense the correct amount of liquid sample. Discard the pipette in a biohazard waste container. Proceed to the “Using the Sofia Analyzer” section to complete the test. Procedure for Survey specimens: a.)Add the Reagent Solution to the Reagent Tube. b.)Gently swirl the Reagent Tube to dissolve its contents. Fill the large, pink (260 µl) fixed volume pipette, or a micropipettor with 260 μl of the patient sample from the Reagent Tube with the patient sample and empty the contents into the Reagent Tube and then discard the pink pipette. Gently swirl the reagent tube to mix. Fill the provided small, clear fixed volume pipette (120 μl) or a micropipettor with 120 μl of the patient sample from the Reagent Tube. Dispense the patient sample into the Cassette sample well. Firmly squeeze the top bulb to empty the contents of the fixed volume pipette into the Cassette sample well (extra liquid in the overflow bulb is OK). NOTE: The fixed volume pipette is designed to collect and dispense the correct amount of liquid sample. Discard the pipette in a biohazard waste container. Proceed to the “Using the Sofia Analyzer” section to complete the test. Using the Sofia® Analyzer. Walk Away / Read Now Modes Walk Away Mode, see Run Test below for documenting patient information. Immediately after filling the cassette with the patient specimen, insert the cassette into the analyzer. The Analyzer automatically times the test development before scanning and displaying the test result. Read Now Mode When the testing is complete, discard the cassette in a biohazard waste container. Read Now Mode, see Run Test below for documenting patient information. Allow the test to develop for a full 15 minutes BEFORE placing in the analyzer. Set a timer for 15 minutes. Then, insert the cassette into the Analyzer. In Read Now Mode, the analyzer will scan and display the test result within one (1) minute. Note: The results will remain stable for an additional 15 minutes after the recommended development time of 15 minutes. When the testing is complete, discard the cassette in a biohazard waste container. Run Test Input the User ID using the handheld barcode scanner or manually enter the data using the keypad. NOTE: If you scan an incorrect barcode, simply rescan using the correct barcode. The previous one will be overwritten with the correct barcode. Input the Patient ID or Order # using the handheld barcode scanner or manually enter the data using the keypad. Press Start Test and the Analyzer drawer will automatically open. Verify that the correct mode, Walk Away or Read Now, has been selected. Insert the cassette into the drawer. Then gently close the drawer. The Sofia® Analyzer will start automatically and display the progress. In Walk Away Mode, the test results will be displayed on the screen in approximately 15 minutes. In Read Now Mode, the test results will be displayed on the screen within 1 minute. See Interpretation of Results.
B. CALCULATIONS:
Not applicable.

7. INTERPRETATION OF RESULTS:

When the test is complete, the results will be displayed on the Sofia® Analyzer screen. The Sofia® Analyzer detects the test line. The test line will not be visible to the user. The Sofia® Analyzer screen will display results for the procedural control as being “valid” or “invalid”, and will individually provide a positive or negative result for both influenza A and influenza B. If the procedural control is “invalid”, or patient testing is invalid retest with a new patient sample and a new cassette. If repeat testing is still invalid report as invalid and verbally suggest viral culture or molecular assay testing. Co-infection with Influenzae A and B is rare. Sofia Influ A+B FIA “dual positive” clinical specimens (influenza A and influenza B positive) should be re-tested with a new patient sample and a new test cassette. If the repeat specimen is also positive for both Flu A and Flu B, report results as inconclusive, and suggest confirmation by viral culture or an FDA-cleared influenza A and B molecular assay.

Reader Display Interpretation

Flu A: Positive	Positive Test for Flu A (influenza A antigen present)

Flu B: Negative

Flu A: Negative	Positive Test for Flu B (influenza B antigen present)

Flu B: Positive

Flu A: Negative	Negative Test for Flu A and Flu B (no antigen detected)

Flu B: Negative

Flu A: Invalid	Result Invalid X 2; report invalid

Flu B: Invalid	suggest viral culture or molecular assay

Procedural Control: Invalid

Flu A: Positive *** Inconclusive; suggest viral culture or molecular assay Flu B: Positive ***

*** Note: Co-infection with Influenzae A and B is rare. Sofia Influenzae A+B FIA “dual positive” clinical specimens (influenza A and influenza B positive) should be re-tested with a new patient sample and a new test cassette. If the repeat specimen is also positive for both Flu A and Flu B, report results as inconclusive, and suggest confirmation by viral culture or an FDA-cleared influenza A and B molecular assay.

NOTE: If the procedural control is “invalid”, or patient testing is invalid retest with a new patient sample and a new cassette. If repeat testing is still invalid report as invalid and verbally suggest viral culture or molecular assay testing.

B. FAILURE/REMEDIAL ACTION:

If the test fails, repeat the procedure with a new patient sample and new cassette.

8. EXPECTED RESULTS: A. REPORTABLE RANGE:

Negative or Positive

B. REFERENCE RANGE:

Negative

C. CRITICALVALUES:

Not applicable

9. REPORTING RESULTS: A. NORMAL VALUES:

Report results in the LIS computer system. Positive : Positive for influenza A and/or influenza B antigen. Negative : Negative for influenza A and/or influenza B antigen. CLEAR : Valid for internal (procedural) control.
10. PROCEDURAL NOTES: NOTE: The Sofia® Analyzer must be sent to Quidel if maintenance is required. The User should not attempt any maintenance except for changing paper, batteries and external cleaning. (See Supervisor Instructions: Installation and Settings for how to change paper and batteries). To reduce the risk of electrical shock: Do not disassemble the Analyzer. The Analyzer contains no Operator serviceable components and warranty will be voided if disassembled. Possible electrical shock: Turn off and unplug the Analyzer prior to cleaning. Do not immerse the Analyzer in liquid. Cleaning Turn off and unplug the Analyzer before cleaning. Use 70% alcohol preps or 0.5% bleach solution to clean the Analyzer. Wipe the external surfaces and Cassette drawer only. DO NOT clean with soap or other solutions. The Sofia® Analyzer screen will display results for the procedural control as being “valid” or “invalid”, and will individually provide a positive or negative result for both influenza A and influenza B. If the procedural control is “invalid” and or patient testing is invalid retest with a new patient sample and a new cassette. If repeat testing is still invalid report as invalid and verbally suggest viral culture or molecular assay testing. Co-infection with Influenzae A and B is rare. Sofia Influenzae A+B FIA “dual positive” clinical specimens (influenza A and influenza B positive) should be re-tested with a new patient sample and a new test cassette. If the repeat specimen is also positive for both Flu A and Flu B, report results as inconclusive, and suggest confirmation by viral culture or an FDA-cleared influenza A and B molecular assay. For in vitro diagnostic use. Do not use the kit contents beyond the expiration date printed on the outside of the box. Use appropriate precautions in the collection, handling, storage, and disposal of patient samples and used kit contents. Follow standard PPO policies when collecting and handling patient samples. Dispose of containers and used contents in accordance with Federal, State and Local requirements. Do not reuse the used cassette, fixed volume pipettes, reagent tubes, solutions, or control swabs. The user should never open the foil pouch of the test Cassette exposing it to the ambient environment until the Cassette is ready for immediate use. Discard and do not use any damaged cassette or material. The Reagent Solution contains a salt solution (saline). If the solution contacts the skin or eye, flush with copious amounts of water. To obtain accurate results, the instructions must be followed. The Calibration Cassette must be kept in the provided storage pouch between uses. Inadequate or inappropriate specimen collection, storage, and transport may yield false test results. Specimen collection and handling procedures require specific training and guidance. Use only the Viral Transport Media recommended. When collecting a nasal swab specimen, use the nasal swab supplied in the Sofia® kit. Do not write on the barcode of the Cassette. This is used by the Sofia® Analyzer to identify the type of test being run and to identify the individual Cassette so as to prevent a second read of the Cassette by the same Sofia® Analyzer. 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 BSL 3+ facility is available to receive and culture specimens. Although this test has been shown to detect cultured avian influenza viruses, including avian Influenza A subtype H5N1 virus, the performance characteristics of this test with specimens from humans infected with H5N1 or other avian influenza viruses are unknown. As the detection reagent is a fluorescent compound, no visible results will form on the test strip. The Sofia® Analyzer must be used for result interpretation. In the United States, influenza is most prevalent during the winter months. During peak periods, up to 30% of specimens tested may be culture positive for influenza. The proportion of influenza A positive specimens compared to influenza B can vary dramatically from year to year, ranging from about 50% to 99%. Recent intranasal vaccination (e.g. Flu Mist) may cause false positive test results. Infection due to influenza A or B cannot be ruled out since the antigen present in the specimen may be below the detection limit of the test. Viral cultures may be ordered on nasal aspirate or swab specimens within 2 hours of collection, if volume is sufficient and if clinically indicated. A. BACKUP FOR INOPERABLE SYSTEM

Suggest viral culture be performed or FDA cleared Influenzae A & B molecular Assay.

B. REFERRAL OF SPECIMENS:

Send specimen to reference laboratory.

C. SUBMISSION/HANDLING OF REFERRAL SPECIMENS:

Submit specimen to reference testing area for handling.

11. LIMITATIONS AND INTERFERING SUBSTANCES:

The test is not intended to detect influenza C antigens.

A negative test is presumptive and it is recommended these results be confirmed by virus culture or an FDA-cleared influenza A and B molecular assay.

Negative results do not preclude influenza virus infections and should not be used as the sole basis for treatment or other management decisions.

The test is intended for professional and laboratory use only.

The contents of this kit are to be used for the qualitative detection of influenza type A and B antigens from a nasal swab, nasopharyngeal swab, and nasopharyngeal aspirate/wash specimens.

This test detects both viable (live) and non-viable influenza A and B. Test performance depends on the amount of virus (antigen) in the specimen and may or may not correlate with virus culture results performed on the same specimen.

The clinical performance of the Sofia® Influenza A+B FIA for nasopharyngeal aspirate/wash samples has not been established in patients 60 years of age and older and may not be consistent with the clinical performance obtained with younger patients.

A negative test result may occur if the level of antigen in a sample is below the detection limit of the test or if the sample was collected or transported improperly.

Failure to follow the Test Procedure may adversely affect test performance and/or invalidate the test result.

Test results must be evaluated in conjunction with other clinical data available to the physician.

Positive test results do not rule out co-infections with other pathogens.

Positive test results do not identify specific influenza A virus subtypes.

Negative test results are not intended to rule in other non-influenza viral or bacterial infections.

Children tend to shed virus more abundantly and for longer periods of time than adults. Therefore, testing specimens from adults will often yield lower sensitivity than testing specimens from children.

Positive and negative predictive values are highly dependent on prevalence. False negative test results are more likely during peak activity when prevalence of disease is high. False positive test results are more likely during periods of low influenza or activity when prevalence is moderate to low.

Individuals who received nasally administered influenza A vaccine may have positive test results for up to three days after vaccination.

Monoclonal antibodies may fail to detect, or detect with less sensitivity, influenza A viruses that have undergone minor amino acid changes in the target epitope region.

If differentiation of specific influenza A subtypes and strains is needed, additional testing, in consultation with state or local public health departments, is required.

Samples contaminated with whole blood >4% v/v or mucin >0.5% v/v may interfere in the interpretation of the test. Visually bloody or overly viscous samples should not be used.

The performance of this test has not been evaluated for use in patients without signs and symptoms of respiratory infection.

12. METHOD VALIDATION:

June 2013 .

13. REFERENCES:

Murphy B.R. and Webster R.G., 1996, Orthomyxoviruses, pp. 1397-1445. In: Fields Virology, 3rd edition, B.N. Fields, D.M. Knipe, P.M. Howley, et al. (eds.), Lippincott-Raven, Philadelphia.

CDC, Key Facts About Seasonal Influenza. www.cdc.gov/flu/keyfacts.htm accessed 7/2011.

Biosafety in Microbiological and Biomedical Laboratories, 5th Edition. U.S. Department of Health and Human Services, CDC, NIH, Washington, DC (2007).

Bartlett J. Management of Respiratory Tract Infections. 2nd Ed, 1999: 149-169.

ASSISTANCE

If you have any questions regarding the use of this product, please call Quidel’s Technical Support Number (800) 874-1517 (toll-free in the U.S.A.) or (858) 552-1100, Monday through Friday, between 7:00 a.m. and 5:00 p.m., Pacific Time, U.S.A. If outside the United States contact your local distributor or technicalsupport@quidel.com.

For additional Quick Reference Instructions in your language, please see our website quidel.com, or contact Quidel Technical Support (858) 552-1100 or your local distributor for a copy delivered free of charge.

Policy Approval:

Approved by: Cathy Turnwald – Micro Technical Specialist – 01/31/2017

Approved by: Christine Wilmoth – Lead Micro Technical Specialist – 02/03/2017

Approved by: Dr. Kirk M. Griffith – Micro Medical Director – 02/04/2017