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Chikungunya e dengue: aspetti diagnostici
Alma Mater Studiorum – Università Di Bologna
Dept Hematology & Oncology “L. e A. Seragnoli”Unit of Microbiology
Regional Reference Center for Emerging Infections - CRREM
“S.Orsola-Malpighi” University Hospital Bologna - Italy
VITTORIO [email protected]
Chikungunya• Spherical virions, about 70 nm diameter with
envelope• Nucleocapsid icosaedric (40nm, nucleoprotein C).• Envelope: spikes (glycoproteins gpE1 e gpE2)• ssRNA (+), fully sequenced
Chikv_Usuv_Vs_220710
Chikungunya
‘Chikungunya’ is a Makonde word meaning ‘that which bends up’ and refers to the contorted posture of infected patients suffering from severe joint pain
Chikv_Usuv_Vs_220710
•Chikv inoculated by infected mosquito•Asymptomatic period (2-12 dd)•Acute clinical onset:
•Flu-like symptoms (fever, chills, headache, vomiting)•Joints pain•Mialgia•Cutaneous rash
•7 days: recovery•Persistent joints pain – arthralgias•No vaccine or specific therapy•
Pathogenesis of Chikv
Chikv_Usuv_Vs_220710
Log
RNA
cop
ies/
mL
Viremia
IgMIgG
Days after symptoms onset0 6
Symptoms
- 3/7
10
5
3
Pathogenesis of Chikv
Chikv_Usuv_Vs_220710
Log
RNA
cop
ies/
mL
Viremia
IgMIgG
Days after symptoms onset0 6
Symptoms
- 3/7
10
5
3
Laboratory diagnosis of Chikv
Real time RT PCR EIA /IFA IgM
Virus isolation EIA /IFA IgG
ENIVD 09 / 10
Dengue and dengue haemorrhagic fever emergence
100.000.000 cases/year
250.000 cases DHF
Mortality 2,5-5%
ENIVD 09 / 10
Dengue cases registered by DengueNet
854,452
2002-2006
ENIVD 09 / 10
Imported dengue in Europe
ENIVD-TropNet Dengue Working Group; unpublished results
DENV1 DENV1
DENV2
DENV1 DENV2 DENV3 DENV4
DENV1 DENV2 DENV3
DENV1 DENV2
DENV2 DENV3 DENV1
DENV3
DENV2 DENV4
DENV3 DENV2
Dengue infection
Dengue fever lasts 3 to 9 days, is self-limiting, and is rarely associated with serious sequelae.
Clinical features: •sudden onset of systemic toxicity,
• fever, •headache, vomiting, severe myalgia
•bone pain •maculopapular or morbilliform rash on the trunk
• recrudescence of fever • lymphadenopathy•granulocytopenia• thrombocytopenia.
Dengue hemorrhagic fever
Clinical features:• fever, rash, and anorexia (3 to 5 days)
Followed by a shock phase with
•hepatomegaly•hypotension, •hemorrhagic diathesis•complement activation • thrombocytopenia•diffuse capillary leak•hemoconcentration, t•hrombocytopenia, •disseminated intravascular coagulation.
Dengue primary and secondary infection
“Origin antigenic sin”
Virus Isolation
Time of collection after onset of symptom: 1-5 days
Specimen: serum, plasma, PBMC and autopsy tissues
(liver, lung, lymph nodes, thymus, bone marrow)
Storage of specimen: 4/8°C for 24h, -80°C for longer periods
Cell culture: • mosquito cell lines C6/36 (Ae. albopictus), AP61 (Ae. pseudoscutellaris)
• mammalian cell lines(Vero, LLCMK2, BHK21)
Confirmation of viral: isolation by IFA or PCR
Time to result: 1-2 weeks
Clinical specimens may also beinoculated by intracranial route in suckling mice or intrathoracicinoculation of mosquitos
Molecular diagnosis
1 2 3 N P 1 2 3 N P
3 N 3 N 4 N 2 4 4 N 2 4
Domingo et al., Dengue Bulletin 2004
13
PScaramozzino et al., JCM 2001
Pan Flaviviruses RT-PCR (Target: NS5)
Specific nested RT-PCR DENV (1-2-3-4 types) (Target: E/NS1 )
I II
I III II
DENV-1 DENV-2
Modified from Gurukumar et al., J Virol 2009
Real-time RT-PCR (Target: 3’UTR)
Haemoagglutination-inhibition test (HI)
acute-serum
convalescent serum
Limitscross-reactivity with other circulating flavivirusesNo discrimination between two immunoglobulin isotypes
Time of collection after onset of symptom: 5 days or moreSpecimen: serum, blood on filter paper, saliva (not urine)Antigen: envelope proteinFalse positives: malaria, leptospirosis and past dengue infectionCross-reactivity with JEV, SLEV and WNV no major problem It is not possible to identify dengue serotypes…
IgM antibody-capture enzyme-linked immunoassorbent assay (MAC-ELISA)
Time of collection: acute-sera 1-5 days, convalescent after 15 days
Specimen: plasma, serum, blood on filter paper
Antigen: E/M antigen
Cross-reactivity within flaviviruses serocomplex group
Identification of primary or secondary infection
IgG antibody-capture enzyme-linked immunoassorbent assay (GAC-ELISA)
Distinction between primary and secondary dengue virus infection
Primary dengue infection: IgM/IgG OD ratio is greater than 1.2 (using patient’s sera at 1/100 dilution) or 1.4 (using patient’s sera at 1/20 dilutions)Secondary infection: : IgM/IgG OD ratio is less than 1.2 or 1.4
IgM/IgG ratio
OD ratio >1.2-1.4 OD ratio <1.2-1.4
Immunofluorescence Assay (IFA)
Mammalian cell lines VeroE6
Attention!False positives: cross-
reactivity with othercirculating flaviviruses or YFV17D strain vaccine
Infected cells on slides
Microneutralization ASSAY
Add Sera2-fold dilutions
100 TCID50/well
Add Virus
18H 37C
Add susceptible Cells
1-2H 37C
- - - -+ + + Ctr
ls
CPE
Unsperger et al. Emerg Infect Dis. 2009
Pierro et al., Clinical Microbiology and Infection, 2011. In press.
“Luck generally helps prepared minds”
Louis Pasteur (1822-1895)
CRREM•Francesca Cavrini•Paolo Gaibani•Anna Pierro•Giada Rossini
•Maria Paola Landini•Matthias Niedrig & Maria Rosaria Capobianchi