Crimean-Congo Haemorrhagic Fever

DIAGNOSIS

Hervé ZellerNational Reference Center - WHO Collaborating Centre for Arboviruses and Viral Haemorrhagic Fevers, Institut Pasteur, Lyon

Viral Haemorrhagic Fevers

Flaviviridae(dengue, yellow fever,

Groupe TBE)

Arenaviridae(Lassa, Junin, Machupo, Guanarito)

Viral Haemorrhagic Fevers

Flaviviridae(dengue, yellow fever,

Groupe TBE)

Filoviridae(Ebola, Marburg)

Arenaviridae(Lassa, Junin, Machupo, Guanarito)

Viral Haemorrhagic Fevers

Flaviviridae(dengue, yellow fever,

Groupe TBE)

Filoviridae(Ebola, Marburg)

Arenaviridae(Lassa, Junin, Machupo, Guanarito)

Bunyaviridae(CCHF, RVF,

Hantaviruses)

Viral Haemorrhagic Fevers

Flaviviridae(dengue, yellow fever,

Groupe TBE)

EnveloppedRNA viruses

Family Genus VIRUS DISTRIBUTION

Flaviviridae Flavivirus Yellow Fever Africa South AmericaDengue 1,2,3,4. Tropical areas

Omsk HF Russia Alkhurma Saudi Arabia

Kyasanur Forest HF India

Bunyaviridae Phlebovirus Rift Valley Fever Africa, Saudi Arabia

Nairovirus Crimean-Congo HF Africa, Eurasia

Hantavirus Hantan Dobrava Puumala Eurasia Sin Nombre, Andes Americas

Hemorrhages are inconstant :

Emerging part of the iceberg

…Most frequentlyasymptomatic infections +

++

Biosafety Issues Related to Haemorrhagic Fever Viruses

• Criteria for classification– Disease severity– Transmissibility to laboratory workers– Availability of treatment– Availability of vaccine

• Classification BSL 1 to BSL4

CCHF VIABILITY

SENSITIVITY TO DESINFECTANTS:

sodium hypochlorite 2%, glutaraldehyde 2%, formaldehyde

SENSITIVE TO DESSICATION

INACTIVATION : IRRADIATIONUVTEMPERATURE : 1 hour 60°C

not complete inactivationbeta propiolactone 4°C

not complete inactivation

Triton X100

S M

L

N

G1

G2

10 nm

Nairovirus structure

L

CCHF Nairovirus genome _____________________________________________

Segment Nucleotides Amino acids Protein_____________________________________________

S 1659-1712 442-482 N

M 4888 1551 G1 G2 NSm?

L 12255 4036 L?_____________________________________________

VHF SUSPECT CASE

VHF SUSPECT CASE

Malaria – Hepatitis – Typhoidis – ToxicosisSepticemia – Leptospirosis

Rickettsiosis… 

VHF SUSPECT CASE

Epidemiological data, risk evaluationbiological analysis, differential diagnostic

Malaria – Hepatitis – Typhoidis – ToxicosisSepticemia – Leptospirosis

Rickettsiosis… 

VHF SUSPECT CASE

Contact between clinicians and biologists

Epidemiological data, risk evaluationbiological analysis, differential diagnostic

Malaria – Hepatitis – Typhoidis – ToxicosisSepticemia – Leptospirosis

Rickettsiosis… 

CCHF : laboratory data

LEUCOPENIA, particularly neutropeniaTHROMBOCYTOPENIA

Hematocrite increases early then falls down

ASL, AST levels increases

Proteinuria and hematuriaMild azotemia, bilirubine increase

Viral detection: blood specimen– RT-PCR (nested)– Cell culture (Vero E6 cells)

CCHF : laboratory diagnosis

Viral detection: (blood specimen)– RT-PCR (nested)– Cell culture (Vero E6 cells)

Antibody detection : (serum sample)- IFA - ELISA IgM (immuno-capture) IgG- NT

CCHF : laboratory diagnosis

CCHF : laboratory diagnosis

Viremia 10-12 days (although afebrile). Can be detected by PCR up to day 16

By day 9 all patients will have IgM or IgG antibody

Information needed : DATE OF ONSET OF FEVER

viremia

5

IgM

RT-PCR

ELISA IgM IgG IFA

CCHF : viral/antibody kinetics

Viral isolation

0 10

IgG

16

IgM duration: 2-3 months up to 6 months…

Primers for RT-PCR on S segment

100 300 500 700

3'5'

F2 R3

Fragments amplifiés: F2 / R3 (536 bp) , F3/R2 (259 bp)

Séquences des amorces: F2 5' TGG ACA CCT TCA CCA AAC TC 3' R3 5' GAC AAA TTC CCT GCA CCA 3' F3 5' GAA TGT GCA TGG GTT AGC TC 3' R2 5' GAC ATC TTC CCT GCA CCA 3'

670135

Segment S ARN CCHF

F3 R2

290 550

From J. Smith, 1990

RT-PCR /Southern blot hybridization/antibody : retrospective study

From Burt et al,J Virol Methods 1998, 70:129-137

Day of illnessPCR + Virus +

PCR + Virus -

PCR - Virus +

PCR - virus - Ab +

Total tested

3 1 1 1 3

4 1 1 1 2

5 3 2 3 5

6 5 3 4 3 8 15

7 5 4 8 9

8 1 2 4 7 7

9 5 1 5 6

10 3 3 3

11 1 2 1 1 5 5

12 1 1 2 2

13 1 4 3 8 8

14 4 1 3 8 8

15 2 1 3 3

16 2 1 3 3

18 1 1 1

Total 18 34 6 22 65 80

RT-PCR /Southern blot hybridization/IFA antibody : 26 samples from 19 patients from day 3-12 of illness

From : Burt et al, J Virol Methods 1998, 70:129-137

day of illness

PCR + Virus +

PCR + Virus -

PCR - Virus +

PCR - virus -

IFA Ab +

Total tested

3 1 1

4 1 1

5 1 1

6 4 1 2 5

7 3 1 1 5 5

8 6 1 2 8 9

9 2 2 2

10

11 1 1 1

12 1 1 1

Total 14 1 3 8 19 26

Hyalomma sp.

ticks

RT-PCRViral isolation

DUGBE

AP 92

AnD 15786

ArD 8194

ArTeh 193-3

HD 49199

ArMg 951

C 68031

ArB 604

HD 38562

ArD 39554

ArD 97264

ArD 97268

HAZARA

3 2 1

1 1 1

1 3 1

1 2 3

2 2 4

2 2 3

2 2 3

2 2 2

2 2 2

2 2 2

1 3 1

1 3 1

Grèce

Sénégal

Sénégal

Sénégal

Sénégal

Mauritanie

Mauritanie

Iran

Madagascar

Chine

Rép. Centrafr.

Burkina Faso

ORIGINEPROFIL RFLP

(100) (100)

(96)

(100)

(100)

(99)

(100)

(84)(57)

536 pb amplicons of the S fragment of CCHF genome using primers CSDR3/CSDF2. RFLP with Hinf I, Hae III, and Alu I endonucleases

Rapport IP Dakar 1993

Phylogenetic analysis of 46 partial sequences (219 bp) of the S segment of CCHF virus

Turkey 2003

Phylogenetic analysis of 46 partial sequences (219 bp) of the S segment of CCHF virus.

Seven major genetic groups.

Strains from the Middle and Far East and from different African regions cluster in clearly separated groups.

TURKEY 2003 Preliminary data: 96-98% homology with strains from the Balck Sea area and Kosovo

KOSOVO AF404507; STAVROPOL AF481802 ; DROSDOV U88412 ; ROSTOV AY277672

Drostein et al, J Clin Microbiol 2002, 40 1122

National Reference Center - WHO Collaborating Centre for Arboviruses and Viral Haemorrhagic Fevers, Institut Pasteur, Lyon

Marie-Claude Georges

Isabelle Schuffenecker

Ingrid Marendat

Séverine Murri

Hervé Zeller

BSL 4

BSL 3