Martha Thompson, MPH Viral Isolation Team Leader Medical Virology Group Laboratory Services Section

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Martha Thompson, MPHViral Isolation Team
LeaderMedical Virology GroupLaboratory Services
SectionTX DSHS

• Influenza Surveillance
• Viral Isolation Laboratory
• TX DSHSJuly 23, 2008

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Viral Isolation

• 2007-2008 Season
• Laboratory Diagnostics Influenza
• Influenza testing Viral Isolation Lab
• Cell Culture
• Immunofluorescence
• Hemagglutination/HA Inhibition
• Levels of Identification
• Specimen rejection criteria
• Summary of isolates sent to CDC

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• Influenza A specimens not subtyped (2)
• B strains identified as B/Shanghai/361/2002-like
  (B/Yamagata) by the viral isolation laboratory

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Laboratory Diagnostic Testing Influenza
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Rapid EIA Kits

• Advantages
• Rapid and on-site testing
• Impact patient management
• Simple
• CLIA waved

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Limitations

• Typing/Results
• Flu only
• A or B
• No subtyping
• Variation between kits
• Storage conditions
• Acceptable specimens (includes type and time of
  collection)
• Must follow manufacturer instructions
• Less sensitive than viral culture or molecular
• False negatives
• PPV and prevalence in the community affect test
  performance

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• These limitations affect test performance
• Patient management
• Use positive and negative predictive values to
  assess test performance
• PPV Probability of disease in a patient with a
  positive test result
• PNV Probability of no disease in a patient with
  a negative test result

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Test Performance
Disease
Test Result
Sensitivity TP/TPFN Specificity TN/TNFP
PPV TP/TPFP PVN TN/TNFN
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Positive predictive value
Prevalence20
Disease
Test Result
Predictive Value Positive TP/TP FP
380/38064 85.6
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Positive predictive value
Prevalence 1
Disease
Test Result
Predictive Value Positive TP/TP FP
19/1980 19.1
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Conclusion

• When prevalence is low, the PPV is low and chance
  of getting a false positive increases
• Confirm with culture during off season

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Other Methods

• DFA
• Quick TAT
• No culture available for further studies
• Serology
• Positive results can be obtained even after viral
  shedding has stopped
• Acute/convelescent serum requireddelay in
  diagnosis
• No culture available for further studies

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Real Time RT-PCR

• Advantages
• Rapid
• Sensitive/Specific
• High throughput can be obtained
• Identification of highly pathogenic strains of
  avian influenza possible

• Disadvantages
• Costly
• Risk of cross contamination
• Variability among protocols means variability
  among sensitivity/specificity rates
• No isolate available for further studies

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Cell Culture

• Confirm virus is infectious
• Antigenic characterization
• Vaccine Studies
• Antiviral resistance testing
• Important for surveillance
• Slower TAT
• 2-10 days

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Immunofluorescence (IFA)

• Indirect test
• Antibody to Flu A and B antigens
• Fluorescent tag
• A, B, or Neg
• If positive continue with subtyping
• Reagents in WHO kit

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Hemagglutination/HA Inhibition

• Antisera to neutralize antigens
• Blood as an indicator, agglutinates to antigen
• Antigenic characterization

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2007-2008 WHO Influenza Reagent Kit

• Antisera
• Level of identification
• A(H3)
• A(H1)
• B/Shanghai/361/2002-like
• B/Malaysia/2506/2004-like
• Isolates to CDC
• Beginning, middle, and late season
• Patients who received vaccine
• Anything unusual
• Unable to subtype

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WHO Summary Weeks Ending Oct 6, 2008 May 17,
2008
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Vaccine Strains

• 2007-2008 Vaccine Strains
• A/Solomon Islands/3/2006 (H1N1)-like
• A/Wisconsin/67/2005 (H3N2)-like
• B/Malaysia/2506/2004-like (B/Victoria)
• A/Brisbane/10/2007 is a variant form of
  A/Wisconsin/67/2005 strain
• All B strains identified by VI lab were
  B/Shanghai/361/2002-like (B/Yamagata)

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Specimen Rejection Criteria

• Meet regulatory standards
• Optimal specimen for testing
• Expired transport media
• Wooden sticks/Calcium alginate
• Inhibitors to virus preservatives
• Cotton swabs
• First AND Last name on specimen AND submission
  form
• 1 Specimen 1 Submission form
• DATE of COLLECTION

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Contact Information

• martha.thompson_at_dshs.state.tx.us
• Phone [phone]
• Fax [phone]
• Viral Isolation Laboratory
• 512-458-7111 x2452

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Useful Links

• Resource Manual for Seasonal and Pandemic
  Influenza http//www.dshs.state.tx.us/comprep/pand
  emic/flu20outreach20manual2012-28-2007.pdf
• Laboratory Services Section http//www.dshs.state
  .tx.us/lab/