Diagnostic Tests for Influenza: the Tortoise and the Hare

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Diagnostic Tests for Influenza the Tortoise and
the Hare

• Arthur E. Crist, Jr., Ph.D.
• Director, Clinical Microbiology
• Ph. [phone]
• E-mail acrist_at_wellspan.org

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Influenza Virus - Schematic
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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

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Cell Culture
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Will cell culture replace fertilized eggs?
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Cell Culture - CPE
Uninfected
Infected
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Hemadsorption
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Hemadsorption DFA
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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

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Fong, C.K.Y., et. al. 2000. J. Clin. Microbiolo.
38 4660-4662
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Huang, V.T., et al. 2000. J. Clin. Microbiol. 38
422-423
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Performance of RT-PCR, Shell Vial, and Tube
Culture for Influenza
Zitterkopf, N.L., et. al. 2006. J. Clin.
Microbiol. 44 3366-3367
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Comparison of Culture, Serology and PCR
Diagnostic Tests
Concordance of positive results with three
test methods
Serology (61)
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116
418 (43)
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97
RT-PCR (70)
Culture (55)
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Direct and Indirect FA TestsDirect Specimen
Testing or Culture Confirmation
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Bartels/Intracel

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

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Chemicon

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

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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

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DAKO
Flu A

• ImagenTM
• Monoclonal Antibodies
• Direct Procedure
• Conjugated Ab - 15 min
• Approved for culture confirmation and direct
  specimen detection

Flu B
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Diagnostic Products

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

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FA - Advantages

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

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FA - Disadvantages

• Subjective read
• High complexity test
• Fluorescent microscope
• Highly trained personnel
• Longer turnaround times

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Rapid Influenza Assays

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

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CLIA Status
Moderate Complexity Directigen Flu A Directigen
Flu AB Flu OIA Waived Status QuickVue
Influenza ZstatFlu
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Comparison of Rapid Tests
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Comparison of Rapid Tests
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Directigen Flu A Test Directigen Flu AB Assay
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ZstatFlu
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ZstatFlu
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Biostar Flu OIA
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Biostar OIA
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QuickVue Influenza
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Comparison of Rapid Tests
Prevalence rates were all gt18
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Performance Characteristics
Test Sensitivity Specificity
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)
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Performance Characteristics
Test Sensitivity Specificity
QuickVue Influenza 73-81 (A/B) 95 (A)
QuickVue Influenza AB 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) 98-100 (A/B)
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Rapid (POC)Tests Advantages

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

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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

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Percent Positive By Specimen Type
Covalciuc, K.A., et al. 1999. J. Clin.Microbiol.
37 3971-3974
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Patient Age and Onset of Testing
Steininger, C., et al. 2002. J. Clin. Microbiol.
40 2051-2056
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Correlation between number of DFA-positive cells
and EIA result




EIA result No. of samples with indicated no. of cells positive by DFAa No. of samples with indicated no. of cells positive by DFAa No. of samples with indicated no. of cells positive by DFAa No. of samples with indicated no. of cells positive by DFAa No. of samples with indicated no. of cells positive by DFAa No. of samples with indicated no. of cells positive by DFAa No. of samples with indicated no. of cells positive by DFAa
EIA result lt5 5-10 11-25 50-100 gt100 gt1,000 Total

Negative 8 1 3 2 1 0 15
Positive 0 1 2 2 5 6 16
Total 8 2 5 4 6 6 31
a No samples were in the category of 25 to 50
positive cells.
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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

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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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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) 60
  specimens that were flu EIA positive sent for
  confirmatory testing. Only one of them positive
  by RT-PCR

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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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"Don't brag about your lightning pace, for Slow
and Steady won the race!"
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A sculpture of the Tortoise and the Hare by Nancy
Schön. Located at Copley Square, it represents
the finish line of the Boston Marathon