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Dengue diagnostic
Instituto Medicina Tropical “Pedro Kourí” , IPK
Centro Colaborador OPS/OMS para el estudio del dengue y su vector
19 November 2013
Current dengue diagnostic
Relda updating
Flaviviridae family
Flavivirus genus
v
C
E
M
RNAss
Dengue complex (Den1-4)
Enveloped viruses
Intact virus is needed for viral isolation
How to detect a
dengue virus infection?
Detection of virus detection or
some component of the virus
or some product of virus
replication
Human response
to virus infection
Viral isolation
RNA
Antigen detection (NS1)
Antibody
(IgM, IgG, IgE, IgA)
T cell reponse
Diagnosis usefulness
Clinical
diagnostic
Surveillance
Research
Diagnostic
for prognostic
Case diagnostic & management
Differential diagnostic
Severe & fatal cases
Supporting Integrated surveillance
Early warning of transmission
Outbreak detection & characterization
Virus characterization
Entomological surveillance
Vaccine & antiviral studies
Pathogenesis studies
Clinical & epidemiological studies
Detection & evaluation of early
prognostic markers of severity
Clinical
diagnostic
Surveillance
Methods
Sample
Early diagnosis
(acute infection,
<4 days of fever
onset)
+++
++
RNA detection
NS1 detection
Serum
collected
first 4 days
Early
convalescent
diagnosis (4-10
days)
++
+++
IgM detection
High IgG titers
Serum
collected at
days 5-6 of
onset
Late confirmed
diagnosis (longer
period)
+
+
•IgG
seroconversion
•Viral isolation
Paired sera
Serum
collected
first 4 days
Laboratory diagnostic methods employed
for diagnosis of dengue infection
OPPORTUNITY
DIRECT METHODS
Virus Isolation
Genome
detection
INDIRECT METHODS
Antigen
detection
Serology
IgM
Serology
IgG
CONFIDENCE
Guzman et al., Nature Rev, 2011
Serum
Necropsy pieces
Sample conditions:
< 5 days onset
4°C (storage at –70°C)
Sterile
Clinic & Epidemiological information
Rapid Transportation
Molecular diagnosis (RT/PCR, Real time
PCR
RT-PCR
Antigen
detection
histochemical studies
(NS1,
immuno
Dengue
1 2 3 4 1-4
Guzman, et al., 05; Sydow et al., 2000; Kao et al., 01; Rosario et al., 05; Vordam, 2005
Diagnosis (including fatalities)
Molecular surveillance
Entomological surveillance
Molecular epidemiology
Pathogenesis studies
Amplification of target nucleic acid sequences
Rapid & sensititive method for Den identification & early detection
Several protocols (need for proper evaluation)
Several gene targets
New developments, RT/PCR & realtime RT/PCR
CDC DENV1-4 realtime PCR assay
•Platform similar to flu
•Identification of the four serotypes
•Sensitivity & Specificity of 98% compared to IgM
seroconversion
•Tested in three field sites
•Singleplex or multiplex format (equivalent sensitivity)
Santiago et al., PLOS NTD 2013
DENV multiplex rRT-PCR more sensitive than FDA-approved
CDC DENV-1-4 assay. 97.4% versus 87.1%, sensitivity
(tested in 199 samples from Nicaragua & Sri Lanka
Waggoner et al., JC) Microbiol 2013
NS1 protein, possible early diagnostic and prognostic marker
Possible early diagnostic (days 1 to 6, 87-60% detection) (M Flammand)
Prognostic marker (>600ng/ml in 72h in patients at risk of DHF) (P. Young)
Sera 1-7 days (6 countries) From confirmed cases. Sensitivity: 50-62%;
Specificity: 90-100%.
% of positive samples to both IgM & NS1 by day of illness .
IgM & NS1: 82% positive diagnostic in the first 4 days.
Guzman et al., Plos NTD, 2010
NS1
ELISA
NS1 lateral flow
rapid test
Duong et al., Plos
NTD 11
57%
NT
Frey et al., Plos NTD
11
NT
Tondutawat et al.,
SEA JTMH 11
75%
70%
McBride et al., DMID
09
73%
NT
•Lower sensitivity in Ab presence
Rocha et al., Plos
NTD
72%
NT
•Sensitivity variation according
serotype
Hang et al., Plos NTD
09
82%
72%
Huang, . J MII 12
NT
68%
Ferrz et al., JCV 13
NT
94-81% (three
lateral flow tests)
Sandoval et al., Rev
Cubana Med Trop,
2012
NT
58% NS1
100% with IgM and
IgG
Dussart et al., Plos
NTD 09
87%
81%
•Several commercial ELISA &
rapid tests
•Specificity 95-100%
69%
•Higher sensitivity at first 3-4
days
•Sensitivity variation according
gold standard
•Sensitivity variation in 1ry & 2d
infection.
•A negative sample does not
preclude the infection
NS1: fatal dengue diagnosis
Sensitivity
ELISA
PanBio
35%
74 tissues from fatal dengue cases
Cases diagnosed by VI, PCR, serology
ELISA
Biorad
61%
Rapid test
Biorad
91%
Liver 71%
Kidney 100%
Brain 80%
Spleen 67%
compared to the other methods
Rocha Queiroz et al., PLos NTD, 2011
NS1:quantification?
•Higher viral load as detected by realtime PCR & NS1,
in dengue cases with negative IgM sera, p< 0.0001
De La Cruz Hernández, JCV 2013
•Higher levels of NS1 in DHF compared to DF by
DENV-1. Higher levels in primary than in secondary
infection by DENV-1 & 2. de la Cruz-Hernández et
al., AJTMH 2013
NS1: further evaluations
•Good sensitivity (more than 95%) in 2300 samples from
dengue surveillance. Bisordi et al., Rev Inst Med Trop Sao
Paulo. 2011
•Comparison of rapid test for NS1, IgM and IgG test at
primary and national lab levels (sensitivity 86%, specificity
84% at primary level and 94%, 90.% reference lab. Andries
et al., Plos NTD 2012.
•Low NS1 sensitivity in an outbreak in Santos, Brazil
(37%). Felix et al., CVI 2013
Serological Diagnosis
Standard test: IgM detection
Simple
Large numbers of sera
Routine diagnosis
Widest extended
Rapid diagnosis
ELISA (most common format)
Recent infection
Serum
Saliva
Filter paper
Detected in >=90% after 5-6 days
up to 30 days. Dengue-cross reactive
>=5-6 days of fever onset
Many commercial kits IgM
Chromatographic Tests
ELISA
Cassette
AuBioDOT
Dip Stick
Mondesire, 2005
TDR/PDVI Evaluation of Commercially Available Anti–
Dengue Virus Immunoglobulin M Tests
Emerging Infectious Diseases 2009, 15: 436-440
9 kits
Sensitivity: 21-99%
Specificity: 77-98%
Best systems:
ELISA: Focus, PanBio, Standard
Possible diagnostic value in serum
(Talarmin et al, 1998; Balmaseda et al, 2003; Vázquez et al,
2005). Yap et al., Plos NTD 11: 36% in Primary cases versus
100% in secondary cases in the first three days
Possible severity marker
(Koraka et al, 2001)
Possible severity marker
(Pavri, 1977-79; Koraka et al, 2001)
Sero-epidemiological studies
Vaccine studies
Pathogenesis studies
Dengue IgG Abs: past infection
Greatest virus serotype specificity.
Most of studies based on IgG neutralizing Ab determination.
Ab specificity increases over time.
Low level of cross reactivity in primary infection (criteria).
Nt Abs to two viruses: secondary infection.
Low cross reaction with other flavivirus?
Original antigenic sin.
Easy to perform and to extend
Reproducible, fast
Specific of serotype & flavivirus
Non expensive
ELISA test?
System that correlate with in vivo situation?
Guidelines for plaque reduction neutralization testing, IVR/WHO,
2009
Variation in dengue PRNT: systematic review, Rainwater-Lovett et al., BMC 2012
Differences in neutralization %, 3 cell lines, 12 virus concentrations and 51
strains, differences in Ab titers in primary & secondary infection.
New developments: microneutralization, ELISA/NS1, Raji-DC-SIGN cells and flow
cytometry for measuring DC-SIGN (CD 209) expression, Mattia et al., Plos One 2011, Li
et al., PlosOne 2011
Mosquito inoculation (adults
sensitivity, insectary facilities)
or
larvae)
(higher
Mosquito cell line (C636) (routine & standard diagnosis)
Other cell lines & newborn mice
Shell vial application, Rodriguez-Roche et al., 2002
Indirect Immunofluorescence Assay (Monoclonal antibodies)
PCR
ELISA
RT-PCR
Flow cytometry (anti NS1 ab, 10 hrs earlier than IF)
Dengue
1 2 3 4 1-4
Guzman, et al., 05; Sydow et al., 2000; Kao et al., 01; Rosario et al., 05; Vordam, 2005
Highly suggestive
Confirmed
IgM in a single
serum
IgG in a single
serum with HI titer
>=1280 by HI
PCR+
Virus isolation
IgM seroconversion
IgG seroconversion
in paired sera or
fourfold increase
NS1 ????
WHO dengue guidelines, 2009
•Rapidity, low cost, availability of
reagents, early diagnostic
•Protocols & kit evaluation
•Flavivirus & arbovirus diagnosis
•Diagnostic of fatal cases
•Samples
•Entomological surveillance
•Prognostic markers
•Quality control, GLP
New developments
•Differential arbovirus diagnostic
(rRt/PVR & realtime PCR for dengue &
other arboviruses)
•Evaluation of other clinical samples
(serum, salaiva, urine por serological &
virological diagnostic)
•NS1 detection in CSF, necropsy samples,
mosquitoes
•Prognostic markers (viremia)
Future lines of diagnostic???
New developments, NS1
A sensor tip based on carbon nanotube-ink printed
electrode for the dengue virus NS1 protein , Moraes et al.,
Biosens Bioelectron 2013
Micro-Spot with Integrated Pillars (MSIP) for NS1
detection. Amplify detection in five times, Gunda et al.,
Biomed Microdevices. 2013
Immunosensor for (NS1 detection based on carbon
nanotube-screen printed electrodes (CNT-SPE), Dias et
al., Biosens Bioelectron. 2013 , sensitivity 85%
Dengue Lab surveillance
Mostly based on IgM detection + V. isolation & RT/PCR
Most of cases classified as highly suggested
Challenge of NS1 introduction
Need of diagnostic algorithm evaluation & recommendation
Need of external quality control
Reagent availability
Need of proper commercial kit evaluation
Role of syndromic surveillance
Standardized reporting
Personnel training
Improved sample collection, shipping & distribution
Biosafy and GLP
Primary level
District level
Reference
centers
Virus culture
X
Nucleic acid
detection
X
Ag detection
X
X
Serology
X
X
X
X
Rapid tests
X
WHO Guidelines 2009
Need of stronger & real integration to integral surveillance & analysis
Good & bad practices in dengue diagnostic
Good
Bad
When to use a test?
Taking care the purpose of
the study before making a
selection of the test
Use inappropriate test
conducing
misinterpretation
How to use a test?
Follow up the
manufacture’s
recommendations
Not following them
Lab issues
Quality system instituted
Results not reliable
No quality control
No records
Unvalidated kit
Contamination etc
WHO, 2009
Relda
Organización
Panamericana
de la Salud
EGI-dengue & Lab
surveillance
Vigilancia
epidemiológica
Comunicación
social
Entomología
Estrategia de
Gestión Integrada
Medio
Ambiente
Laboratorio
Atención al
paciente
OPS
RELDA
•30 „países
•7 CCOMS
•Labs. nacionales
•1 LAB EURO (ISC III)
Reunion de
DengueNet,
Congreso
Mundial de
dengue
La Habana, Cuba
Antecedentes
Taller de buenas
1° Reunión de
prácticas de
CCOMS de dengue
laboratorio .
Buenos Aires ,
TDR/OMS/OPS
Argentina
8°Curso Internacional
de dengue
Panamá, Panamá
La Habana, Cuba
Reunión de CCOMS
Organización
Panamericana
de la Salud
En el marco del Simposio internacional y
el 9° Curso Internacional de dengue, La
Habana, Cuba
20
12
20
11
20
10
Reunión de CCOMS y
Laboratorios
Nacionales de
referencia de dengue
Panamá, Panamá
20
09
20
08
20
07
20
06
20
05
20
04
Taller de diagnóstico
y caracterización de
virus dengue
Tegucigalpa,
Honduras
Habana,
Curso Dengue, Agosto, 09
Relda, La Habana, agosto de 2009
Se acuerda oficializar la red
Se define comité consultivo RELDA (5 CCs), 4
coordinadores subregionales y labs nacionales
Preparación plan actividades
OBJETIVOS RELDA
•
•
•
•
Fortalecer las capacidades técnicas & científicas de
los laboratorios de dengue de la región
Normalizar protocolos de laboratorio, evaluación
estuches y métodos de diagnósticos, intercambio
reactivos referencia
Apoyar la implementación de sistema de control de
calidad en los laboratorioss
Integrar toda la capacidad científica & técnica
disponible en la región para responder
adecuadamente a brotes & epidemias
Estructura organizativa y funcional de
RELDA
Estructura organizativa y funcional de la RELDA
COORDINADOR
SECRETARÍA TÉCNICA
Programa Regional de Dengue OPS/OMS
Dra. Guadalupe Guzmán,
IPK, Cuba
COMITÉ TÉCNICO CONSULTIVO
NML
Canada
CDC
Puerto Rico
Dr. Harvey Arstob
Dr. Elizabeth Hunsperger
Canadá
Puerto Rico
NORTE AMÉRICA
NML (CAN) –
CDC (PUR)
IPK
Cuba
Dr. Delfina Rosario
Dr. Susana Vázquez
Dr. Pedro Vasconcelos
Dr. Ana Cecilia Cruz
Cuba
COORDINADORES SUBREGIONALES
MÉXICO Y CENTRO AMÉRICA
InDRE (MEX) – INCIENSA (COR)
CANADA: NML
COSTA RICA: INCIENSA
PUERTO RICO:
CDC Dengue Branch
EL SALVADOR: ULC
USA: CDC- Atlanta
IEC
Brazil
Brasil
ANDINA Y CONO SUR
INHRR (VEN) – LCSP (PAR)
INEVH
Argentina
Dr. Delia Enria
Dr. Silvana Levi
Argentina
EL CARIBE
PASTEUR (FRG) -JAMAICA
BOLIVIA: CENETROP
BELIZE: CML
COLOMBIA: INS
CAREC: COUNTRY
MEMBERS
GUATEMALA: LNS
ECUADOR
HONDURAS: LNV
PERÚ: INS
MÉXICO: InDRE
VENEZUELA: INHRR
NICARAGUA: CNDR
ARGENTINA
PANAMÁ: ICGES
BRAZIL: IEC
FRENCH GUYANA:
PASTEUR
GUYANA: NPHRL
OTRAS REGIONES - OMS
JAMAICA
EURO
WPRO
SEARO
MARTINICA: CHU
ESPAÑA ISC III
CHILE: ISP
REP. DOMINICANA:
LNSPDD
PARAGUAY: LCSP
SURINAME: CLPH
URUGUAY
Instituto de Medicina Tropical
"Pedro Kourí" (IPK), La Havana.
Cuba
Centros para el Control y la Prevención
de las Enfermedades (CDC),Oficina para
Dengue, Puerto Rico
Centro
Epidemiológico del
Caribe ( CAREC) ,
Puerto España,
Tirinidad y Tobago
CENTROS
COLABORADORES
PARA DENGUE
Instituto Evandro Chagas (IEC),
Belem, Brasil
OPS/OMS
INEVH “Dr. Julio I.
Maiztegui”- Pergamino,
Argentina
Red de Laboratorios de Dengue de las Américas
RELDA
hacia el fortalecimiento de la Estrategia de Gestión Integrada para la prevención y control del dengue
estatutos de la relda
Organización Panamericana de la Salud (OPS)
Organización Mundial de la Salud (OMS)
Programa Regional de Dengue
Centros Colaboradores de la OPS/OMS para dengue
Laboratorios Nacionales de Referencia de Salud Pública de las Américas
Actividades realizadas (2010-2013)
Encuesta laboratorios Relda
Proficiencia serológica
Encuesta laboratorios Relda
2011-12
Argentina, Bolivia, Chile, Colombia, Costa Rica, Cuba, España,
Ecuador, Guyana, Guatemala, Honduras, Mejico, Nicaragua,
Salvador, Panamá, Puerto Rico (CDC). (16)
Espacio OK
12/15
Proficiencia
10/14
Equipamiento OK
8 OK
6 problemas
Investigación
14/14
BSL-2
15
Red labs.
14/15 (2-181)
BSL-3
3/15
Control calidad
interno
14/16
Entrenamiento
Bioseguridad
9/15
Control
internacional
7/9
Entrenamiento diagnóstico
13/14
Plan contingencia
11/14
Auditoría interna
8/14
MSc, PhD
14/14
Encuesta laboratorios Relda
2011-12
IgM
16/16
IgG
15/16
AV-tipificación
RT/PCR
Realtime RT/PCR
16/16
15/16
9/16
IH
11/16
InmunoHistoquímica
PRNT
5/16
8/16
NS1
10/15
Algoritmo
diagnóstico
Banco células
Banco sueros
Banco cepas
Prod.
reactivos
7/8
13/14
13/14
4/6
6/14
Encuesta laboratorios Relda
2011-12
•Necesidad reactivos dengue y otros arbovirus
(antigenos y monoclnales)
•Capacitación: control calidad, bioinformática, filogenia,
neutralización, prod. reactivos
•Investigación: evaluación estuches comerciales,
epidemiológicas, epidemiologia molecular, patogenia
•Proficiencia internacional: CCs de la región y Red
Europea Virus Importados
Proficiencia serológica dengue IgM
2010-2011
Invitados- 20 Laboratorios
Enviados – 16 países
Perdidos por problemas de Recepción (3)
Respuesta de Laboratorios – 12
Coincidencia IgM entre el 90-100%
Actividades realizadas (2010-2013)
1ra y 2da reunión del Grupo Referencia
Dengue, TDR/OMS, Cuba, Panamá
Preparación proyecto CYTED como apoyo
a RELDA
Reunión Relda, 2011.
Actividades realizadas (2010-2013)
Preparedness and Response
for Chikungunya Virus Introduction
in the Americas
Actividades realizadas (2010-2013)
Technical report:
Flavivirus Diagnostic Algorithm for the Americas
region
Recommendations from the “Consensus building meeting:
Standardization of flavivirus diagnostic testing protocol and
development of Chikungunya laboratory diagnostic capacity for the
Americas” in Pergamino, Argentina, August 2011.
Updated in March, 2013.
Actividades realizadas (2010-2013)
Participación red de laboratorios
(TDR/DVI/OPS/OMS) para evaluación estuches
comerciales (tres evaluaciones concluidas)
Reporte TropiKa (Dengue research network meets in Cancun;
DENGUE IN THE AMERICAN REGION. AN UPDATE,
Participación proyectos multicéntricos (DENCO,
DENFREE, IDAMS (en curso), otros)
Actividades realizadas (2010-2013)
Proyecto DENCO (UE/TDR/OMS/OPS).
Evaluación NS1 (Plos NTD, 2010)
Evaluación estuches comerciales de IgM
(TDR/PDVI/OPS/OMS).
Emerging Infectious Diseases 2009
Actividades realizadas (2010-2013)
Capacitación en diagnóstico molecular
para INCIENSA/febrero 2011/CDC
dengue Branch
Evaluaciones de EGI en el período 2011-
2013
Actividades realizadas (2010-2013)
Implementación EGI/dengue
a) Guyana, febrero 2011 (CAREC)
b) Trinidad Tobago, mayo 2011 (CAREC)
•
Curso dengue, La Habana, agosto 2011
a) Minicurso CHIK
Actividades realizadas (2010-2013)
•
Asesoría Instituto Pasteur Guyana (2012)
•
II Reunión Internacional de Monitoreo de la
Implementación de las Estrategias Nacionales de
Gestión Integradas para la Prevención y Control del
Dengue en las América, Argentina 2012
•
Curso Internacional Intervención Integral dengue,
Colombia 2012
Actividades realizadas (2010-2013)
Proyecto vigilancia en el marco de la
vacunación para dengue. Panamá 2013
Proyecto mapa genómico dengue
• Mapa virológico
• Mapa genómico
• Mecanismos moleculares asociados al
virus, el individuo y su respuesta
inmune en la gravedad por dengue
• Papel de otros flavivirus
• Factores del hospedero incluyendo su
genética, co-morbilidades entre otras
Posible Algoritmo para Diagnóstico Dengue
Panama, 2013, OPS
Actividades realizadas (2010-2013)
Curso Internacional de dengue agosto
2013
XIII Curso dengue, agosto 2013, La Habana
Actividades Prácticas
Taller de BPL, OPS/IPK/Universidad del Valle, agosto
2013, La Habana.
41 participantes
de 20 países de la región
Actividades en curso
Enviado a revisión al programa regional: Pautas
de la vigilancia de laboratorio de dengue. Utilidad
del diagnóstico de dengue”
En curso metanálisis NS1
Proyecto vigilancia/vacuna/mapa genómico
Iniciativas Internacionales
Aspectos a fortalecer
Intercambio entre los laboratorios y los CCs
Reactivos de referencia incluyendo otros virus
Algoritmo diagnóstico en el marco de dengue y otros
agentes para la vigilancia. Valor NS1
Control Calidad
Capacitación continua
Gestión calidad
Mayor interacción en los sistemas de vigilancia. Papel
del laboratorio.
Habana, 2014
VIII Congreso Cubano de
Microbiología & Parasitología
V Congreso Nacional de Medicina
Tropical
III Seminario Internacional de
VIH/sida en Cuba
Octubre 14-16, 2014
www.eventospalco.com