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Diagnostic in infectia cu
virusuri gripale
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Orientare clinica
• febra (3-5 zile), mialgie, cefalee, astenie (>2
saptamani), tuse neproductiva, faringita, rinoree.
• Gripa nu se diagnosticheazaclinic!!!
Simptomatologie asemanatoare cu infectii determinate de:
Mycoplasma pneumoniae, adenovirusuri, virusulrespirator sincitial, rhinovirusuri, virusuri paragripale,
si Legionella spp.
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Forma clinicã Etiologie viralãcomunã
Etiologie viralãrarã
Guturai,Rhinofaringite
Rhino,coronavirusuri
v. gripale,Enterovirusuri(Cox)
Laringo-traheobronsite
VRS, V.paragripale
Adeno-, v.rujeolic
Bronsite V. gripale V. paragripale
Pneumonii V. gripale,SARS
V. gripeiaviare,Micoplasme
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ETIOLOGIE
POSIBILA
Produs
Patologic
IZOLARE Dg. DIRECT
=detectie
directa
Dg.
INDIRECT
=serologie
V GRIPALE(A, B, C) SNF (ENF)
ANF
ATB
•OGE
•Culturi
celulare
DA –
PCRsi IF
DA –
studiiepidemiolog
ice
DIAGNOSTICUL DE GRIPA
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Produse patologice : a)Spalatura nazo faringiana (SNF) =
Exsudat nazo faringian
Recoltare.
N- tampoane sterile cu coada introduse in cornetul nazal inferior,secretii de la nivelul peretelui posterior al nazofarinxului.
F - tampon prin cavitatea bucala pentru secretii de pe peretele
lateral/posterior al orofaringelui si din orice arie adiacenta cuinflamatie, exudat sau ulceratie.
se evita atingerea tamponului de limba, obraji sau buze pentru evitareacotaminarii.
Stocare si transport.
optim imediat dupa recoltare (cand nu este posibil: -70 C, cu transport
pe zapada carbonica)Prelucrare.
tamponul sau secretiile recoltate sunt dispuse intr-un tub steril cu circa15 ml mediu minim o ora, timp necesar elutiei virusului. centrifugarela 2500 - 15 minute, la 4° C. Supernatantul este indepartat sisedimentul este resuspendat in 3-4 ml TFS sau mediu. In final
depozitul de celule reluat in TFS serveste pentru izolare virala saupentru efectuarea de frotiuri ce vor fi testate in imunofluorescenta.
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Produse patologice :
a) exsudat nazo faringian
b) aspiratul nazofaringian se obtine folosind unspeculum nazal prin administrarea a 3 ml ser fiziologic in
nari cu o seringa atasata la un tub de plastic.
c) lavaj traheobronsic se obtine prin bronhoscopiein conditii speciale de catre personal calificat.
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Directii de diagnostic
• IZOLARE – OGE/ c.c .
•
DETECTIE RAPIDA –
RIF / Rt PCR
• DIAGNOSTIC SEROLOGIC
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1.-culturi primare de rinichide maimuta rhesus.
-linia continua epiteliala de
rinichi de caine(MDCK).
2. pe OGE- 9-11 zile,
inoculat – intraamniotic
– intraalantoidian.
Izolare virala – diagnostic de electie
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V. GRIPAL ESTE NONCITOPATOGEN!!
Tipuri v gripal: A, B, C
Subtipuri: A(H1N1)
A(H3,N2)… Dupa HA si NA
•16 tipuri de HA
•
9 tipuri de NA
Matrice
Nucleocapsida helicoidalaARNss
HA - hemaglutinina
transcriptaza
anvelopa
NA - neuraminidaza
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Demonstrarea izolarii v.gripale
pe culturi de celule - HEMADSORBTIE
• 48 ore dupa inocularea
v. gripal se incearca
detectia virusului prin
hemadsorbtie, obisnuit
cu eritrocite de cobai
• Hematiile adsorbite
specific rozeteaza la
periferia celulelor
infectate.
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Etape:
•Ag. viral - dilutii binare
•suspensie de hematii
•Se incubeaza la temperatura camerei - 30 minute
•Martorul reactiei contine suspensie hematii
Rol RHA:
Demonstrarea prezentei virusului hemaglutinant
Titrarea antigenului hemaglutinant
UHA
DEMONSTRAREA PREZENTEI
VIRUSULUI IN OGE
REACTIA DE HEMAGLUTINARE -RHA
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IDENTIFICAREA V. GRIPALE
R. HEMAGLUTINO-INHIBARE
RHAIEtape:
•Ag. viral hemaglutinant •Dilutii binare din serurile imune de referinta (Ac antivirali
cunoscuti)- 60 min la 37°C.•Sistem indicator - suspensia de hematii.•Se incubeaza 60 min la temperatura camerei
Reactia de identificare a virusurilor gripale
UHAI
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DIAGNOSTIC SEROLOGIC
RHAI = Tulpini de referinta + ser pacient
•diagnostic retrospectiv,
•recoltarea de probe de serpereche
•cresteri semnificative (de
peste 4 ori) a titrului Ac
specifici
•sau determinarea
rezultatului vaccinarii
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OMS - Global Influenza
Surveillance Network
2012-2013 Emisfera de Nord
•
A / California/7/2009 (H1N1) -like virus;• A / Victoria/361/2011 (H3N2)-like virus;
• B / Wisconsin/1/2010-like virus.
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Reactia de Imunofluorescenta directa
(RIF)
test rapid, reproductibil, ieftin
detecteaza ANTIGENE VIRALE cu anticorpi marcati
cu fluorocromi
ID
PP
Anticorpi marcati cu
fluoresceina sau rhodamina
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Reactia de Imunofluorescenta
(RIF)
Aplicatii – DIAGNOSTIC IN•Infectiile herpetice
•Infectiile respiratorii (Gripa)
•Rabia
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Teste moleculare
NAT (nucleic acid tests)
RT PCR
• Swine flu real-time PCR kit
• Quick Diagnosis of Flu Strains Possible with New
Microchip Test - FluChip,• Inexpensive Test Detects influenza Infections Quickly
and Accurately – MChip
• FDA Approves New Laboratory Test To Detect HumanInfections With Avian Influenza A/H5 Viruses (
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DIAGOSTIC IN VIROZERESPIRATORII
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VIROZE RESPIRATORII
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Diagnostic in infectia cu
virusuri gripale
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Orientare clinica
febra (3-5 zile), mialgie, cefalee, astenie(>2 saptamani), tuse neproductiva,faringita, rinoree.
Gripa nu se diagnosticheaza clinic.
Simptomatologie asemanatoare cu infectii determinate de:
Mycoplasma pneumoniae, adenovirusuri,
virusul respirator sincitial, rhinovirusuri,virusuri paragripale, si Legionella spp.
ORTHOMIXOVIRUSURI V GRIPALE A B C
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ORTHOMIXOVIRUSURI-V. GRIPALE A, B, C
myxa-mucus
tip A, B, C : NP, M
subtipuri: HA si NA – 15 tipuri de HA si 9 de NA
Matrice
Nucleocapsida helicoidala
ARNss
HA - hemaglutinina
transcriptaza
anvelopa
NA - neuraminidaza
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Produse patologice :
a) spalatura nazo faringiana se obtine cuajutorul unor tampoane cu coada (sterile)introduse in cornetul nazal inferior.
b) aspiratul nazofaringian se obtine folosind
un speculum nazal prin administrarea a 3 ml serfiziologic in nari cu o seringa atasata la un tub deplastic.
c) lavaj traheobronsic se obtine prinbronhoscopie in conditii speciale de catre personal
calificat
Pentru a mari sansa de izolare probele se recolteazain primele 72 ore de la debutul infectiei.
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Produse patologice
Exsudat naso-faringian/SNF Recoltare. tampon steril - recoltare secretii de pe peretele
posterior si lateral al orofaringelui si din orice arie adiacenta cuinflamatie, exudat sau ulceratie. Se evita atingerea tamponuluide limba, obraji sau buze pentru evitarea contaminarii;
recoltarea nazala se obtine cu ajutorul unui tampon cu coadaintrodus in cornetul nazal inferior.
Stocare si transport. Optim se inoculeaza imediat duparecoltare. Daca aceasta nu este posibil se ingheata la -70 C sise transporta pe zapada carbonica
Prelucrare. Tamponul este dispus intr-un tub steril cu circa 15ml mediu de transport pentru virusuri si 2% SFV minim o ora,timp necesar elutiei virusului. Tubul se centrifugheaza la 2500g, 15 minute, la 4 C. Supernatantul este indepartat sisedimentul este resuspendat in 3-4 ml TFS sau mediu decultura. Spalarea celulelor se poate repeta pentru indepartareaurmelor de mucus din secretii. In final depozitul de celule reluatin TFS serveste pentru izolare virala sau pentru efectuarea defrotiuri ce vor fi testate in imunofluorescenta cu intreaga baterie
de seruri antivirale disponibile.
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Diagnosticul de laborator al gripeiDirectii de diagnostic
Izolare virala + identificare
Detectie directa antigene virale- RIF
acid nucleic- PCR
Diagnostic serologic
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1.-culturi primare de rinichide maimuta rhesus.
-linia continua epitelialade rinichi de caine (MDCK- Madin-Darby CanineKidney).
2. pe ou de gaina embrionat,de 9-11 zile, inoculatintraamniotic sauintraalantoidian.
Izolare virala – diagnostic de electie
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Hemadsorbtiademonstreaza izolarea v.gripalepe culturi de celule
Virusurile gripale nu suntcitopatogene.
La 48 ore dupa inocularea v.gripal se incearca detectia
virusului prin hemadsorbtie,obisnuit cu eritrocite decobai la 4 C.
Hemadsorbtia rezulta dinincorporarea in membrana
plasmatica celulara a HAvirale. Hematiile adsorbitespecific rozeteaza laperiferia celulelor infectate.
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1.-culturi primare de rinichi demaimuta rhesus.
-linia continua epiteliala derinichi de caine (MDCK -Madin-Darby CanineKidney).
2. pe ou de gainaembrionat, de 9-11 zile,inoculat intraamnioticsau intraalantoidian.
Izolare virala – diagnostic de electie
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Izolarea pe OGE
Izolarea se dem. prin proprieratea v.gripale de a aglutina hematii
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REACTIA DE HEMAGLUTINARE
Capacitatea unor virusuri de a aglutinahematiile unor specii de animale
Virusurile care poseda capacitatea de a
aglutina hematii se numesc virusurihemaglutinante (rubeola, rujeola, urlian,unele adenovirusuri, enterovirusuri, gripalesi paragripale)
Punerea in contact a unor dilutii succesive,binare de antigen hemaglutinant cuhematiile corespunzatoare virusuluiincriminat
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Etape:
•Ag. viral - dilutii binare
•suspensie de hematii
•Se incubeaza la temperatura camerei - 60 minute
Rol RHA:
Demonstrarea prezentei virusului hemaglutinant
Titrarea antigenului hemaglutinant inaintea reactiei de hemagutino-inhibare
UHA
DEMONSTRAREA PREZENTEI VIRUSULUIIN OGEREACTIA DE HEMAGLUTINARE
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CLASIFICAREA V. HEMAGLUTINANTE
I. HA structurala care distruge R V gripale, paragripale
II. HA structurala care nu distruge R Arbovirusuri, v rubeolos
III. HA non-structurala poxvirusuri
IDENTIFICAREA V GRIPALE
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IDENTIFICAREA V. GRIPALER. HEMAGLUTINO-INHIBARE
Tehnica reactiei:
dilutii binare din ser imun de referintaag. viral hemaglutinant - 60 min la 37°C.sistem indicator - suspensia de hematii.Se incubeaza 60 min la temperatura camerei
Reactia de identificare a virusurilor gripale
UHAI
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Diagnosticul de laborator al gripeiDirectii de diagnostic
Izolare virala + identificare
Detectie directa antigene virale- RIF
acid nucleic- PCR
Diagnostic serologic
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Imunofluorescenta directa –
test rapid, reproductibil, ieftin
Diagnostic rapid in
gripa,
infectie herpetica,
rabie.
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Reactia deImunofluorescentadirecta
Amprenta se va usca bine:
pp. amprentat direct pe lama
Imersie in acetona la 2-8°C timp de 10
minute si uscare.
ID
PP
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Reactivii trebuie sa fie la temp.camerei
Incubare cu anticorp fluorescent (30 min la 37° C)
Spalare
Citire la MO in lumina UV
ID
PP
Anticorpi monoclonali marcati
cu ITCF
anti epitopi specifici de la
nivelul anvelopei,
ex. anti H1N1; anti H3N3
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Di ti l d l b t l i i
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Diagnosticul de laborator al gripeiDirectii de diagnostic
Izolare virala + identificare
Detectie directa antigene virale- RIF
acid nucleic- PCR
Diagnostic serologic
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Diagnosticul serologic
Necesita recoltarea de probe de ser pereche de laacelasi pacient.
Nu se practica in mod curent decit in caz de epidemii,pentru diagnostic retrospectiv.
Diagnosticul necesita demonstrarea unei cresterisemnificative (de peste 4 ori) a titrului Acspecifici antivirali intre proba de faza acuta, recoltatala o saptamana de la debutul bolii si cea dinconvalescenta, recoltata 2-3 saptamani mai tarziu.
Metoda preferata de evidentiere a anticorpilor antivirali specifici este hemaglutinoinhibarea.
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DIAGNOSTIC SEROLOGIC
RHAI = Tulpini de referinta + ser pacient
•diagnostic retrospectiv,
•recoltarea de probe de ser
pereche•cresteri semnificative (de
peste 4 ori) a titrului Ac
specifici
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Pacient cu febra, cefalee, astenie,tuse neproductiva. Ce produsepatologice utilizati?
Cum faceti diagnosticul rapid?
Pe ce substraturi se poate faceizolarea virala?
Este diagnosticul serologic de primaintentie in aceasta viroza?
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Precizati tipul de reactie reprezentata in sirurile de godeuri C, D, Esi principiul tehnicii.Pentru ce viroze tehnica aceasta este de electie si ce simptomeclinice prezinta pacientul?
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Precizati tipul de reactie si infectiile viralecare pot fi diagnosticate cu aceasta.
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Cum demonstrati izolarea unuivirus hemaglutinant?
Cum se face identificareavirusurilor hemaglutinante?