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 SAMBRIvittorio.sambri@unibo.it

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ENIVD-TropNet Dengue Working Group; unpublished results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