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Crimean-Congo Haemorrhagic Fever DIAGNOSIS

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

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  1. Crimean-Congo Haemorrhagic Fever DIAGNOSIS Hervé Zeller National Reference Center - WHO Collaborating Centre for Arboviruses and Viral Haemorrhagic Fevers, Institut Pasteur, Lyon

  2. Flaviviridae (dengue, yellow fever, Groupe TBE) Viral Haemorrhagic Fevers

  3. Flaviviridae (dengue, yellow fever, Groupe TBE) Viral Haemorrhagic Fevers Arenaviridae (Lassa, Junin, Machupo, Guanarito)

  4. Flaviviridae (dengue, yellow fever, Groupe TBE) Viral Haemorrhagic Fevers Arenaviridae (Lassa, Junin, Machupo, Guanarito) Filoviridae (Ebola, Marburg)

  5. Flaviviridae (dengue, yellow fever, Groupe TBE) Viral Haemorrhagic Fevers Arenaviridae (Lassa, Junin, Machupo, Guanarito) Envelopped RNA viruses Bunyaviridae (CCHF, RVF, Hantaviruses) Filoviridae (Ebola, Marburg)

  6. 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

  7. Hemorrhages are inconstant : Emerging part of the iceberg …Most frequently asymptomatic infections +++

  8. 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

  9. 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

  10. Nairovirus structure G1 M S N G2 L L 10 nm

  11. CCHFNairovirus genome _____________________________________________ Segment Nucleotides Amino acids Protein _____________________________________________ S 1659-1712 442-482 N M 4888 1551 G1 G2 NSm? L 12255 4036 L? _____________________________________________

  12. VHF SUSPECT CASE

  13. VHF SUSPECT CASE Malaria – Hepatitis – Typhoidis – Toxicosis Septicemia – Leptospirosis Rickettsiosis…

  14. VHF SUSPECT CASE Malaria – Hepatitis – Typhoidis – Toxicosis Septicemia – Leptospirosis Rickettsiosis… Epidemiological data, risk evaluation biological analysis, differential diagnostic

  15. VHF SUSPECT CASE Malaria – Hepatitis – Typhoidis – Toxicosis Septicemia – Leptospirosis Rickettsiosis… Epidemiological data, risk evaluation biological analysis, differential diagnostic Contact between clinicians and biologists

  16. CCHF : laboratory data LEUCOPENIA, particularly neutropenia THROMBOCYTOPENIA Hematocrite increases early then falls down ASL, AST levels increases Proteinuria and hematuria Mild azotemia, bilirubine increase

  17. CCHF : laboratory diagnosis Viral detection: blood specimen • RT-PCR (nested) • Cell culture (Vero E6 cells)

  18. 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

  19. 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

  20. 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…

  21. Primers for RT-PCR on S segment From J. Smith, 1990

  22. RT-PCR /Southern blot hybridization/antibody : retrospective study From Burt et al, J Virol Methods 1998, 70:129-137

  23. 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

  24. Hyalomma sp. ticks RT-PCR Viral isolation

  25. 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

  26. Turkey 2003 Phylogenetic analysis of 46 partial sequences (219 bp) of the S segment of CCHF virus

  27. 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

  28. 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

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