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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,
Institut Pasteur, Lyon, [email protected]
Session 7.
The etiological agents and laboratory diagnosis
Conventional diagnostic methods
Filoviridae(Ebola, Marburg)
Arenaviridae(Lassa, Junin,
Machupo, Guanarito)
MOLECULAR DIAGNOSTICS OF VIRAL HEMORRHAGIC FEVERS
Source: WHO
Bunyaviridae(Crimean-Congo Hemorrhagic
Fever, Rift valley fever)
Flaviviridae(Yellow fever, dengue, Alkhurma, Kyasanur,
Omsk...)
Family Genus VIRUS LOCATION
Flaviviridae Flavivirus Yellow Fever Africa S. AmericaDengue 1,2,3,4. Omsk HF, Alkhurma Russia, S. ArabiaKyasanur Forest Disease India
Bunyaviridae Phlebovirus Rift Valley Fever Africa S. Arabia
Nairovirus Crimean-Congo Hem. F. Africa, Eurasia
Hantavirus Hantan Dobrava Puumala EurasiaSin Nombre Andes Americas
Arenaviridae Arenavirus Lassa AfricaJunin ArgentinaMachupo/Chapare BoliviaGuanarito VenezuelaSabia Brasil
Filoviridae Filovirus Marburg AfricaEbola Africa
Niger
ABIDJANACCRA
OUAGUADOUGOU
LASSA fever
cases/year ? 100,000(s) ?
1-5% mortality rate18% of survivors with partial deafness
NigeriaGuinea
Liberia
SierraLeone
Mastomys natalensis
Asymptomatic infection
Mild febrile illnessFever, arthralgia, myalgia, asthenia
Hemorrhages :
Minor: gingivitis, epistaxis, purpura
Severe : melena, hematemesis
Mortality:
<1 - 80% of hospitalised patients.
VIRAL HEMORRHAGIC FEVERS
Clinical suspicion of VHF
Clinical suspicion of VHF
Malaria - Hepatitis –Typhoid - Toxicosis -Septicemia - Leptospirosis -Rickettsiosis…
Clinical suspicion of VHF
Epidemiological contextRisk assessment
Malaria - Hepatitis –Typhoid - Toxicosis -Septicemia - Leptospirosis -Rickettsiosis…
Clinical suspicion of VHF
Dialogue between Clinicians and Biologists
Epidemiological contextRisk assessment
Malaria - Hepatitis –Typhoid - Toxicosis -Septicemia - Leptospirosis -Rickettsiosis…
DIAGNOSIS OF VHF
Samples
Whole blood: plasma EDTA serum
Urine (Lassa,… )SalivaCSFBiopsy: skin (Filoviridae)
organs (liver, kidney, lungs… )Fixation (formaldehyde 10%)
Transportation: 4°C UN 3373 regulations
Laboratory Information
Contact prior sending biological materials
Onset of disease
Origins of the patient:- Activities- Travels within the last 2 months :countries, region(s)date
Immunization data:- Yellow fever- Hepatitis- Others
DIAGNOSIS OF VHF
UN 3373No biohazard
label required
UN 2814“Infectious substances”biohazard label
FICHE DE RENSEIGNEMENTS Arboviroses/ Fièvres hémorragiques virales
HÔPITAL Médecin : Service: Tél n°: Fax sécurisé Biologiste : Tél n : Fax : IDENTIFICATION DU PATIENT : Nom: Prénom:
Date de naissance : ___/___/____ Sexe : M F Domicile (commune, dépt) : DATE DE DEBUT DES SYMPTÔMES: : _____/___/____
hospitalisation : non oui date: ____/___/___ PRINCIPAUX SIGNES CLINIQUES :
- fièvre - myalgies - céphalées - arthralgies - nausées - méningite - douleurs abdominales - encéphalite - diarrhées troubles oculaires - éruption signes hémorragiques: préciser autres:
NOTIONS DE VOYAGES DANS LE MOIS PRECEDENT :
PAYS : LIEUX: DATES: Date du retour :
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 : ___/___/____
BIOLOGIE
Paludisme: date : ___/___/____ Goutte épaisse : nég pos Détection d’antigène : nég pos
Autres diagnostics demandés : leptospirose : nég pos Hématocrite: ALAT : Taux plaquettes: ASAT :
PRELEVEMENTS : DATE : __/___/____
Etat fébrile lors du prélèvement : oui : °C non non précisé
sang : sérum LCR : urines : biopsie : REMARQUES : Arboviroses suspectées :
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)
Lassa 4Junin Machupo Sabia Guanarito 4Ebola, Marburg 4
Hantaan, Seoul, Dobrava, Andes 3Puumala 2
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
Filtration of the inoculum on 0,22 µm
Inoculation of cell cultures :
Visualization of cytopathogenic effect
Detection / Identification byCell culture
Mosquito cell culture : for dengue, YF, RVF… )
Mammal cell culture
Titration : plaque assays : pfu/ml
Indirect diagnosis
IMMUNOFLUORESCENCE ASSAYS
Immunofluorescence
Multiple labeling methods
Test ELISA
IgM / IgG
Immuno -chromatographyStrip assays
Serological diagnosis limitations
Commercial assays availability
Specific antibodies
hyperimmune mouse ascitic fluid (ethic concerns)(TG 180 sarcoma)
Rabbit (or other… ) antibodies
Constraint: biosafety regulation in animal facilities
Monoclonal antibodies
Some examples
Direct diagnosisviral ARN viral
D 0-5 (PCR)
Dengue : biological diagnosisin humans
----------->NS1 Ag
Clinical signs
Viral RNA
D1
D2
D3
D4
Dengue : Conventional nested RT-PCR
Positive controls
1ère étape
2ème étape
Sensibilité par sérotype : DEN1 : 10 pfu/ml ; DEN2 : 0.1 pfu/ml ; DEN3 : 0.1 pfu/ml ; DEN4 : 1 pfu/ml.
D1 482
D4 392D3 290
D2 119
D 511 bp
Adapted from : Nabeth P, et al, EID, 2004
Crimean Congo Hem Fever: geographic distribution
Viral isolation
S M
L
N
G1
G2
10 nm
L
Segment Nucleotides Amino Proteinacids
___________________________________________
S 1659-1712 442-482 N
M 4888 1551 G1 G2 NSm
L 12255 4036
Crimean-Congo Hemorrhagic fever
CCHF : RT-PCR on S segment*
Control +Single round
536 pb
Nested
236 bp300200
500
Control +
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)
Ebola/Marburg, Lassa virus; Crimean-Congo HF; Rift valley fever; Yellow fever, Dengue; Flavivirus universal
• 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).
ARENAVIRUSES3
Arenavirus phylogeny
Source: CDC
Old world arenavirusesLassaMobalaIppyLCM
New world arenaviruses
Clade BAmapari 1964 Oryzomys capito Brazil
Neacomys gulanae
Guanarito 1989 Sigmodon alstoni Venezuela
Machupo 1963 Calomys callosus Bolivia
Junin 1958 Calomys musculinus ArgentinaTacaribe 1956 Artibeus literatus VenezuelaSabia 1990 ? BrazilChapare 2004 Bolivia
Clade APirital 1995 Sigmodon alstoni VenezuelaPichinde 1965 Oryzomys albigularis ColombiaFlexal 1975 Oryzomys spp. BrazilParana 1965 Oryzomys buccinatusTamiami 1964 Sigmodon hispidus USAWhite waterArroyo 1995 Oryzomys albigularis USA
Clade COliveros 1990 Bolomys obscurus ArgentinaLatino 1965 Calomys callosus BoliviaConventional PCR : Demby A.H. et Al.J (1994) J.Clin.Microbiol.32 :2898-2903
Source: C Drosten
•ARN FP6•Sensitivity : 10-3 10-4
•ARN Lassa AV•Sensitivity : 2 103 pfu/ml 2 104 pfu/ml
•ARN Lassa Josiah •Sensitivity : 10-5 10-6
L Gene GPC Gene
Old-World Arenavirus
(Gel Detection)
NP 35 GP L30 2440EBO 3’ 5’
NP 35 GP L30 2440MBG 3’ 5’
RNA 19 kd
RNP complex made of viral proteins NP, VP35, VP30, L,matrix space made of VP40 and VP24viral envelope and surface protein GP
FILOVIRUSES : Ebola Zaire/Sudan/Cote d'Ivoire/RestonMarburg
2
09DRC99
01KEN87
BAT349/99/246
02DRC99
BAT349/99/50
01GER67
01KEN80
BAT349/99/69
BAT349/99/135
01ZIM75
BAT349/99/90
BAT349/99/116
BAT349/99/93
01DRC99
BAT349/99/385
03DRC99
25DRC00
23DRC00
BAT349/99/202
05DRC99
BAT349/99/259
28DRC00
BAT349/99/161
BAT349/99/383
31DRC00
100
99
85
92
84
99 85
68
80
0.01 substitutions/site
DRC Durba Watsa 1999 2000 MBG outbreak :
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
PCR Ebo/Mbg NEGATIVE (Filo A/B, GP....)CCHF POSITIVEYF NEGATIVEDEN NEGATIVELASSA NEGATIVE
Ag capture : nd
Serology IgM/IgG :
CCHF IgM positivity IgG negativeEBO/MBG LAS IgM/IgG negativeYF, DEN IgG positive (weak)
Cell cultivation: negative (2 blind passages)
Second specimen taken on March 05 2008 : 20 days post onset of fever.
CCHF IgM IgG positive
External quality assurance : ENIVD task
Comparative testing of the same samples by different laboratories
• Syndromic diagnostics requires broad- range screening
• Novel post-PCR approaches are soon available
However
• Recent successes in virus identification relied on cell culture :
• No practicable universal amplification system is currently available for patient samples
• Virus culture indispensable
Conclusions (1)
– Diagnostics should go beyond detection–therapy monitoring
– Ecology and transmission studies
– Lack of assay standardisation
–Limited availability of virus strains– Limited sequence information (ie. Ebola Bundibugyo… .)
– No commercial kits for exotic viruses
Conclusions (2)
Contributors
• National Reference//WHO Collaborating Centre for Arboviruses and Hemorrhagic fevers
Severine Murri, Nadege Mollard, Stéphanie Michel,
Isabelle Schuffenecker
• And the : Clinicians and biologists
- NGO’s : MSF … .
- ENIVD : European Network for Viral Imported Diseases
