Blood Products Advisory Committee Meeting Harold S. Gaithersburg, MD December 14, 2010 Harold S. Margolis, MD Chief, Dengue Branch Centers for Disease.

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

NS1IgM

Virus Virus

NS1

Primary Infection

Secondary Infection

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

0 1 2 3 4 5 6 7 8 9 10

Incubation

IgM

IgG Anti-DENVnot 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

Reference Tested BioRad Panbio

Pos Neg Sensitivity* Specificity Sensitivity Specificity

Blacksell et al, 2008 38 54 63.2 100 -- --

Dussart et al, 2008 222 48 87.4 100 60.4 97.9

Zainah et al, 2009 314 219 90.4 99.5 -- --

Hang et al, 2009 125 13 82.0 100 -- --

Lapphra et al, 2008 171 64 63.2* 98.4 -- --

Kumarasamy et al, 2006

213 354 97.3 100 -- --

McBride, 2009 91 -- 73.6 -- 63.7 --

Bessoff et al, 2008 208 45 83.2 100 64.9 97.8

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 2009 15(3):436-440

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= 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

NS1Commercial Tests Evaluated

NS1 Microplate ELISAs− PanBio− BioRad− Standard Diagnostics

NS1 Rapid Diagnostic Tests − CTK− PanBio− BioRad− Standard Diagnostics

Preliminary Results: NS1 ELISAAmericas Panel

Kit Sensitivity Specificity Positive Predictive Value

Negative Predictive Value

A 64.3% 90.8% 85.7% 74.8%

B 82.9% 87.9% 85% 86.1%

C 75.9% 100% 100% 83.2%

ComparisonsPrevious and Current Evaluation

Bessoff et al., 2008

Current Evaluation 2010Kit Sensitivity Specificity Positive

Predictive Value

Negative Predictive Value

A 64.3% 90.8% 85.7% 74.8%

B 82.9% 87.9% 85% 86.1%

Kit Sensitivity Confidence Intervals

A 83.2% 77.5%-87.7%

B 64.9% 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