Viral Hemorrhagic Fevers Istanbul, 27-28 June 2008 Hervé Zeller Unit of Biology of Emerging Viral infections, National Reference and WHO Collaborating Centre for Viral Hemorrhagic Fevers, Institut Pasteur, Lyon, France [email protected]Session 7. The etiological agents and laboratory diagnosis Conventional diagnostic methods
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Viral Hemorrhagic Fevers Istanbul, 27-28 June 2008
Hervé Zeller
Unit of Biology of Emerging Viral infections,National Reference and WHO Collaborating Centre for Viral Hemorrhagic Fevers,
VACCINATIONS : Fièvre jaune (YF) non oui Date : ___/___/____ Encéphalite japonaise (JE) non oui Date : ___/___/____ Encéphalite à tique (TBE) non oui Date : ___/___/____ Hépatite A non oui Date : ___/___/____ Hépatite B non oui Date : ___/___/____ Typhoïde non oui Date : ___/___/____
Viral Identification : •Conventional RT-PCR (one round or nested/semi-nested) PCR•Real-time PCR•Antigen capture : •Isolation : long process and biosafety issues
Serology :Never a confirmatory assay but sometimes the unique way to be used Inactivation of specimens
Delay : variable according to techniques : few hours to several days
DIAGNOSIS OF VHF
Laboratory regulations
Virus Biosafety classification
Dengue 3 2 (USA,… )Yellow fever 3Crimean-Congo 4 3 in some countriesRift valley fever 3Omsk, Kyasanur forest 3 4 (USA, Canada)
CITERIA Severity of diseaseTransmission to laboratory techniciansAvailability of treatment / vaccine
BSL 4
BSL 3
Viral detection techniques
Direct detectionviral genomeantigenviral particleisolation
Indirect detectionImmune response
Antigen capture
RT-PCR
IgG
IgM
Isolation
Serology :ELISA IgM IgG, IFA…
Onset ofdisease
80 4 1612
Viremia Dengue, Yellow fever, Rift
Viremia Arenavirus, Filovirus, Crimean-Congo
days
Detection, Identification, Quantification
1. Cell culture
2. Immunochemistry
3. Molcular Biology
DIRECT DETECTION
RT-PCR
DNA c elongation
RNA to amplify
ONE-STEP RT-PCRThe one-step procedure performs first-strand cDNA synthesis and PCR in one tube using RNA specific primers.
STANDARD TWO-STEP RT-PCRIn the two-step procedure, first-strand synthesis is performed using oligo(dT) or random primers. A small amount of cDNA is then used in subsequent PCR.
MODIFIED TWO-STEP RT-PCRIn the modified two-step procedure, specific PCR primers and enzymes are added directly to the first-strand cDNA synthesis reaction for amplification by PCR.
Real-Time PCR
• Advantages:
– rapid response : avoid some biosafety issues...
– quantitative approach: • follow –up of virus load
• treatment monitoring (ribavirin for some viruses :
side effects : hemolysis, teratogenicity, rigor)
– Epidemiological studies
• Difficulties
– adequate primers
– Inhibitors
– Contimination (nested… .)
Molecular diagnosis
Support
Viral Ag
Signal
Chromogenic Substrate
Antigen detection
peroxydase/phosphatase binded Specific Ab
Antigen capture with monoclonal antibodies immunohistochiemistry on organs…
Necessity of specific antibodies
ELISA: Antigen Capture
to identify
Exemple : Ag NS1 dengue�
Other formats : strip rapid test
Isolation = GOLD STANDARD technique
VIRAL ISOLATION
Viral Culture = amplification of potentially infectious pathogens
Allows a complete identification of the agent and further studies en terms of pathogenicity, antiviral sensitivity,
Contraints : laboratory/animal facility of biosafety 3 or 4
Use :in primary intention ? NO In combination when unknown cause: YES
From J Smith, 1990 ; Rodriguez et al, AJTMH 1997 , 57:512
Sensibility : 10-5
CT : 37,25
Linearity : 10-3
Coefficient : 3.75 Efficiency : 80-90 %
CCHF v irus NP gene RT-PCR
y = 3,754x + 18,049
15,00
17,00
19,00
21,00
23,00
25,00
27,00
29,00
31,00
0 1 2 3 4
Dilutions
CT
Gamme ARN fp6
Linéaire (Gamme ARN fp6)
CCHF Virus NP gene RT-PCR
y = -3,1521x + 28,961
15,00
20,00
25,00
30,00
35,00
40,00
-3 -2 -1 0 1 2 3 4
log de concentration
CT
Gamme ARN CCHF IbAn
Linéaire (Gamme ARNCCHF IbAn)
Sensibility : 0,01 pfu/ml
CT : 35,08
Linearity : 0,01 pfu/ml
Coefficient : -3,15 Efficiency : 90-100 %
qPCR : idem or more sensitive than conventional PCR
(for conventional PCR : no assays under de 0,1 pfu/ml)
ARN FP6 ARN IbAn 10200
CCHF qPCR evaluation
Phylogenetic analysis of 46 partial sequences (219 bp) of the S segment of CCHF virusJ Clin Microbiol 2002, 40 1122
W. AFRICAIRAN
GREECE
UA EMIRATESPAKISTAN
CHINAKAZAKSTAN
southern RUSSIAKOSOVO
C S AFRICA
W C S AFRICAUA EMIRATES
MADAGASCAR
Gambia Belgium, Nov 2001 = suspicion Yellow fever
• female Belgian patient, 47 y.o• hospitalized on day 3 of fever• AST 48,620 U/l, ALT 22,500 U/l on admission• hepatic encepalopathy/hepatorenal syndrome• severe generalized hemorrhage• death on day 7
Diagnosis : neg within 4 hours (spiked inhibition control failed)
• Pre-dilution of patient plasma in negative plasma• Re-extraction/retesting
– 4.00 h: YFV-PCR + / Universal flavivirus PCR +
• Inoculation on Vero/and C6- 36 cells: positive direct IFA after 3 days (one day after patient deceased)
Pitfalls in molecular diagnostics
Source: C Drosten
ArenaviridaeArenaviridae
Arenaviruses associated with human disease
Virus Origin of Name Year DistributionLassa Town, Nigeria 1969 West Africa
Junin Town, Argentina 1957 South AmericaMachupo river, Bolivia 1962 South AmericaGuanarito area, Venezuela 1989 South AmericaSabia Town, Brazil 1990 South AmericaChapare Bolivia 2004 South America
LCMV Clinical disease 1933 Worldwide
ArenavirusArenavirusImage source: C.S. Goldsmith and M. Bowen (CDC).
Phylogenic sequences 302-nucleotide fragments of MBG VP35 gene
from 12 bats in Durba Mine
from human patients in the Durba
from previous outbreaks of the disease (DRC = Democratic Republic of the Congo; GER = Germany; KEN = Kenya; ZIM = Zimbabwe).
Swanepoel et al, EID Dec 2007
•Conventional PCR Filo A-B:•Gabon 2001:
» Sensitivity RT : 50 pfu/ml
» Sensitivity Nested : 5 10-2 pfu/ml
•Gulu : » Sensitivity RT : 1,5 102 pfu/ml
» Sensitivity Nested : 1,5 pfu/ml
•Musoke : » Sensitivity : 2,5 pfu/ml
» Sensitivity : 2,5 pfu/ml
Ebola / Marburg L Gene
Sensitivity : 10-4
CT : 28,66
Linearity : 10-4
Coefficient : 3,36 Efficiency : 100 %
Marburg virus GP gene
y = 3,363x + 15,905
15,00
17,00
19,00
21,00
23,00
25,00
27,00
29,00
31,00
0 1 2 3 4
Dilutions
CT
Gamme ARN fp6 Dilué
Linéaire (Gamme ARNfp6 Dilué)
Sensitivity : 2,5 103 pfu/ml
CT : 36,53
Linearity : 2,5 103 pfu/ml
Coefficient : -3 Efficiency : 90 %
Marburg virus GP gene
y = -3,042x + 46,929
15,00
20,00
25,00
30,00
35,00
40,00
2 3 4 5 6 7 8
Log de concentration
CT
Gamme ARN Dilué
Linéaire (Gamme ARN Dilué)
ARN FP6 ARN Musoke
qPCR, is a good technique for detection but still less sensitive than the conventional PCR Filo A-B (sensitivity Nested 2,5 pfu/ml)
Marburg qPCR(FP6 project)
A man, 26 years-old, was admitted in Lubutu hospital, (North Kivu) on February 18, 2008.
He reported some activity linked to a mine in Bisie 25 kms from his village Biruwe (Walikale area, North Kivu province).
Onset of disease in February 14 with fever and headache.
On day2, he presented a diarrhea with hemorrhages, on day 3 epistaxis and important gum bleeding and hematuria and on day 4 strong asthenia.
He got a Malaria treatment for one day (Quinine) and traditional medication
He was seen to two primary health care units before arriving at the hospital on day 5 in motorbike driven by his friend.
Palliative treatment was instaured ans symptoms disappeared progressively.
Whole blood specimen taken on Feb 18, 2008
A RECENT CASE
Kinshasa
Lyon ß Brussels
Lubutu
TRANSPORTATION ISSUES
Specific measures were taken :
Increase of standard procedures for the entire medical staff, and training of care nurses with personal form Kisangani in the hospital and awarness in the
primary heaklth care units
Isolation of the patient in a dedicated area.
His friend who was attending him from the begining and the motobike driver were kept in isolation in the hospital
Contact tracing in the villages.....
MSF
MEANWHILE LOCALLY
BIOLOGICAL DIAGNOSIS
PCR Ebola /MarburgCCHFYFDENLASSA
Ag capture :
Ebola/MarburgCCHF
Serology IgM/IgG
Cell cultivation :
BIOLOGICAL DIAGNOSIS
Transportation: one week at RT : Lumbutu à Kisangani à Kinshasa à Brussel à Lyon