Pedro Fernando C. VasconcelosInstituto Evandro Chagas, SVS/MS
Centro Colaborador OPAS/OMS em Arbovírus
INCT para Febres Hemorrágicas Virais
Laboratório de Referênciia Nacional de Arbovírus
Ananindeua, Pará State, Brazil
Laboratory diagnosis to Chikungunya virus infections
Chikungunya Virus
• Family Togaviridae, genus Alphavirus• Enveloped, single stranded RNA virus
• Plus-sense, unsegmented genome of 11.5-11.8 kilobases
• First isolated from human
serum during outbreak in
Tanganyika - 1953
• Distinct biological and
transmission patterns between
African and Asian strains
Classical clinical presentationFever
Severe joint pain
Rash (maculopapular)
Chikungunya Virus
• Family Togaviridae, genus Alphavirus• Enveloped, single stranded RNA virus
• Plus-sense, unsegmented genome of 11.5-11.8 kilobases
• First isolated from human
serum during outbreak in
Tanganyika - 1953
• Distinct biological and
transmission patterns between
African and Asian strains
Classical clinical presentationFever
Severe joint pain
Rash (maculopapular)
Phylogenetic Tree of Alphaviruses
Période d’incubation 2
( 5 à 7 jours)
Cycle extrinsèque
chez le moustique
(10 jours)
Virémie= Période à risque de
contamination d'un moustique
5-7 joursPériode d’incubation 1
(5 à 7jours)
Arrivée d’une personne
infectée, en début
d’incubation
Survenue d’un cas
secondaire
Début des signes cliniquesContamination secondaire d’une
personne résidant en Martinique
Risk of introduction and dissemination of
Chikungunya virus
Chikungunya no continente americano:
Até 15/08/2014: 585.798 casos, 37 fatais
ChikungunyaFever in Brazil
More than 600 suspect cases
~60 importaed cases
The majority of notified cases were people coming from Haiti and
diagnosed in São Paulo State;
Cases were also reported in AM, PA, RR, CE, PE, MG, GO, DF, PR
and RS;
41 autocthonous cases
15 cases from Oiapoque (Amapá State)
26 cases from Feira de Santana (Bahia State)
23 under investigation (16 BA; 4 RR; 3 PA)
Transmission by the Aedes aegypti mosquitoes;
All cases were classic Chikungunya fever;
The nucleotide sequencing followed by phylogeny of two imported cases and
another of local transmission showed the Asian genotype was responsible for
transmission in Brazil; the same genotype occurring in the Caribbean region.
Chikungunya Virus
Diagnostic Assays
DAYS POST ONSET
1 2 3 4 5 6 7 8 9 10-14 to -2 0
IgM
IgG
ELISA
P/N#pfu/ml106
Simplified Depiction of CHIK Human Viremia & Immune Response
2
20
Neutralizing Ab
CHIKviremia
Current Laboratory Testing Strategy
for CHIKV and other Arboviruses• Human Infection
– Acute antibody (IgM) in serum and/or csf.
• IgM ELISA or Microsphere Immunoassay
• Confirmation by PRNT
– Seroconversion in paired specimens by
• IgG ELISA, HI and/or 4-fold rise in neutralizing antibody by PRNT
– Detection of virus and/or viral RNA in serum and/or csf.
• Real time RT-PCR, Consensus RT-PCR, or virus isolation
• Environmental Surveillance
– Detection of virus and/or viral RNA in mosquito vectors or amplifying hosts.
• Real time RT-PCR, Consensus RT-PCR, or virus isolation
IgM Cross-Reactivity of Human CHIK
Cases With Other Alphaviruses
CHIK IgM Capture ELISA (P/N)
CHIK RR ONN VEE MAY EEE
12.3 1.5 15.3 0.89 1.9 1.2
10.6 1.2 13.9 1.1 1.9 1.3
14.5 1.7 17.3 1.1 3.1 0.89
20.4 0.92 20.7 1.5 7.1 1.8
26.6 4.9 27.4 2.2 1.7 1.5
21.2 1.5 24.7 2.2 1.2 1.6
15.3 1.5 8 1.6 1.9 2.2
31.3 1.5 24.6 1.7 2.9 1.8
22 1.6 15.9 1.8 1.4 1.4
18.8 0.59 13.1 1.2 1.5 1.1
34.3 3.7 22.6 1.6 2.8 9.1
CDC CHIK Real-Time RT-PCR Assay
• Developed 4 primer/probe sets
• Detects all CHIK genotypes (WA, SCEA, & Asia)
• Analytical sensitivity; 25 copies/reaction
Genetic Lineages of Chikungunya Virus
• Chikungunya virus is within the Semliki Forest Virus complex.
• Chikungunya virus is genetically classified into 3 clusters;
– Asian, West African, and Central/South/East African.
– 2006 Indian Ocean epidemic caused by CHIK virus from the CSEA cluster.
- 2014 American continent epidemic is caused by CHIKV Asian genotype
Serological Testing Algorithm
for Chikungunya Virus Infection
single acute
patient serum
IgM Capture ELISA
IgG ELISA
RT-PCR (<10day)
IgM POS
PRNT
IgM NEG
(<10day)
No
Interpretation
IgM NEG
(>10day)
NEG
RT-PCR or
Isolation POS
POSPOS
Environmental Testing Algorithm
for Chikungunya Virus Detection
Mosquito
pool
Real-time RT-PCR
Consensus RT-PCR &Nucleic acid sequencing
Virus isolation
CHIK Case Definition
• Laboratory evidence of an acute CHIKV
infection
1. Detection of CHIK viral RNA or virus, or
2. Detection of anti-CHIKV IgM antibodies (Ab), or
3. Four-fold increase in neutralizing Ab titers
between acute and convalescent samples
Distribuição dos Casos Importados de Chikungunya por Estado no Brasil segundo a Procedência
1 12
15
2 2
41
1
1
1
1
1
Amapá
Ceará
Distrito Federal
Góias
Maranhão
Pará
Paraná
São Paulo
Rio de Janeiro
Rio G
rande do Sul
0
2
4
6
8
10
12
14
16
Haiti
Rep. Dominicana
Guiana Francesa
Guadalupe
Comments• Chikungunya virus represents a serious concern to
Brazil and other dengue endemic countries due to:
– Presence of both vectors in high density levels
– Completely naïve population to CHIKV living in country
– Increase in the international travel to/from the CHIKV endemic/epidemic countries
– Disease is completely unknown to Brazilian physicians
– Several diseases, mainly viral, mimic CHIK fever
– Limited laboratory capacity to confirm CHIKV infection
CHIK GUIDELINES LINKS
• Guidelines for Prevention and Control of Chikungunya fever (SEARO/WHO)
http://www.searo.who.int/LinkFiles/Chikungunya_SEA-CD-182.pdf
• Guidelines on Clinical Management of Chikungunya fever (SEARO/WHO)
http://www.searo.who.int/LinkFiles/Publication_guidelines_on_cli_mgmt_chikungunya_fvr-(cd-180).pdf
• Mission Report, Italy CHIK Outbreak. Joint ECDC/WHO visit for a European risk assessment
http://www.afpmb.org/bulletin/vol27/071020_CHK_report.pdf
• Programme de surveillance, d’alerte et de gestion du risque d’émergence du virus Chikungunya dans les départements français d’Amérique
http://www.martinique.sante.gouv.fr/documents/accueil/cire/chikungunya/prog_action_anti_chik.pdf
Acknowledgements
CDC PAHO
Ann Powers Otávio Oliva
Barbara Johnson
Roger Nasci IEC
Rob Lanciotti Sueli G. Rodrigues
Guajará Bay and Ver-o-peso Market, Belém
IV Simpósio Pan Americano de Pesquisa em Dengue, Belém, Pará, Brasil,
19-23/10/2014
INSTITUTO EVANDRO CHAGAS1936-2011
New Campus of Instituto Evandro Chagas in Ananindeua/Pará.
Arbovirology and Hemorrhagic FeversINSTITUTO EVANDRO CHAGAS1936-2011
Arbovirology and Hemorrhagic FeversINSTITUTO EVANDRO CHAGAS1936-2011
Arbovirology and Hemorrhagic FeversINSTITUTO EVANDRO CHAGAS1936-2011
Arbovirology and Hemorrhagic Fevers
New Campus of Instituto Evandro Chagas in Ananindeua/Pará.
INSTITUTO EVANDRO CHAGAS1936-2011
Arbovirology and Hemorrhagic Fevers
New Campus of Instituto Evandro Chagas in Ananindeua/Pará.
INSTITUTO EVANDRO CHAGAS1936-2011
Arbovirology and Hemorrhagic Fevers
New Campus of Instituto Evandro Chagas in Ananindeua/Pará.
INSTITUTO EVANDRO CHAGAS1936-2011
Arbovirology and Hemorrhagic Fevers
New Campus of Instituto Evandro Chagas in Ananindeua/Pará.
INSTITUTO EVANDRO CHAGAS1936-2011
Arbovirology and Hemorrhagic Fevers
www.iec.pa.gov.br
Obrigado!
New Campus of Instituto Evandro Chagas in Ananindeua/Pará.
INSTITUTO EVANDRO CHAGAS1936-2011
Arbovirology and Hemorrhagic Fevers
New Campus of Instituto Evandro Chagas in Ananindeua/Pará.
INSTITUTO EVANDRO CHAGAS1936-2012
Arbovirology and Hemorrhagic Fevers
Top Related