LP 6,7 - Diagnosticul in Infectie Cu HIV

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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.