Influenza Surveillance Viral Isolation Laboratory TX DSHS July 23, 2008 Martha Thompson, MPH Viral Isolation Team Leader Medical Virology Group Laboratory Services Section TX DSHS.
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Influenza Surveillance Viral Isolation Laboratory TX DSHS July 23, 2008 Martha Thompson, MPH Viral Isolation Team Leader Medical Virology Group Laboratory Services Section TX DSHS 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 2007-2008 Influenza Summary: Viral Isolation Laboratory 2000 1600 1570 1200 800 546 426 400 219 118 0 Total Specimens A H1 Influenza A specimens not subtyped (2) B strains identified as B/Shanghai/361/2002-like (B/Yamagata) by the viral isolation laboratory H3 B Total 1570 % Pos for Influenza 49% Influenza A 35% Influenza B 14% 2007 - 2008 Influenza Summary: Viral Isolation Laboratory 200 180 160 140 120 100 80 60 40 20 0 Total specimens received A H3 A H1 MMWR Week Ending 05/06/2008 04/06/2008 03/06/2008 02/06/2008 01/06/2008 12/06/2007 11/06/2007 10/06/2007 A Unknown B Laboratory Diagnostic Testing: Influenza TAT Rapid ~30 minutes Serology ~2 Weeks DFA 2 hrs Culture 2 – 10 days Molecular/PCR 4 hrs – 1 day Rapid EIA Kits Advantages • Rapid and on-site testing • Impact patient management • Simple • CLIA waved 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 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 Test Performance Disease Test Result Positive Negative Sensitivity = TP/TP+FN Specificity = TN/TN+FP Present Absent True Positive (TP) False Positive (FP) False Negatives (FN) True Negatives (FN) PPV= TP/TP+FP PVN = TN/TN+FN Positive predictive value: Prevalence=20% Test Result Disease Present Absent Positive 380 64 Negative 20 1536 Predictive Value Positive = TP/TP + FP = 380/380+64 = 85.6% Positive predictive value: Prevalence = 1% Positive Test Result Negative Disease Present Absent 19 80 1 1900 Predictive Value Positive= TP/TP + FP = 19/19+80 = 19.1% Conclusion When prevalence is low, the PPV is low and chance of getting a false positive increases Confirm with culture during off season 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 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 Cell Culture Confirm virus is infectious Antigenic characterization Vaccine Studies Antiviral resistance testing Important for surveillance Slower TAT • 2-10 days 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 Hemagglutination/HA Inhibition Antisera to neutralize antigens Blood as an indicator, agglutinates to antigen Antigenic characterization 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 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) LOW 2 B/FLORIDA/04/2006-LIKE 6 B/FLORIDA/04/2006-LIKE LOW 1 Did not test 17 Unable to grow virus; confirmed by PCR as A/H1 1 Unable to grow virus; confirmed by PCR as A/H3 8 Total 68 Vaccine Strains 2007-2008 Vaccine Strains A/Solomon Islands/3/2006 (H1N1)-like χ A/Wisconsin/67/2005 (H3N2)-like 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) 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 Contact Information [email protected] Phone 512-458-7594 Fax 512-458-7293 Viral Isolation Laboratory 512-458-7111 x2452 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 Laboratory Services Section http://www.dshs.state.tx.us/lab/