Crimean-Congo Haemorrhagic Fever
DIAGNOSIS
Hervé Zeller
National Reference Center - WHO Collaborating Centre for
Arboviruses and Viral Haemorrhagic Fevers, Institut Pasteur, Lyon
Flaviviridae
(dengue, yellow fever,
Groupe TBE)
Viral Haemorrhagic Fevers
Flaviviridae
(dengue, yellow fever,
Groupe TBE)
Viral Haemorrhagic Fevers
Arenaviridae
(Lassa, Junin, Machupo, Guanarito)
Flaviviridae
(dengue, yellow fever,
Groupe TBE)
Viral Haemorrhagic Fevers
Arenaviridae
(Lassa, Junin, Machupo, Guanarito)
Filoviridae
(Ebola, Marburg)
Flaviviridae
(dengue, yellow fever,
Groupe TBE)
Viral Haemorrhagic Fevers
Arenaviridae
(Lassa, Junin, Machupo, Guanarito)
Envelopped
RNA viruses
Filoviridae
(Ebola, Marburg)
Bunyaviridae
(CCHF, RVF,
Hantaviruses)
Family
Genus
VIRUS
DISTRIBUTION
Flaviviridae
Flavivirus
Yellow Fever
Dengue 1,2,3,4.
Africa South America
Tropical areas
Omsk HF
Alkhurma
Russia
Saudi Arabia
Kyasanur Forest HF
India
Phlebovirus
Rift Valley Fever
Africa, Saudi Arabia
Nairovirus
Crimean-Congo HF
Africa, Eurasia
Hantavirus
Hantan Dobrava Puumala Eurasia
Sin Nombre, Andes
Americas
Bunyaviridae
Hemorrhages are
inconstant :
Emerging part of the
iceberg
…Most frequently
asymptomatic 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 :
IRRADIATION
UV
TEMPERATURE : 1 hour 60°C
not complete inactivation
beta propiolactone 4°C
not complete
inactivation
Nairovirus structure
G1
N
G2
M
S
L
L
10 nm
CCHF Nairovirus genome
_____________________________________________
Segment Nucleotides Amino acids Protein
_____________________________________________
S
1659-1712
M
4888
442-482
N
1551
G1 G2 NSm?
L
12255
4036
L?
_____________________________________________
VHF SUSPECT CASE
VHF SUSPECT CASE
Malaria – Hepatitis – Typhoidis – Toxicosis
Septicemia – Leptospirosis
Rickettsiosis…
VHF SUSPECT CASE
Malaria – Hepatitis – Typhoidis – Toxicosis
Septicemia – Leptospirosis
Rickettsiosis…
Epidemiological data, risk evaluation
biological analysis, differential diagnostic
VHF SUSPECT CASE
Malaria – Hepatitis – Typhoidis – Toxicosis
Septicemia – Leptospirosis
Rickettsiosis…
Epidemiological data, risk evaluation
biological analysis, differential diagnostic
Contact between clinicians and biologists
CCHF : laboratory data
LEUCOPENIA, particularly neutropenia
THROMBOCYTOPENIA
Hematocrite increases early then falls down
ASL, AST levels increases
Proteinuria and hematuria
Mild azotemia, bilirubine increase
CCHF : laboratory diagnosis
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
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
CCHF : viral/antibody kinetics
IgM
IgG
viremia
0
5
10
RT-PCR
16
Viral isolation
ELISA IgM IgG
IgM duration: 2-3 months up to 6 months…
IFA
Primers for RT-PCR on S segment
Segment S ARN CCHF
100
300
500
700
5'
3'
135
290
550
F2
670
R3
F3
R2
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 A AA TTC CCT GCA CCA 3'
F3 5' GAA TGT GCA TGG GTT AG C TC 3'
R2 5' GAC ATC TTC CCT GCA CCA 3'
From J. Smith, 1990
RT-PCR /Southern blot hybridization/antibody : retrospective study
Day of illness
PCR +
Virus +
PCR +
Virus -
3
1
1
4
1
1
5
3
6
5
3
7
5
4
8
1
2
9
5
10
3
11
1
12
13
1
PCR Virus +
PCR virus -
Ab +
1
Total
tested
3
1
2
2
3
5
3
8
15
8
9
4
7
7
1
5
6
3
3
1
5
5
1
1
2
2
4
3
8
8
3
8
8
2
4
1
14
4
1
15
2
1
3
3
16
2
1
3
3
1
1
1
22
65
80
18
Total
18
34
6
From Burt et al,
J Virol Methods
1998, 70:129137
RT-PCR /Southern blot hybridization/IFA antibody :
26 samples from 19 patients from day 3-12 of illness
day of
illness
3
PCR +
Virus +
PCR +
Virus -
PCR Virus +
PCR virus -
IFA
Ab +
1
4
Total
tested
1
1
1
5
1
6
4
1
7
3
1
8
6
1
9
1
2
5
1
5
5
2
8
9
2
2
2
10
11
1
1
1
12
1
1
1
8
19
26
Total
14
1
3
From :
Burt et al, J Virol
Methods 1998,
70:129-137
Hyalomma sp.
ticks
RT-PCR
Viral isolation
536 pb amplicons of the S fragment of CCHF genome using primers
CSDR3/CSDF2. RFLP with Hinf I, Hae III, and Alu I endonucleases
PROFIL
RFLP
ORIGINE
321
Grèce
AnD 15786
111
Sénégal
ArD 8194
131
Sénégal
ArTeh 193-3
131
Iran
HD 49199
131
Mauritanie
ArMg 951
123
Madagascar
C 68031
224
Chine
ArB 604
223
Rép. Centrafr.
HD 38562
223
Burkina Faso
ArD 39554
222
Mauritanie
ArD 97264
222
Sénégal
ArD 97268
222
Sénégal
DUGBE
AP 92
(100)
(100)
(96)
(57)
(84)
(100)
(99)
(100)
(100)
HAZARA
Rapport IP Dakar 1993
Turkey 2003
Phylogenetic analysis of 46 partial sequences
(219 bp) of the S segment of CCHF virus
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 3
BSL 4