Diagnostic Tests for Influenza: the Tortoise and the Hare Arthur E. Crist, Jr., Ph.D. Director, Clinical Director, Clinical Microbiology Microbiology Ph. (717) 851-2393 Ph. (717) 851-2393 E-mail: [email protected]
Mar 27, 2015
Diagnostic Tests for
Influenza: the Tortoise and
the Hare
Arthur E. Crist, Jr., Ph.D.Director, Clinical MicrobiologyDirector, Clinical Microbiology
Ph. (717) 851-2393Ph. (717) 851-2393E-mail: [email protected]: [email protected]
Influenza Virus - Schematic
17
Laboratory Diagnosis of Influenza
Retrospective, requires paired sera>2 weeksSerology
Widely available, requires little expertise15-30 minutesAntigen Detection
(Rapid EIA-like)
Requires reading expertise & IF microscope2-4 hoursAntigen Detection
(IF)
Becoming gold standard(?), requires expertise & expensive equipment
2-4 hoursMolecular(RT-PCR)
Still gold standard(?), requires expertise, provides virus for studies
1-10 daysCulture
CommentsTime to ResultsTestMethod
Conventional Cell CultureConventional Cell Culture
– Inoculation of specimens into cell culture: Inoculation of specimens into cell culture: Madin-Darby canine kidney cell line Madin-Darby canine kidney cell line (MDCK), primary rhesus monkey kidney (MDCK), primary rhesus monkey kidney cells (RMK), Vero cells, MRC-5 human cells (RMK), Vero cells, MRC-5 human diploid embryonic lung fibroblastsdiploid embryonic lung fibroblasts
– If CPE, hemadsorption (HAD) with 0.5% If CPE, hemadsorption (HAD) with 0.5% suspension guinea pig red blood cells suspension guinea pig red blood cells followed by DFA if positivefollowed by DFA if positive
– In the absence of CPE, “blind” (HAD) at In the absence of CPE, “blind” (HAD) at days 2 and 7 followed by FA if positivedays 2 and 7 followed by FA if positive
Cell CultureCell Culture
Will cell culture replace fertilized Will cell culture replace fertilized eggs?eggs?
Uninfected Infected
Cell Culture - CPECell Culture - CPE
Hemadsorption
Hemadsorption Hemadsorption +DFA+DFA
Shell Vial Cell CultureShell Vial Cell Culture
The use of shell vials for more rapid The use of shell vials for more rapid isolation and identification of Influenzaisolation and identification of Influenza
MDCK, RMK, mink lung cells (Mv1Lu)MDCK, RMK, mink lung cells (Mv1Lu)
R-Mix: Mv1Lu and A549 cellsR-Mix: Mv1Lu and A549 cells
CentrifugationCentrifugation– shell vials inoculated, incubated, and “blind” shell vials inoculated, incubated, and “blind”
stained at day 1 and 5 for Influenza A and B stained at day 1 and 5 for Influenza A and B
Fong, C.K.Y., et. al. 2000. J. Clin. Microbiolo. 38: 4660-4662
Huang, V.T., et al. 2000. J. Clin. Microbiol. 38: 422-423
Performance of RT-PCR, Shell Vial, Performance of RT-PCR, Shell Vial, and Tube Culture for Influenzaand Tube Culture for Influenza
Zitterkopf, N.L., et. al. 2006. J. Clin. Microbiol. 44: 3366-3367
Comparison of Culture, Serology and Comparison of Culture, Serology and PCR Diagnostic TestsPCR Diagnostic Tests
Concordance of positive results with three testConcordance of positive results with three testmethodsmethods
11
418 (43%)
116
41
97 428
Serology Serology (61%)(61%)
RT-PCR RT-PCR (70%)(70%)
Culture Culture (55%)(55%)
Direct and Indirect FA Tests:Direct and Indirect FA Tests:Direct Specimen Testing or Culture ConfirmationDirect Specimen Testing or Culture Confirmation
Bartels/IntracelBartels/Intracel
Monoclonal AntibodiesMonoclonal Antibodies
Indirect ProcedureIndirect Procedure– 11oo Antibody - 30 min Antibody - 30 min– Conjugate - 30 minConjugate - 30 min
Approved for culture Approved for culture confirmation and direct confirmation and direct specimen detectionspecimen detection
ChemiconChemicon
Light DiagnosticsLight Diagnostics
Monoclonal AntibodiesMonoclonal Antibodies
Direct - 30 minDirect - 30 min
Approved for culture Approved for culture confirmation and direct confirmation and direct specimen detectionspecimen detection
ChemiconChemicon
SimulFluorSimulFluorTMTM
Dual color fluorescence: Flu A Dual color fluorescence: Flu A and Flu B detection in one welland Flu B detection in one well
Direct - 30 minDirect - 30 min
Approved for culture Approved for culture confirmation and direct confirmation and direct specimen detectionspecimen detection
DAKODAKO
ImagenImagenTMTM
Monoclonal AntibodiesMonoclonal Antibodies
Direct ProcedureDirect Procedure
Conjugated Ab - 15 minConjugated Ab - 15 min
Approved for culture Approved for culture confirmation and direct confirmation and direct specimen detectionspecimen detection
Flu B
Flu A
Diagnostic ProductsDiagnostic Products
Monoclonal AntibodiesMonoclonal Antibodies
Direct ProcedureDirect Procedure– 15 min - Package insert15 min - Package insert– 30 min works better30 min works better
Approved for direct Approved for direct specimen and culture specimen and culture confirmation testingconfirmation testing
FA - AdvantagesFA - Advantages
Relatively inexpensiveRelatively inexpensive
Amenable to batch testingAmenable to batch testing
Rapid (1-2 hrs)Rapid (1-2 hrs)
Able to assess specimen qualityAble to assess specimen quality
FA - DisadvantagesFA - Disadvantages
Subjective readSubjective read
High complexity testHigh complexity test
Fluorescent microscopeFluorescent microscope
Highly trained personnelHighly trained personnel
Longer turnaround timesLonger turnaround times
Rapid Influenza AssaysRapid Influenza Assays
Enzyme Immunoassay (EIA)Enzyme Immunoassay (EIA) Directigen Flu A and Directigen Flu A+B Directigen Flu A and Directigen Flu A+B
(Becton Dickinson) (Becton Dickinson) Endogenous Viral-Encoded Assay (EVEA)Endogenous Viral-Encoded Assay (EVEA) ZstatFlu (ZymeTx)ZstatFlu (ZymeTx)
Optical Immunoassay (OIA)Optical Immunoassay (OIA) Flu OIA (Biostar)Flu OIA (Biostar)
Lateral-Flow Immunoassay (LFIA)Lateral-Flow Immunoassay (LFIA) QuickVue Influenza (Quidel)QuickVue Influenza (Quidel)
CLIA StatusCLIA Status
Moderate ComplexityModerate Complexity
Directigen Flu ADirectigen Flu A
Directigen Flu A+BDirectigen Flu A+BFlu OIAFlu OIA
Waived StatusWaived Status
QuickVue InfluenzaQuickVue Influenza
ZstatFluZstatFlu
Test Specimen VTM? Swabs Directigen
Flu A NP wash/ aspirate (pref) NP & Throat swab, OK
OK Rayon or dacron. Can purchase separately
Directigen Flu A+B
NP wash/ aspirate (pref) NP, nasal, & throat swab OK
OK Rayon or dacron. Can purchase separately
ZstatFlu Throat swab No Foam-tipped. Included in kit
Flu OIA Nasal aspirate, NP swab, throat swab, sputum
No Included in kit. Can purchase separately
QuickVue Influenza
Nasal swab, wash, aspirate
No Foam tipped. Included in kit
Comparison of Rapid TestsComparison of Rapid Tests
Comparison of Rapid TestsComparison of Rapid Tests
Test Steps* Assay Time*
(minutes)
Hands Time*
(minutes)
Specimen Storage
Directigen Flu A 10 15-20 15-20
2-8C / No defined limit
Directigen Flu A+B 10 15-20 7
2-8C / No defined limit
ZstatFlu 6 30 10 0-40C / 24 hr.
FLU OIA 10 35-40 10-15 2-8C / 24 hr.
QuickVue Influenza 3 10-15 5 2-8C / 1 hr.
Directigen Flu A TestDirectigen Flu A Test Directigen Flu A+B Assay Directigen Flu A+B Assay
ZstatFluZstatFlu
ZstatFluZstatFlu
Biostar Flu OIABiostar Flu OIA
Biostar OIABiostar OIA
QuickVue InfluenzaQuickVue Influenza
Comparison of Rapid TestsComparison of Rapid Tests
Test
Sensitivity
Specificity Directigen
Flu A 67-92% 88-97%
Directigen Flu A+B
(A) 77-95%
(B) 71-88%
(A) 90-91%
(B) 98-100%
ZstatFlu 57-65% 98-100%
FLU OIA 62-88% 52-80% QuickVue Influenza 73-81% 96-99%
Prevalence rates were all >18%
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Laboratory Diagnosis of Influenza Rapid EIA-Like Tests
A & BNon-WaivedClearview Flu A/B
A & BNon-waivedDirectigen EZ Flu A + B
A & BNon-waivedFlu OIA A/B
A / BNon-waivedFlu OIA
A & BNon-waivedDirectigen Flu A & B
A & BWaivedBinaxNOW Influenza A & B
A & BWaived/Non-waivedNOW Flu A and NOW Flu B
ANon-waivedDirectigen Flu A
Antigen DetectedCLIA StatusTest
19
Laboratory Diagnosis of Influenza Rapid EIA-Like Tests (continued)
A & BNon-WaivedSAS Influenza A and Influenza B
A & BNon-waivedXpect Flu A & B
A & BWaivedQuickVue Influenza A + B
A / BWaivedQuickVue Influenza
A / BWaivedZstatFlu
Antigen DetectedCLIA StatusTest
Performance CharacteristicsPerformance Characteristics
TestTest SensitivitySensitivity SpecificitySpecificity
Directigen Directigen
Flu AFlu A
91% (A)91% (A) 95% (A)95% (A)
Directigen Directigen
Flu A & BFlu A & B
86% (A) 81% (B)86% (A) 81% (B) 91% (A) 99.5% (B)91% (A) 99.5% (B)
Flu OIAFlu OIA 62-88% (A/B)62-88% (A/B) 52%-80% (A/B)52%-80% (A/B)
Flu OIA A/BFlu OIA A/B 62%-88% (A/B)62%-88% (A/B) 52%-80% (A/B)52%-80% (A/B)
NOW Flu A & BNOW Flu A & B 78%-82% (A)78%-82% (A)
58-71% (B)58-71% (B)
92-94% (A) 97% (B)92-94% (A) 97% (B)
Performance CharacteristicsPerformance Characteristics
TestTest SensitivitySensitivity SpecificitySpecificity
QuickVueQuickVue InfluenzaInfluenza
73-81% (A/B)73-81% (A/B) 95% (A)95% (A)
QuickVueQuickVue Influenza A&BInfluenza A&B
72-77% (A) 72-77% (A)
73-82% (B)73-82% (B)
91% (A)91% (A)
99.5% (B)99.5% (B)
SAS InfluenzaSAS Influenza A A & Influenza B& Influenza B
76% (A)76% (A)
91% (B)91% (B)
98% (A)98% (A)
100% (B)100% (B)
Xpect Flu A &Xpect Flu A & BB 92% (A)92% (A)
98% (B)98% (B)
100% (A)100% (A)
100% (B)100% (B)
ZstatFluZstatFlu 58-65% (A/B)58-65% (A/B) 98-100% (A/B)98-100% (A/B)
Rapid (POC)Tests: AdvantagesRapid (POC)Tests: Advantages
Rapid turn around timeRapid turn around time
Rapid outbreak identificationRapid outbreak identification
Cost-effective (?)Cost-effective (?)
Widespread testing availableWidespread testing available
Less expertise requiredLess expertise required
Optimize antibiotic and antiviral usageOptimize antibiotic and antiviral usage
Rapid Tests: ConcernsRapid Tests: Concerns
Specimen adequacySpecimen adequacyNo viral isolates available for further No viral isolates available for further characterizationcharacterizationLoss of surveillance dataLoss of surveillance dataLive, attenuated intranasal vaccine can Live, attenuated intranasal vaccine can produce positive results (culture positive produce positive results (culture positive too!)too!)Variable performance characteristicsVariable performance characteristicsResult interpretation- poor positive Result interpretation- poor positive predictive values during low prevalencepredictive values during low prevalence– Improper treatment choices Improper treatment choices
Percent Positive By Specimen TypePercent Positive By Specimen Type
Covalciuc, K.A., et al. 1999. J. Clin.Microbiol. 37: 3971-3974
Patient Age and Onset of TestingPatient Age and Onset of Testing
Steininger, C., et al. 2002. J. Clin. Microbiol. 40: 2051-2056
a No samples were in the category of 25 to 50 positive cells.
EIA resultNo. 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 1 0 15
Positive 0 1 2 2 5 6 16
Total 8 2 5 4 6 6 31
Correlation between number of DFA-positive cells and EIA result
Predictive ValuePredictive Value
The probability of the presence or The probability of the presence or absence of disease given the test resultabsence of disease given the test result– PPV is the probability of disease in a patient with PPV is the probability of disease in a patient with
a positive test resulta positive test result– NPV is the probability of not having disease when NPV is the probability of not having disease when
the test result is negative.the test result is negative.
Determined by Determined by – sensitivity and specificity of the testsensitivity and specificity of the test– prevalence of disease in the population prevalence of disease in the population
being testedbeing tested
Hypothetical Influenza Test PerformanceHypothetical Influenza Test Performance
Prevalence = 1 in 5Prevalence = 1 in 5– Sensitivity = 95%Sensitivity = 95%– Specificity = 96%Specificity = 96%
– PPV = 85.6%PPV = 85.6%– NPV = 96%NPV = 96%
Prevalence = 1 in 100Prevalence = 1 in 100– Sensitivity = 95%Sensitivity = 95%– Specificity = 96%Specificity = 96%
– PPV = 19.2%PPV = 19.2%– NPV = 99.9%NPV = 99.9%
Which test is best? Know the test performance characteristics and the epidemiology of the disease
False Positive EIA’sFalse Positive EIA’s
Tampa, FL - 16 Flu A positives by EIA in Tampa, FL - 16 Flu A positives by EIA in the period from August to the end of the period from August to the end of October – none confirmedOctober – none confirmed
NY State Dept. of Health (December NY State Dept. of Health (December 2006) 2006) 60 specimens that were flu EIA positive sent for confirmatory testing. Only one of them positive by RT-PCR
Rapid Tests: Optimizing UseRapid Tests: Optimizing Use
Educate clinicians on predictive values Educate clinicians on predictive values & limitations of test results& limitations of test resultsConfirm early, late and out-of-season Confirm early, late and out-of-season positivespositivesConfirm peak-season negatives, if Confirm peak-season negatives, if applicableapplicableUse prevalence indicators to decide:Use prevalence indicators to decide:– When to testWhen to test– When to qualify resultWhen to qualify result– When to confirm resultsWhen to confirm results
"Don't brag about your lightning pace, "Don't brag about your lightning pace,
for Slow and Steady won the race!"for Slow and Steady won the race!"
A sculpture of the Tortoise and the Hare by Nancy A sculpture of the Tortoise and the Hare by Nancy Schön. Located at Copley Square, it represents the Schön. Located at Copley Square, it represents the
finish line of the Boston Marathonfinish line of the Boston Marathon