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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