1. Martha Thompson, MPH Viral Isolation Team Leader Medical Virology Group Laboratory Services Section TX DSHS Influenza Surveillance Viral Isolation Laboratory TX DSHS July 23, 2008

2. Viral Isolation 2007-2008 Season 2007-2008 Season Laboratory Diagnostics: Influenza Laboratory Diagnostics: Influenza Influenza testing: Viral Isolation Lab Influenza testing: Viral Isolation Lab Cell CultureCell Culture ImmunofluorescenceImmunofluorescence Hemagglutination/HA InhibitionHemagglutination/HA Inhibition Levels of IdentificationLevels of Identification Specimen rejection criteria Specimen rejection criteria Summary of isolates sent to CDC Summary of isolates sent to CDC

3. Influenza A specimens not subtyped (2) B strains identified as B/Shanghai/361/2002-like (B/Yamagata) by the viral isolation laboratory Total1570 % Pos for Influenza 49% Influenza A35% Influenza B14%

5. Laboratory Diagnostic Testing: Influenza TAT Rapid~30 minutes Serology~2 Weeks DFA2 hrs Culture2 – 10 days Molecular/PCR4 hrs – 1 day

6. Rapid EIA Kits Advantages Advantages Rapid and on-site testingRapid and on-site testing Impact patient managementImpact patient management SimpleSimple CLIA wavedCLIA waved

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

8.  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

9. Test Performance PresentAbsent Positive True Positive (TP) False Positive (FP) Negative False Negatives (FN) True Negatives (FN) Disease Test Result Sensitivity = TP/TP+FN Specificity = TN/TN+FP PPV= TP/TP+FP PVN = TN/TN+FN

10. PresentAbsent Positive 38064 Negative 201536 Disease Test Result Predictive Value Positive = TP/TP + FP = 380/380+64 = 85.6% Prevalence=20% Positive predictive value:

11. PresentAbsent Positive 1980 Negative 11900 Disease Test Result Prevalence = 1% Predictive Value Positive = TP/TP + FP = 19/19+80 = 19.1% Positive predictive value:

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

13. 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 required—delay in diagnosis No culture available for further studies

14. 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

15. Cell Culture Confirm virus is infectious Confirm virus is infectious Antigenic characterization Antigenic characterization Vaccine Studies Vaccine Studies Antiviral resistance testing Antiviral resistance testing Important for surveillance Important for surveillance Slower TAT Slower TAT 2-10 days2-10 days

16. 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

17. Hemagglutination/HA Inhibition Antisera to neutralize antigens Antisera to neutralize antigens Blood as an indicator, agglutinates to antigen Blood as an indicator, agglutinates to antigen Antigenic characterization Antigenic characterization

18. 2007-2008 WHO Influenza Reagent Kit 2007-2008 WHO Influenza Reagent Kit Antisera Antisera Level of identification Level of identification A(H3)A(H3) A(H1)A(H1) B/Shanghai/361/2002-likeB/Shanghai/361/2002-like B/Malaysia/2506/2004-likeB/Malaysia/2506/2004-like Isolates to CDC Beginning, middle, and late season Patients who received vaccine Anything unusual Unable to subtype

19. WHO Summary: Weeks Ending Oct 6, 2008 – May 17, 2008 A/SOLOMON ISLANDS/03/2006-LIKE (H1N1)8 A/BRISBANE/10/2007-LIKE (H3N2)22 A/WISCONSIN/67/2005-LIKE (H3N2)3 A/WISCONSIN/67/2005-LIKE (H3N2) LOW2 B/FLORIDA/04/2006-LIKE6 B/FLORIDA/04/2006-LIKE LOW1 Did not test17 Unable to grow virus; confirmed by PCR as A/H11 Unable to grow virus; confirmed by PCR as A/H38 Total68

20. Vaccine Strains 2007-2008 Vaccine Strains A/Solomon Islands/3/2006 (H1N1)-like χ χ A/Wisconsin/67/2005 (H3N2)-like χ χ 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)

21. Specimen Rejection Criteria Meet regulatory standards Optimal specimen for testing Expired transport media Expired transport media Wooden sticks/Calcium alginate Wooden sticks/Calcium alginate Inhibitors to virus: preservativesInhibitors to virus: preservatives Cotton swabs Cotton swabs  First AND Last name: on specimen AND submission form  1 Specimen = 1 Submission form  DATE of COLLECTION

22. Contact Information martha.thompson@dshs.state.tx.us Phone 512-458-7594 Fax 512-458-7293 Viral Isolation Laboratory 512-458-7111 x2452

23. Useful Links Resource Manual for Seasonal and Pandemic Influenza http://www.dshs.state.tx.us/comprep/pandemic/f lu%20outreach%20manual%2012-28-2007.pdf http://www.dshs.state.tx.us/comprep/pandemic/f lu%20outreach%20manual%2012-28-2007.pdf Laboratory Services Section http://www.dshs.state.tx.us/lab/