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 Experience with Dengue Virus Antigen Tests

Dengue Diagnostics 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

‘Acute’ Dengue – A Diagnostic Challenge NS1 IgM Virus NS1 Primary Infection Secondary Infection Fever Day Incubation IgM IgG Anti-DENV not helpful for diagnosis of acute disease

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

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

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

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

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

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

Evaluation of Commercial IgM anti-DENV ELISA Kits  Evaluation Panel (n=350) − 181 confirmed dengue − 169 challenge specimens  7 Participating Laboratories EID (3):

NS1 Antigen Detection Kit Evaluation, 2010 Panel Composition (n=471) 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= ) − 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

NS1Commercial Tests Evaluated  NS1 Microplate ELISAs − PanBio − BioRad − Standard Diagnostics  NS1 Rapid Diagnostic Tests − CTK − PanBio − BioRad − Standard Diagnostics

Preliminary Results: NS1 ELISA Americas Panel KitSensitivitySpecificityPositive Predictive Value Negative Predictive Value A64.3%90.8%85.7%74.8% B82.9%87.9%85%86.1% C75.9%100% 83.2%

Comparisons Previous and Current Evaluation Bessoff et al., 2008 Current Evaluation 2010 KitSensitivitySpecificityPositive Predictive Value Negative Predictive Value A64.3%90.8%85.7%74.8% B82.9%87.9%85%86.1% KitSensitivityConfidence Intervals A83.2%77.5%-87.7% B64.9%58.2%-71.1%

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

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