Diagnostic Tests for Influenza: the Tortoise and the Hare

1. Diagnostic Tests for Influenza: the Tortoise and the Hare Arthur E. Crist, Jr., Ph.D. Director, Clinical Microbiology Ph. (717) 851-2393 E-mail: acrist@wellspan.org

2. Influenza Virus - Schematic

4. Conventional Cell Culture • Inoculation of specimens into cell culture: Madin-Darby canine kidney cell line (MDCK), primary rhesus monkey kidney cells (RMK), Vero cells, MRC-5 human diploid embryonic lung fibroblasts • If CPE, hemadsorption (HAD) with 0.5% suspension guinea pig red blood cells followed by DFA if positive • In the absence of CPE, “blind” (HAD) at days 2 and 7 followed by FA if positive

5. Cell Culture

6. Will cell culture replace fertilized eggs?

7. Cell Culture - CPE Uninfected Infected

8. Hemadsorption

9. Hemadsorption +DFA

10. Shell Vial Cell Culture • The use of shell vials for more rapid isolation and identification of Influenza • MDCK, RMK, mink lung cells (Mv1Lu) • R-Mix: Mv1Lu and A549 cells • Centrifugation • shell vials inoculated, incubated, and “blind” stained at day 1 and 5 for Influenza A and B

11. Fong, C.K.Y., et. al. 2000. J. Clin. Microbiolo. 38: 4660-4662

12. Huang, V.T., et al. 2000. J. Clin. Microbiol. 38: 422-423

13. Performance of RT-PCR, Shell Vial, and Tube Culture for Influenza Zitterkopf, N.L., et. al. 2006. J. Clin. Microbiol. 44: 3366-3367

14. Comparison of Culture, Serology and PCR Diagnostic Tests Concordance of positive results with three test methods Serology (61%) 41 11 116 418 (43%) 8 42 97 Culture (55%) RT-PCR (70%)

15. Direct and Indirect FA Tests:Direct Specimen Testing or Culture Confirmation

16. Bartels/Intracel • Monoclonal Antibodies • Indirect Procedure • 1o Antibody - 30 min • Conjugate - 30 min • Approved for culture confirmation and direct specimen detection

17. Chemicon • Light Diagnostics • Monoclonal Antibodies • Direct - 30 min • Approved for culture confirmation and direct specimen detection

18. Chemicon • SimulFluorTM • Dual color fluorescence: Flu A and Flu B detection in one well • Direct - 30 min • Approved for culture confirmation and direct specimen detection

19. DAKO • ImagenTM • Monoclonal Antibodies • Direct Procedure • Conjugated Ab - 15 min • Approved for culture confirmation and direct specimen detection Flu A Flu B

20. Diagnostic Products • Monoclonal Antibodies • Direct Procedure • 15 min - Package insert • 30 min works better • Approved for direct specimen and culture confirmation testing

21. FA - Advantages • Relatively inexpensive • Amenable to batch testing • Rapid (1-2 hrs) • Able to assess specimen quality

22. FA - Disadvantages • Subjective read • High complexity test • Fluorescent microscope • Highly trained personnel • Longer turnaround times

24. Rapid Influenza Assays • Enzyme Immunoassay (EIA) Directigen Flu A and Directigen Flu A+B (Becton Dickinson) • Endogenous Viral-Encoded Assay (EVEA) ZstatFlu (ZymeTx) • Optical Immunoassay (OIA) Flu OIA (Biostar) • Lateral-Flow Immunoassay (LFIA) QuickVue Influenza (Quidel)

25. CLIA Status Moderate Complexity Directigen Flu A Directigen Flu A+B Flu OIA Waived Status QuickVue Influenza ZstatFlu

26. Comparison of Rapid Tests

27. Comparison of Rapid Tests

28. Directigen Flu A Test Directigen Flu A+B Assay

29. ZstatFlu

30. ZstatFlu

31. Biostar Flu OIA

32. Biostar OIA

33. QuickVue Influenza

34. Comparison of Rapid Tests Prevalence rates were all >18%

37. Performance Characteristics

38. Performance Characteristics

40. Rapid (POC)Tests: Advantages • Rapid turn around time • Rapid outbreak identification • Cost-effective (?) • Widespread testing available • Less expertise required • Optimize antibiotic and antiviral usage

41. Rapid Tests: Concerns • Specimen adequacy • No viral isolates available for further characterization • Loss of surveillance data • Live, attenuated intranasal vaccine can produce positive results (culture positive too!) • Variable performance characteristics • Result interpretation- poor positive predictive values during low prevalence • Improper treatment choices

42. Percent Positive By Specimen Type Covalciuc, K.A., et al. 1999. J. Clin.Microbiol. 37: 3971-3974

43. Patient Age and Onset of Testing Steininger, C., et al. 2002. J. Clin. Microbiol. 40: 2051-2056

44. Correlation between number of DFA-positive cells and EIA result a No samples were in the category of 25 to 50 positive cells.

45. Predictive Value • The probability of the presence or absence of disease given the test result • PPV is the probability of disease in a patient with a positive test result • NPV is the probability of not having disease when the test result is negative. • Determined by • sensitivity and specificity of the test • prevalence of disease in the population being tested

46. Hypothetical Influenza Test Performance • Prevalence = 1 in 5 • Sensitivity = 95% • Specificity = 96% • PPV = 85.6% • NPV = 96% • Prevalence = 1 in 100 • Sensitivity = 95% • Specificity = 96% • PPV = 19.2% • NPV = 99.9% Which test is best? Know the test performance characteristics and the epidemiology of the disease

48. False Positive EIA’s • Tampa, FL - 16 Flu A positives by EIA in the period from August to the end of October – none confirmed • NY State Dept. of Health (December 2006) 60 specimens that were flu EIA positive sent for confirmatory testing. Only one of them positive by RT-PCR

50. Rapid Tests: Optimizing Use • Educate clinicians on predictive values & limitations of test results • Confirm early, late and out-of-season positives • Confirm peak-season negatives, if applicable • Use prevalence indicators to decide: • When to test • When to qualify result • When to confirm results