Martha Thompson, MPHMartha Thompson, MPHViral Isolation Team LeaderViral Isolation Team Leader

Medical Virology GroupMedical Virology GroupLaboratory Services SectionLaboratory Services Section

TX DSHSTX DSHS

Influenza Surveillance Influenza Surveillance Viral Isolation LaboratoryViral Isolation Laboratory

TX DSHSTX DSHSJuly 23, 2008July 23, 2008

Viral IsolationViral Isolation

2007-2008 Season 2007-2008 Season Laboratory Diagnostics: InfluenzaLaboratory Diagnostics: Influenza Influenza testing: Viral Isolation LabInfluenza testing: Viral Isolation Lab

• Cell CultureCell Culture• ImmunofluorescenceImmunofluorescence• Hemagglutination/HA InhibitionHemagglutination/HA Inhibition• Levels of IdentificationLevels of Identification

Specimen rejection criteriaSpecimen rejection criteria Summary of isolates sent to CDCSummary of isolates sent to CDC

2007-2008 Influenza Summary: Viral Isolation Laboratory

1570

546

118

426219

0

400

800

1200

1600

2000

TotalSpecimens

A H1 H3 B

Influenza A specimens not subtyped (2) B strains identified as

B/Shanghai/361/2002-like (B/Yamagata) by the viral isolation laboratory

Total 1570

% Pos for Influenza

49%

Influenza A 35%

Influenza B 14%

2007 - 2008 Influenza Summary: Viral Isolation Laboratory

020406080

100120140160180200

10/0

6/20

07

11/0

6/20

07

12/0

6/20

07

01/0

6/20

08

02/0

6/20

08

03/0

6/20

08

04/0

6/20

08

05/0

6/20

08

MMWR Week Ending

Total specimens received

A H3

A H1

A Unknown

B

Laboratory Diagnostic Testing: Influenza

TAT

Rapid ~30 minutes

Serology ~2 Weeks

DFA 2 hrs

Culture 2 – 10 days

Molecular/PCR 4 hrs – 1 day

Rapid EIA KitsRapid EIA Kits

AdvantagesAdvantages• Rapid and on-site testingRapid and on-site testing• Impact patient managementImpact patient management• Simple Simple • CLIA wavedCLIA waved

LimitationsLimitations

• Typing/Results Typing/Results Flu + onlyFlu + only A or B A or B No subtypingNo subtyping

• Variation between kitsVariation between kits Storage conditionsStorage conditions Acceptable specimens (includes type and time of Acceptable specimens (includes type and time of

collection)collection)Must follow manufacturer instructionsMust follow manufacturer instructions

• Less sensitive than viral culture or molecularLess sensitive than viral culture or molecular False negativesFalse negatives

• PPV and prevalence in the community affect PPV and prevalence in the community affect test performancetest performance

These limitations affect test performanceThese limitations affect test performance

Patient managementPatient management

Use positive and negative predictive values to Use positive and negative predictive values to assess test performanceassess test performance

PPV: Probability of disease in a patient with a PPV: Probability of disease in a patient with a positive test resultpositive test result

PNV: Probability of no disease in a patient with a PNV: Probability of no disease in a patient with a negative test resultnegative test result

Test PerformanceTest Performance

PresentPresent AbsentAbsent

PositivePositiveTrue True

Positive Positive (TP)(TP)

False False Positive Positive

(FP)(FP)

NegativeNegative False False Negatives Negatives

(FN)(FN)

True True Negatives Negatives

(FN)(FN)

Disease

Test Result

Sensitivity = TP/TP+FNSpecificity = TN/TN+FP

PPV= TP/TP+FPPVN = TN/TN+FN

Present Absent

Positive 380 64Negativ

e20 1536

Disease

Test Result

Predictive Value Positive = TP/TP + FP = 380/380+64

= 85.6%

Prevalence=20%

Positive predictive value:

Present Absent

Positive 19 80Negativ

e1 1900

Disease

Test Result

Prevalence = 1%

Predictive Value Positive= TP/TP + FP

= 19/19+80

= 19.1%

Positive predictive value:

ConclusionConclusion

When prevalence is low, the PPV is When prevalence is low, the PPV is low and chance of getting a false low and chance of getting a false positive increasespositive increases

Confirm with culture during off Confirm with culture during off seasonseason

Other Methods

DFA• Quick TAT• No culture available for further studies

Serology• Positive results can be obtained even after

viral shedding has stopped• Acute/convelescent serum required—delay in

diagnosis• No culture available for further studies

Real Time RT-PCRAdvantages

Rapid: Sensitive/Specific* High throughput can be obtained Identification of highly pathogenic

strains of avian influenza possible

Disadvantages Costly Risk of cross contamination Variability among protocols means

variability among sensitivity/specificity rates

No isolate available for further studies

Cell CultureCell Culture

Confirm virus is infectiousConfirm virus is infectious Antigenic characterizationAntigenic characterization Vaccine StudiesVaccine Studies Antiviral resistance testingAntiviral resistance testing Important for surveillanceImportant for surveillance

Slower TATSlower TAT• 2-10 days2-10 days

Immunofluorescence (IFA)Immunofluorescence (IFA)

Indirect testIndirect test Antibody to Flu A and B antigensAntibody to Flu A and B antigens Fluorescent tagFluorescent tag A, B, or NegA, B, or Neg If positive – continue with subtypingIf positive – continue with subtyping Reagents in WHO kitReagents in WHO kit

Hemagglutination/HA InhibitionHemagglutination/HA Inhibition

Antisera to neutralize antigensAntisera to neutralize antigens Blood as an indicator, agglutinates to Blood as an indicator, agglutinates to

antigenantigen Antigenic characterizationAntigenic characterization

2007-2008 WHO Influenza 2007-2008 WHO Influenza Reagent Kit Reagent Kit

AntiseraAntisera Level of identificationLevel of identification

• A(H3)A(H3)• A(H1)A(H1)• B/Shanghai/361/2002-likeB/Shanghai/361/2002-like• B/Malaysia/2506/2004-likeB/Malaysia/2506/2004-like

Isolates to CDC• Beginning, middle, and late season• Patients who received vaccine• Anything unusual• Unable to subtype

WHO Summary: Weeks Ending Oct 6, 2008 – May 17, 2008

A/SOLOMON ISLANDS/03/2006-LIKE (H1N1) 8

A/BRISBANE/10/2007-LIKE (H3N2) 22

A/WISCONSIN/67/2005-LIKE (H3N2) 3

A/WISCONSIN/67/2005-LIKE (H3N2) LOW 2

B/FLORIDA/04/2006-LIKE 6

B/FLORIDA/04/2006-LIKE LOW 1

Did not test 17

Unable to grow virus; confirmed by PCR as A/H1 1

Unable to grow virus; confirmed by PCR as A/H3 8

Total 68

Vaccine Strains

2007-2008 Vaccine Strains A/Solomon Islands/3/2006 (H1N1)-like

χ A/Wisconsin/67/2005 (H3N2)-like

χ B/Malaysia/2506/2004-like (B/Victoria)

A/Brisbane/10/2007 is a variant form of A/Wisconsin/67/2005 strain

All B strains identified by VI lab were B/Shanghai/361/2002-like (B/Yamagata)

Specimen Rejection CriteriaSpecimen Rejection Criteria

Meet regulatory standardsMeet regulatory standardsOptimal specimen for testingOptimal specimen for testing

Expired transport mediaExpired transport media Wooden sticks/Calcium alginateWooden sticks/Calcium alginate

• Inhibitors to virus: preservativesInhibitors to virus: preservatives Cotton swabsCotton swabs

First AND Last name: on specimen AND First AND Last name: on specimen AND submission formsubmission form

1 Specimen = 1 Submission form1 Specimen = 1 Submission formDATE of COLLECTIONDATE of COLLECTION

Contact InformationContact Information

martha.thompson@dshs.state.tx.us

Phone 512-458-7594Phone 512-458-7594

Fax 512-458-7293Fax 512-458-7293

Viral Isolation LaboratoryViral Isolation Laboratory

512-458-7111 x2452512-458-7111 x2452

Useful Links

Resource Manual for Seasonal and Pandemic Influenza http://www.dshs.state.tx.us/comprep/pandemic/flu%20outreach%20manual%2012-28-2007.pdf

Laboratory Services Section http://www.dshs.state.tx.us/lab/