Crimean-Congo Haemorrhagic Fever
DIAGNOSIS
Hervé ZellerNational Reference Center - WHO Collaborating Centre for Arboviruses and Viral Haemorrhagic Fevers, Institut Pasteur, Lyon
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
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
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 dataCCHF : laboratory data
LEUCOPENIA, particularly neutropenia
THROMBOCYTOPENIA
Hematocrite increases early then falls down
ASL, AST levels increases
Proteinuria and hematuria
Mild azotemia, bilirubine increase
Viral detection: blood specimen– RT-PCR (nested)– Cell culture (Vero E6 cells)
CCHF : laboratory diagnosisCCHF : 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 diagnosisCCHF : 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
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
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