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

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Presentation on theme: "Diagnostic Tests for Influenza: the Tortoise and the Hare Arthur E. Crist, Jr., Ph.D. Director, Clinical Microbiology Ph. (717) 851-2393"— Presentation transcript:

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

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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 Uninfected Infected Cell Culture - CPE

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 J. Clin. Microbiolo. 38:

12 Huang, V.T., et al J. Clin. Microbiol. 38:

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 (43%) Serology (61%) RT-PCR (70%) Culture (55%)

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

16 Bartels/Intracel Monoclonal Antibodies Indirect Procedure –1 o 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 SimulFluor TM 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 Imagen TM Monoclonal Antibodies Direct Procedure Conjugated Ab - 15 min Approved for culture confirmation and direct specimen detection Flu B Flu A

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

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

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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%

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37 Performance Characteristics TestSensitivitySpecificity Directigen Flu A 91% (A) 95% (A) Directigen 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) 52%-80% (A/B) NOW Flu A & B 78%-82% (A) 58-71% (B) 92-94% (A) 97% (B)

38 Performance Characteristics TestSensitivitySpecificity 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) 100% (B) ZstatFlu 58-65% (A/B) % (A/B)

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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 a No samples were in the category of 25 to 50 positive cells. EIA result No. of samples with indicated no. of cells positive by DFAa < >100>1,000Total Negative Positive Total Correlation between number of DFA-positive cells and EIA result

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

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48 False Positive EIAs 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) 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

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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

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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. Located at Copley Square, it represents the finish line of the Boston Marathon


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