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Experience with Dengue Virus Antigen Tests

Experience with Dengue Virus Antigen Tests. Blood Products Advisory Committee Meeting Harold S. Gaithersburg, MD December 14, 2010 Harold S. Margolis, MD Chief, Dengue Branch Centers for Disease Control and Prevention San Juan, Puerto Rico, USA. Virus Detection Mosquito inoculation

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Experience with Dengue Virus Antigen Tests

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  1. Experience with Dengue Virus Antigen Tests Blood Products Advisory Committee Meeting Harold S. Gaithersburg, MD December 14, 2010 Harold S. Margolis, MD Chief, Dengue Branch Centers for Disease Control and Prevention San Juan, Puerto Rico, USA

  2. Virus Detection Mosquito inoculation Cell culture (C6/36) Immunofluorescent assay (IFA) Nucleic Acid Amplification Antigen detection Antibody Response IgM anti-DENV IgG anti-DENV - ELISA - PRNT Dengue Diagnostics

  3. Virus Virus IgM NS1 ‘Acute’ Dengue – A Diagnostic Challenge Primary Infection Secondary Infection IgG Anti-DENV not helpful for diagnosis of acute disease IgM NS1 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Fever Day Fever Day Incubation

  4. NS1 Antigen • Highly conserved, soluble glycoprotein • Secreted during virus replication along with envelop proteins/glycoproteins • Present during both 1° and 2° infections • DENV-type specific – detected using MABs • Anti-NS1 produced during infection

  5. NS1 Antigen • Appears as early as day of fever onset • Present in both 1° and 2° infections • Correlated with viremia (Alcon-LePoder et al, 2007) • Possible association with disease severity (Thomas et al., 2010, Libraty et al., 2002, Dussart et al, 2006) • Single specimen diagnostic tool

  6. Comparison: NS1 ELISA Commercial Kits +Only sensitivities for acute samples reported *Sensitivity increased to 72% using immune complex dissociation by acid treatment

  7. Evaluation of Dengue Diagnostic Kits • Joint project of WHO-TDR-PDVI • Initiated 2005 • Network sites established • Asia: Cambodia, Malaysia, Thailand, Vietnam • Americas: Argentina, (Brazil), Cuba, Puerto Rico • Reference Labs established – Thailand, Puerto Rico • Steering Group with annual meetings • Evaluation protocols / SOPs • Biobanking/GCLP workshops • Network lab proficiency assured • Evaluation panels developed

  8. Objectives • To assess the performance and operational characteristics of commercially available IgM anti-DENV and NS1 antigen detection tests for the diagnosis of dengue • To provide data on test performance to WHO member states • To provide advice on the inclusion of tests in WHO bulk procurement scheme

  9. The Process • Companies informed of evaluation scheme by TDR • Companies that expressed interest were invited to participate • Companies signed agreement to participate • Companies sent kits to Reference Labs • Sites obtained permits for import of specimens and test kits • Tests and Evaluation Panels sent to sites from Reference Labs

  10. Evaluation of Commercial IgM anti-DENV ELISA Kits • Evaluation Panel (n=350) • 181 confirmed dengue • 169 challenge specimens • 7 Participating Laboratories EID 2009 15(3):436-440

  11. AMERICAS DENV Positive(n=85) 10 Acute 1°(0-7 days) 75 Acute 2° Challenge Panel 40 Negative (PR) 49 Neg non-endemic areas 21 Related flaviviruses (WN, YF, other) 18 Other febrile illnesses 12 Systemic diseases (lupus, RF) ASIA DENV Positive(n= 76 + 88) 19 Acute 1° 57 Acute 2° 26 Convalescent 1° (>7 days) 62 Convalescent 2° Challenge Panel 13 Related flaviviruses (JE) 59 Other Febrile Illness 10 Systemic diseases NS1 Antigen Detection Kit Evaluation, 2010 Panel Composition (n=471)

  12. NS1Commercial Tests Evaluated • NS1 Microplate ELISAs • PanBio • BioRad • Standard Diagnostics • NS1 Rapid Diagnostic Tests • CTK • PanBio • BioRad • Standard Diagnostics

  13. Preliminary Results: NS1 ELISAAmericas Panel

  14. ComparisonsPrevious and Current Evaluation Bessoff et al., 2008 Current Evaluation 2010

  15. Unexpected Results, NS1 ELISA’s • Company A • low sensitivity to DENV2 • 2/10 false positive for WNV IgM (challenge panel) • Company B • low sensitivity to DENV2 • 7/10 false positive for RA (challenge panel) • 2/3 YFV IgM positive (challenge panel) • Company C • low sensitivity to DENV2

  16. Summary • NS1 antigen detection appears useful for diagnosis of 1° and 2° dengue (symptomatic DENV infection ) • Previous studies showed variability in sensitivity / specificity between commercially available kits • WHO-TDR-PDVI Evaluation – Preliminary Results • Data only from Americas • Suggest variability in sensitivity between kits

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