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

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

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

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

Viral Haemorrhagic Fevers (Lassa, Junin, Machupo, Guanarito) 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 Nairovirus Crimean-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 +++ .Among all the hemmorrhagic fevers, hemorrhages are the smaller part of the iceberg “  Symptoms ” In fact, those diseases are very frequently asymptomatic : either because we are faced with mild infections showing only non hemorrhagic symptoms, or because it is a really asymptomatic infection. I shall give you soon the example of what we have shown in Ebola 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 Triton X100

S M L N G1 G2 10 nm Nairovirus structure

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 – Toxicosis Septicemia – Leptospirosis 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 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 Day of illness PCR + Virus + PCR + Virus - PCR - Virus + PCR - virus - Ab + Total tested 3 1 4 2 5 6 8 15 7 9 10 11 12 13 14 16 18 Total 34 22 65 80 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 day of illness PCR + Virus + PCR + Virus - PCR - Virus + PCR - virus - IFA Ab + Total tested 3 1 4 5 6 2 7 8 9 10 11 12 Total 14 19 26 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 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 2 Grèce Sénégal Mauritanie Iran Madagascar Chine Rép. Centrafr. Burkina Faso ORIGINE PROFIL RFLP (100) (96) (99) (84) (57) Rapport IP Dakar 1993

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

Seven major genetic groups. 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

BSL 3 BSL 4 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