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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
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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 Bunyaviridae (CCHF, RVF, Hantaviruses) Filoviridae (Ebola, Marburg)
Family Genus VIRUS DISTRIBUTION Flaviviridae Flavivirus Yellow Fever Africa South America Dengue 1,2,3,4. Tropical areas Omsk HF Russia Alkhurma Saudi Arabia Kyasanur Forest HF India Bunyaviridae Phlebovirus Rift Valley Fever Africa, Saudi Arabia NairovirusCrimean-Congo HF Africa, Eurasia Hantavirus Hantan Dobrava Puumala Eurasia Sin Nombre, Andes Americas
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 • Triton X100
Nairovirus structure G1 M S N G2 L L 10 nm
CCHFNairovirus 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 – 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 IFA IgM duration: 2-3 months up to 6 months…
Primers for RT-PCR on S segment From J. Smith, 1990
RT-PCR /Southern blot hybridization/antibody : retrospective study From Burt et al, J Virol Methods 1998, 70:129-137
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
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 ORIGINE DUGBE RFLP Grèce AP 92 3 2 1 AnD 15786 1 1 1 Sénégal (100) Sénégal (100) ArD 8194 1 3 1 ArTeh 193-3 Iran 1 3 1 (96) HD 49199 1 3 1 Mauritanie ArMg 951 1 2 3 (57) Madagascar (84) Chine C 68031 2 2 4 Rép. Centrafr. ArB 604 2 2 3 (100) (99) Burkina Faso HD 38562 2 2 3 Mauritanie ArD 39554 2 2 2 (100) Sénégal 2 2 2 ArD 97264 (100) 2 2 2 Sénégal ArD 97268 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