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Louisiana Department of Health & Hospitals | Kathy Kliebert, Secretary

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Influenza Detection and Characterization

Influenza PCR

  • Laboratory Location – Central Laboratory
  • CPT Code – 83891:Isolation or extraction of highly purified nucliec acid, 87501:Infectious agent detection by nucleic acid (DNA or RNA); influenza virus, reverse transcription and amplified probe technique, each type or subtype, 83912: Interpretation and report
  • Synonyms – Flu, H1N1, Influenza
  • Test Description - Multiple target Real Time RT-PCR for Influenza Detection and Further Characterization. InfA and InfB are designed for universal detection of type A and type B influenza viruses. H1, H3 and H5 are designed to specifically detect contemporary human A/H1, human A/H3, and A/H5 (Asian lineage) influenza viruses. pdmInfA and pdmH1 are designed to detect the nucleoprotein gene and hemagglutinin gene RNA from 2009 H1N1 influenza virus. RP is designed to detect the human RNase P gene RNA and is used as an internal specimen control as well as an extraction control

Possible Results

This laboratory will determine the best testing algorithm for your sample based on current reagent availability, epidemiologic data and specimen volume. Available options include:

  • Influenza A/B Typing - (infA, infB and RP) - If Influenza A is detected, the sample will automatically be retested for suptypes
  • Influenza A/B plus Subtyping - (infA, infB, H1, H3, pdmInfA, pdmH1 and RP)
  • Influenza A/H5 Subtyping Assay - (InfA, H5a, H5b, and RP) - Testing for avian influenza A/H5N1 is considered on a case-by-case basis in consultation with the Infectious Disease Epidemiology department for hospitalized or ambulatory patients with: Documented temperature of > 38°C AND one or more of the following: cough, sore throat, shortness of breath, AND History of contact with poultry or a known or suspected case of Influenza A (A/H5N1) in an A/H5N1-affected country within 10 days of symptom onset.

Each Influenza target tested will be listed on the report with a result of Positive or Negative. After each target is listed, a report conclusion will follow. RP will not be printed on the report.

Non-Standard result combinations such as being positive for Influenza A and Influenza B will have INCONCLUSIVE listed as the report conclusion. Additional comments may be added as warranted by the specific result combination.

Reference Range

  • Negative or Not Detected

Specimen Requirements

  • Specimen Type
    • Upper respiratory tract clinical specimens
      • nasopharyngeal swabs [NPS]
      • nasal swabs [NS]
      • nasal aspirates [NA]
      • nasal washes [NW]
      • dual nasopharyngeal/throat swabs [NPS/TS]
    • Lower respiratory tract specimens
      • bronchoalvolar lavage [BAL]
      • bronchial wash [BW]
      • tracheal aspirate [TA]
      • sputum
      • lung tissue
    • viral culture
  • Specimen Container(s) - Viral Transport Media Tubes
  • Minumum accepted volume - approximately 1 mL

Specimen Collection Instructions

If Nasal swabs (NS), nasopharyngeal swabs (NPS), dual nasopharyngeal throat swabs (NPS/TS):

Swabs should be placed and mixed well in a viral transport media tube immediately after collection. Swab specimens should be collected only on swabs with a synthetic tip (such as polyester or Dacron) and an aluminum or plastic shaft. Swabs with cotton tips and wooden shafts are NOT recommended. Specimens collected with swabs made of calcium alginate are NOT acceptable.

If Nasal Aspirates (NA), Nasal Washes (NW), Broncheoalveolar lavage (BAL), Tracheal Aspirate (TA) and Bronchial Wash (BW), sputum:

Aseptically dilute liquid sample with an equal volume of viral transport media. Portion of transport media may be removed so that the volume of the sample equals the volume of the transport media.

Lung tissue or culture:

Add available portion to transport media

Storage and Transport Instructions

Transport specimen to laboratory as soon as possible after collection. If sample will be delivered to the laboratory within 72 hours after collection, hold the specimen at 2-8°C and ship specimen as 2-8°C.

To mimimize the effects of multiple freezing and thawing every attempt should be made to deliver the specimen to the laboratory within 72 hours from collection. If delivery to the laboratory within 72 hours from collection is not possible (i.e. Sample collected on a Friday), freeze the specimen at ≤ -70°C upon collection and ship to the laboratory on dry ice. If the sample is frozen at any point, it must remain and be shipped frozen. Document the date/time of freezing on the specimen submission form. Follow shipping company guidlines for Catergory B transport. Packaging examples are listed bleow.

For delivery in the lab ≤ 72 hours after collection:

Place specimens inside a small Ziploc bag or leak proof plastic canister with enough absorbent to prevent specimen leakage. In a Styrofoam insulated shipping box, layer contents in the following order from bottom to top: ice bricks, Ziploc bag of wet ice, specimen container, second Ziploc bag of wet ice, additional ice bricks. Utilize all the airspace in the insulated shipping box with refrigerant. Tape closed. Lab forms are not to be placed inside the Styrofoam container. They are to be attached to the outside of the Styrofoam container in a HIPPA compliant manner. The container can then be placed into an additional shipping box. Ship overnight delivery to the Central Laboratory in Metairie.

For delivery in the lab >72 hours after collection

Freeze specimens at ≤ -70°C upon collection. When ready for shipment, place specimens inside a small Ziploc bag or leak proof plastic conister with enough absorbant to prevent specimen leakage. Place the specimen container on dry ice inside a Styrofoam container approved for dry ice shipping. Lab forms are not to be placed inside the Styrofoam container. They are to be attahced to the outside of the container in a HIPPA compliant manner. The container can then be placed into an additional shipping box. Ship overnight delivery to the Central Laboratory in Metairie.

Causes for Rejection

  • Samples that do not meet time, temperature or documentation criteria
  • Samples that arrive on expired transport media
  • Sampels from unacceptable specimen sources

Limitations for the Procedure

  • Negative resutls do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.
  • A false negative result may occur if a specimen is improperly collected, transported or handled. False negative results may also occur if amplification inhibitors are present in the specimen or if inadequate numbers of organisms are present in the specimen.
  • Children tend to shed virus more abundantly and for longer periods of time than adults. Therefore, testing specimens from adults will have 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 activity when prevalence is moderate to low.
  • The performance of the assay has not been established in individuals who received nasally administered influenza vaccine. Individuals who received nasally administered influenza A vaccine may have positive test results for up to three days after vaccination.
  • Do not use any reagent past the expiation date.
  • Optimum speimen types and timing for peak viral levels during infections caused by a novel influenza A virus have not been determined. Collection of mulitple specimens from the same patient may be necessay to detect the virus.
  • If the virus mutates in the rRT-PCR target region, a specific novel influenza A virus may not be detected or may be detected less predictably.
  • Inhibitors or other types of interference may produce false negative results.
  • An interference study evaluating the effect of common cold medications was not performed.
  • Test performance can be affected because the epidemiology and pathology of disease caused by a specific novel influenza A virus is not fully known. For example, clinicians and laboratories may not know the optimum types of specimens to collect, and when during the course of infection these specimens are most likely to contain levels of virus that can be readily detected.
  • Detection of viral RNA may not indicate the presence of infectious virus or that influenza is the causative agent for clinical symptoms.
  • The performance of this test has not been established for monitoring treatment of influenza A or 2009 H1N1 influenza infection.
  • The performance of this test has not been established for screening of blood or blood product for the presence of influenza A or 2009 H1N1 influenza infection.
  • The test cannot rule out disease caused by other bacterial or viral pathogens.

Interfering Substances

  • Nasally administered influenza A vaccine may produce positive test results for up to 3 days or longer in some instances
  • Specimens collected with calcium alginate or cotton swabs

References

  • CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel (August 19, 2011)

Additional Information

Currently accepting specimens for Sentinal Sites and from hosptial submitter's with hospitalized patients presenting with influenza like illness.

 

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