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DIAGNOSTIC SEROLOGIC
IN INFECTIA HIV/SIDA
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Este bine sa doresti sa fi diagnosticat la
timp!
TRATAMENT
MONITORIZARE
CRESTE CALITATEA
VIETII
Beneficii
Temeri
CONTAGIOZITATE
SCADE CALITATEA VIETII
AGRAVARE
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1990
Romania
raporteaza 1094
cazuri de SIDA la
copii,reprezentând
jumătate din
totalul de cazuride
SIDA pediatric din
Europa
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ENCEFALOPATIE HIV
Infectie HIV in SNC
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1. DIAGNOSTIC SEROLOGICTESTE DE TRIAJ- sensibilitate inalta
ELISATESTE RAPIDE
TESTE DE CONFIRMARE- specificitate inalta
WESTERN BLOT
RADIOIMUNOPRECIPITAREA (RIPA)
IMUNOFLUORESCENTA INDIRECTA
2. DETECTIA DIRECTA A VIRUSULUI
SAU A PRODUSELOR VIRALEIZOLARE VIRALA
DETECTIA ANTIGENEMIEI p24
DETECTIA ACIDULUI NUCLEIC VIRAL
(ARN HIV PLASMATIC SAU ADN HIV PROVIRAL LIMFOCITAR)
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TESTE DE TRIAJ
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ELISA INDIRECTA
ETAPE
1. Incubare ser pacient 1h la 37ºC
2. Spalare
3. Incubare conjugat (Ac anti IgG uman x enzima)- 1h la 37ºC
4. Spalare
5. Incubare substrat 20 – 30 min la temp camerei
6. Citire la spectrofotometru – DO probe, martori, cut off
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Spalator
automat
Distribuirea probelor cu
pipeta automata
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Spectrofotometru
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ELISA de competitie
ETAPE
Incubare ser pacient si conjugat 1h la 37ºC Spalare
Incubare substrat 20 – 30 min la temp camerei
Citire la spectrofotometru – DO probe, martori,
cut off
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WESTERN BLOT- test imunoblot folosit pentru confirmarea prezentei anticorpilor
in diferite infectii
- se bazeaza pe detectia anticorpilor defalcati pe grupe antigenice
TESTE DE CONFIRMARE
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UTILIZARE
Detectia proteinelor virale
Caracterizarea preparatelor de anticorpi
mono- si policlonali
Determinarea specificitatii raspunsului
imun impotriva antigenelor virale
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PRINCIPIU
Proteinele dintr-un lizat viral sunt separate
electroforetic dupa greutatea moleculara
prin SDS-PAGE (sodium dodecil sulfat)
Transferate pe o membrana de
nitroceluloza
Detectie prin anticorpi marcati radioactiv
(autoradiografie) sau enzimatic
(COLORIMETRIC)
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ELECTROFOREZA
Pe gel de poliacrilamida
Apoi proteinele sunt transferate pe omembrana de nitroceluloza prin difuzie,
electroforeza sau prin afinitateFixarea se face prin simpla absortie
pasiva, prin legaturi covalente pe
membrana diazotata sau prin legarespecifica pe membrana acoperita deanticorpi
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Tehnica
WESTERN BLOT
1. SDS-PAGE
2. BLOT (transfer)
3. DETECTIA
ANTICORPILOR
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WESTERN BLOT – detectia Ac serici
Substrate
Stop
color
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Western blot
in infectia HIV
gp160
gp120
gp41
p66
p31
p24
p17
p10/11
p7/9
p51
p55
Criteriul minim depozitivitate
• 2 env
+
•
1 gag
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ELISA
POZITIV IN DUPLICAT
WESTERN BLOT
NEGATIV=
ABSENTAINFECTIEI HIV
! Ferestraserologica
risc rezidual al
transfuziei = 2/1 milion
NEGATIV
INDETERMINAT
- repeta la 6-8 saptamini
- alt test de confirmare
POZITIV=
INFECTIE HIV
Ag p24- 1.48/1 milion
ADN proviral –
1.48/1 milion
ARN HIV – 1.01/1 milion
DIAGNOSTIC SEROLOGIC HIV
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Test Elisa + in duplicat
WB –
Este posibil acest rezultat?; explicati.
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Pacient 15 ani
test de triaj infectie HIV
Ce test s-a folosit si care sunt urmatorii pasi?
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Diagnostic molecular invirusologie
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Diagnosticul molecular invirusologie
AMPLIFICAREA TINTEI
–PCR
–NASBA (nucleic acid sequenced based
amplification)
AMPLIFICAREA SEMNALULUI
–B-DNA (branched DNA)
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PCRPOLYMERASE
CHAIN
REACTION
Metoda utilizata pentru detectia ADN-ului de orice tip.
-se bazeaza pe obtinerea unui numar mare de catene
tinta prin repetatea unui nr. n de cicluri, ce contin in
principal 3 etape:
- denaturarea
- aditia primerilor la capetele secventei tinta
- extensia primerilor
PCR Amplifies a Targeted
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p gSequence
Target Sequence
DNA Strand
Double Helix
DNA StrandSupercoiledDNA Strand
Chromosome
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PCR 1 Denaturare
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PCR – 1- Denaturare
Secventa tinta
PCR- 2 CUPLAREA PRIMERILOR LA
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PCR- 2 – CUPLAREA PRIMERILOR LA
CAPETELE SECVENTEI TINTA
Target Sequence
Target Sequence
Primer 1Primer 2
Biotin
Biotin
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PCR -3 – Elongarea primerilor sub actiunea
Taq Polimerazei
Secventa tinta
ADN Polimeraza Taq
Secventa tinta
End of the 1st PCR Cycle Results in Two
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End of the 1st PCR Cycle - Results in Two
Copies of Target Sequence
Target Amplification
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Target Amplification
No. of No. AmpliconCycles Copies of
Target
1 2
2 4
3 8
4 16
5 32
6 64
20 1,048,576
30 1,073,741,822
1 cycle = 2 Amplicon
2 cycle = 4 Amplicon
3 cycle = 8 Amplicon
4 cycle = 16 Amplicon
5 cycle = 32 Amplicon
6 cycle = 64 Amplicon
7 cycle = 128 Amplicon
PCR:
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Master Mix
Mg2+
Mn2+
dNTP
Target DNA
Taq DNA
Polymerase
Primers
•forward primer
•reverse primer
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RT- PCR
Reverstranscriere + PCR
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Reverse Transcription - Step 2 –
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Reverse Transcription Step 2
Reverstranscriptaze Catalizeaza extensia
Primerului prin incorporarea nucleotidelor
complementare
E d f R T i ti St 3
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End of Reverse Transcription - Step 3 –
Rezulta sinteza ADNcomplementar (cDNA)
pe secventa tinta ARN
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Safety Gloves, Laboratory Coat
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Safety Glasses
Biohazard Container
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Biohazard Container
0.5% sodium hypochlorite, ethanol,
Biological Safety Hood
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Biological Safety Hood
Vortex Mixer
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Vortex Mixer
Centrifuge and/or Microcentrifuge (min. RCF 12,500 x g),
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Centrifuge and/or Microcentrifuge (min. RCF 12,500 x g),
some tests require a refrigerated, high speed centrifuge (min. RCF 24,000 x g)
Dry-heat Temperature Block
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Dry heat Temperature Block
Incubator (37 C ± 2 C)0
Dedicated Pipettor and Screw-cap Tube Rack,
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•Screw-cap tube racks (to hold Master Mix tubes)
Multichannel Pipettor and Tips
PCR System consumables
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PCR System consumables
(tubes, base, tray and retainer).
Analyser Consumables/parts
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Analyser Consumables/partsD-cups
A-rings, A-ring Holder
Thermal Cycler
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Thermal Cycler PCR System 9600 or System 2400
COBAS AMPLICOR™ Analyser
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COBAS AMPLICOR Analyser
DETECTIA AMPLICONULUI
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DETECTIA AMPLICONULUI
• Electroforeza in gel de agar, colorare
cu bromura de ethidiu si comparare
cu markeri de GM cunoscuta
• Hibridizare cu sonde moleculare
marcate complementare si apoidetectie (+/- spectrofotometrica)
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Hibridizare
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Detectie
spectrofotometrica Add Stop Solution and
Measure Absorbance
Capture
Probe
Microwell Plate
Addition
of Substrate
Tetramethylbenzidine
(TMB) Substrate
Biotin
BSA
BSA
Hydrogen
Peroxide
Avidin-horseradish peroxidase
(Av-HRP conjugate)
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Diagnostic molecular in
infectia HIV
DIAGNOSTIC SEROLOGIC
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DIAGNOSTIC SEROLOGICTESTE DE TRIAJ- sensibilitate inalta
ELISA
TESTE RAPIDE
TESTE DE CONFIRMARE- specificitate inalta
WESTERN BLOT
RADIOIMUNOPRECIPITAREA (RIPA)IMUNOFLUORESCENTA INDIRECTA
DETECTIA VIRUSULUI SAU A PRODUSELORVIRALE
IZOLARE VIRALADETECTIA ANTIGENEMIEI p24
DETECTIA ACIDULUI NUCLEIC VIRAL
(ARN HIV PLASMATIC SAU ADN HIV PROVIRAL LIMFOCITAR)
Utilizarea metodelor moleculare in
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Utilizarea metodelor moleculare in
infectia HIV
• Fereastra serologica
• Nou-nascuti din mame seropozitive HIV
• Initierea si monitorizarea terapiei
antiretrovirale• iv>5-10.000 corelat cu nr. CD4 si status clinic
• iv>30-50.000 indiferent de nr. CD4 si status clinic
• Monitorizarea: reducerea iv sub 50 copii ARN
HIV/ml la 24 sapt
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Precizati tipul dereactie
reprezentata
mai sus si
principiul
tehnicii.
In ce viroze
aceastatehnica este de
electie?
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• MC, 25 ani . Se apreciaza ca se gasestein fereastra serologica pentru infectia HIV.
Ce semnifica aceasta notiune si ce etapa
urmeaza in diagnostic.
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Nou nascut din mama seropozitiva HIV.
Care este testul de electie folosit pentru
diagnosticul infectiei, precizand si etapele
sale.
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• CE TEHNICA ESTE UTILIZATA IN
ETAPA DE CONFIRMARE A INFECTIEIHIV? CE CRITERII TREBUIE
OBLIGATORIU INDEPLINITE PENTRU A
SPUNE CA TESTUL ESTE POZITIV?
CUM SE PROCEDEAZA IN CAZUL UNUI
TEST INDETERMINAT? MENTIONATI
PRINCIPALELE CAI DE TRANSMITERE
ALE INFECTIEI HIV.