1. Diagnostic Tests for Influenza: the Tortoise and the Hare
Arthur E. Crist, Jr., Ph.D.
Director, Clinical Microbiology
Ph. (717)

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 J. Clin. Microbiol. 44:

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
Flu A
ImagenTM
Monoclonal Antibodies
Direct Procedure
Conjugated Ab - 15 min
Approved for culture confirmation and direct specimen detection
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+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
Test
Sensitivity
Specificity
Directigen
Flu A
91% (A)
95% (A)
Flu A & B
86% (A) 81% (B)
91% (A) 99.5% (B)
Flu OIA
62-88% (A/B)
52%-80% (A/B)
Flu OIA A/B
62%-88% (A/B)
NOW Flu A & B
78%-82% (A)
58-71% (B)
92-94% (A) 97% (B)

38. Performance Characteristics
Test
Sensitivity
Specificity
QuickVue Influenza
73-81% (A/B)
95% (A)
QuickVue Influenza A&B
72-77% (A)
73-82% (B)
91% (A)
99.5% (B)
SAS Influenza A & Influenza B
76% (A)
91% (B)
98% (A)
100% (B)
Xpect Flu A & B
92% (A)
98% (B)
100% (A)
ZstatFlu
58-65% (A/B)
98-100% (A/B)

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 J. Clin.Microbiol. 37:

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

44. Correlation between number of DFA-positive cells and EIA result
No. of samples with indicated no. of cells positive by DFAa
<5
5-10
11-25
50-100
>100
>1,000
Total
Negative 8 1 3 2 15
Positive 5 6 16 4 31
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

53. "Don't brag about your lightning pace, for Slow and Steady won the race!"

54. A sculpture of the Tortoise and the Hare by Nancy Schön
A sculpture of the Tortoise and the Hare by Nancy Schön. Located at Copley Square, it represents the finish line of the Boston Marathon