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8/7/2019 congo fever
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Crimean-Congo Haemorrhagic Fever
DIAGNOSIS
Herv ZellerNational Reference Center - WHO Collaborating Centre for
Arboviruses and Viral Haemorrhagic Fevers, Institut Pasteur, Lyon
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Viral Haemorrhagic Fevers
Flaviviridae
(dengue, yellow fever,
Groupe TBE)
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Arenaviridae
(Lassa, Junin, Machupo, Guanarito)
Viral Haemorrhagic Fevers
Flaviviridae
(dengue, yellow fever,
Groupe TBE)
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Filoviridae
(Ebola, Marburg)
Arenaviridae
(Lassa, Junin, Machupo, Guanarito)
Viral Haemorrhagic Fevers
Flaviviridae
(dengue, yellow fever,
Groupe TBE)
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Filoviridae
(Ebola, Marburg)
Arenaviridae
(Lassa, Junin, Machupo, Guanarito)
Bunyaviridae
(CCHF, RVF,
Hantaviruses)
Viral Haemorrhagic Fevers
Flaviviridae
(dengue, yellow fever,
Groupe TBE)
Envelopped
RNA viruses
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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
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Hemorrhages are
inconstant :Emerging part of the
iceberg
Most frequentlyasymptomatic infections
+++
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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
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CCHF VIABILITY
SENSITIVITY TO DESINFECTANTS:
sodium hypochlorite 2%, glutaraldehyde 2%,
formaldehyde
SENSITIVE TO DESSICATION
INACTIVATION :
IRRADIATION
UV
TEMPERATURE : 1 hour 60C
not complete inactivation
beta propiolactone 4C
not complete
inactivation
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S M
L
N
G1
G2
10 nm
Nairovirus structure
L
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CCHF Nairovirus genome
_____________________________________________
Segment Nucleotides Amino acids Protein
_____________________________________________
S 1659-1712 442-482 N
M 4888 1551 G1G2 NSm?
L 12255 4036 L?
_____________________________________________
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VHF SUSPECT CASE
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VHF SUSPECT CASE
Malaria Hepatitis Typhoidis Toxicosis
Septicemia Leptospirosis
Rickettsiosis
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VHF SUSPECT CASE
Epidemiological data, risk evaluation
biological analysis, differential diagnostic
Malaria Hepatitis Typhoidis Toxicosis
Septicemia Leptospirosis
Rickettsiosis
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VHF SUSPECT CASE
Contact between clinicians and biologists
Epidemiological data, risk evaluation
biological analysis, differential diagnostic
Malaria Hepatitis Typhoidis Toxicosis
Septicemia Leptospirosis
Rickettsiosis
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CCHF : laboratory dataCCHF : laboratory dataLEUCOPENIA, particularly neutropenia
THROMBOCYTOPENIA
Hematocrite increases early then falls down
ASL, AST levels increases
Proteinuria and hematuria
Mild azotemia, bilirubine increase
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Viral detection: blood specimen
RT-PCR (nested)
Cell culture (Vero E6 cells)
CCHF : laboratory diagnosisCCHF : laboratory diagnosis
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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 diagnosisCCHF : laboratory diagnosis
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CCHF : laboratory diagnosisViremia 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
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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
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Primers for RT-PCR on S segment
100 300 500 700
3'5'
F2 R3
Fragments amplifis: F2 / R3 (536 bp) , F3/R2 (259 bp)
Squences 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
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RT-PCR /Southern blot hybridization/antibody : retrospective study
From Burt et al,
J Virol Methods
1998, 70:129-
137
Day of illness
PCR +
Virus +
PCR +
Virus -
PCR -
Virus +
PCR -
virus - Ab +
Total
tested
3 1 1 1 34 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 79 5 1 5 6
10 3 3 3
11 1 2 1 1 5 5
12 1 1 2 2
13 1 4 3 8 814 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
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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 ofillness
PCR +Virus +
PCR +Virus -
PCR -Virus +
PCR -virus -
IFAAb +
Totaltested
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
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Hyalomma sp.ticks
RT-PCR
Viral isolation
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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
Grce
Sngal
Sngal
Sngal
Sngal
Mauritanie
Mauritanie
Iran
Madagascar
Chine
Rp. 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 Dakar1993
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Phylogenetic analysis of 46 partial sequences
(219 bp) of the S segment of CCHF virus
Turkey 2003
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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
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National Reference Center - WHO Collaborating Centre for
Arboviruses and Viral Haemorrhagic Fevers, Institut Pasteur, Lyon
Marie-Claude Georges
Isabelle Schuffenecker
Ingrid Marendat
Sverine Murri
Herv Zeller
BSL 4
BSL 3