immunologic methods in diagnosis -...
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Immunologic Methods in Diagnosis of HIV Infection
M P i Ph DM Parsania, Ph.D.Tehran Medical Sciences Branch, Islamic Azad
UniversityUniversity
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R t i (f il R t i id ) l d i l t d d (+) RNA i
RetroviridaeRetroviruses (family Retroviridae) are enveloped, single stranded (+) RNA viruses that replicate through a DNA intermediate using reverse transcriptase. This large and diverse family includes members that are oncogenic, are associated with a variety of immune system disorders, and cause degenerative y y , gand neurological syndromes.
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Retrovirus ClassificationFamily: RetroviridaeFamily: Retroviridae
Genus Features Examples1. Alpharetrovirus Simple,
OncoAvian leucosis virus, RSV
2. Betaretrovirus Simple, Mouse Mammary Tumor Onco Virus
3. Gammaretrovirus Simple, Onco
Murine leukemia virus (Moloney, Harvey)y y
4. Deltaretrovirus Complex, Onco
Bovine Leukemia, Human T Cell Leukemia (HTLV)( )
5. Epsilonretrovirus Complex, Onco
Walleye Dermal Sarcoma
6 Lentivirus Complex HIV Visna EIAV6. Lentivirus Complex HIV, Visna, EIAV
7. Spumavirus Complex Simian Foamy Virus
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• RNA virus, 120nm in diameter
Family : Retroviridae
• Envelope gp160; gp120 & gp41
Subfamily : Lentivirus
gp41
• Icosahedral symmetry
N l id• Nucleocapsid
Outer matrix protein (p17)
M j id t i ( 24)Major capsid protein (p24)
Nuclear protein (p7)
Di l id RNA i h l• Diploid RNA with several copies of reverse transcriptasetranscriptase
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HIV Structure
surface transmembrane
matrix protein
capsid protein
nucleocapsid protein
RTIntegraseprotease
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HIV Genome• 9200 nucleotides (HIV 1):• 9200 nucleotides (HIV‐1):
• env ‐ gp160 (gp120:outer membrane part, gp41: t b t)transmembrane part)
• gag core proteins – p55, p17 and p24
• pol – p66 (protease), p31,p51 (integrase/endonuclease)
EARLY Accessory Genes LATEEARLY Accessory Genes LATEtat ‐ trans activator of transcriptionnef – negative regulatory factorif i l i f ti it f tvif ‐ viral infectivity factor vpr‐ viral protein Rrev‐ regulator of viral protein expression vpu‐ viral protein U vpx – HIV ‐ 2
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Viral ReplicationViral Replication• Methods of transmission:
– Sexual transmission, presence of STD increases likelihood of transmission.
– Sharing contaminated needles (IV drug users).– Transplantation of infected tissues or organs.Exposure to infected blood or blood products– Exposure to infected blood or blood products.
– Use of contaminated clotting factors by hemophiliacs.– Mother to fetus, perinatal transmission variable,Mother to fetus, perinatal transmission variable, dependent on viral load and mother’s CD 4 count.
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Viral ReplicationViral Replication
• First step, HIV attaches to susceptible host cell.First step, HIV attaches to susceptible host cell.– Site of attachment is the CD4 antigen found on a variety of cells
• helper T cells• macrophages• monocytesy• B cells• microglial brain cells• intestinal cells• intestinal cells
– T cells infected later on.
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HIV ‐ Life Historyh
HIV ‐ Life HistoryhCCR5 ‐ the co receptorCCR5 ‐ the co receptor
HIVHIV
chemokineMutant
CD4
CD4CD4
CCR5CCR5
CCR5
macrophage
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Virion interaction with CD4 receptor and CXCR4 co‐receptorreceptor
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Early Phase HIV InfectionEarly Phase HIV Infection
• In early phase HIVIn early phase HIV infection, initial viruses are M‐tropic. Their envelope glycoprotein gp120 is able to bind to CD4 l l dCD4molecules and chemokine receptors called CCR5 found oncalled CCR5 found on macrophages
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Late Phase HIV Infection
• In late phase HIVIn late phase HIV infection, most of the viruses are T‐tropic, having gp120 capable of binding to CD4 and CXCR4 f dCXCR4 found on T4‐lymphocytes.
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Retroviruslife cycle:
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HIV and AIDSThe cellular and immunological picture - The course of the diseaseThe cellular and immunological picture The course of the disease
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HIV Diagnostic Testing
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Purpose of HIV TestingPurpose of HIV Testing
To identify asymptomatic individuals y y pTo diagnose HIV infection in those who practice high risk behaviorTo prevent secondary transmissionDonor screening for blood & tissue productsF h l i M di l t T t tFor prophylaxis, Medical management, TreatmentFor epidemiological surveillanceTo diagnose clinically suspected casesTo diagnose clinically suspected cases
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Laboratory diagnosis of HIV infectionLaboratory diagnosis of HIV infection
• Direct demonstration of infective agent Direct demonstration of infective agent- Virus isolation- virus culture- Antigen detection- P24 detectiong- viral nucleic acid detection- PCR
• Indirect demonstration of infective agent- Anti -HIV antibody detection
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Initial and Supplemental HIV Testspp
• Initial Test• Initial Test- Enzyme Immunoassay (EIA)
Ch il i t I (CIA)- Chemiluminescent Immunoassay (CIA)
• Supplemental Tests• Supplemental Tests- Western blot
I di I fl A (IFA)- Indirect Immunofluorescence Assay (IFA)- Qualitative HIV-1 RNA
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Spectrum of Immunologic Methods in Diagnosis of HIV Infection
HIV diagnosis (Antibody/Antigen testing)
Enzyme Immunoassays (EIAs) Rapid testsW t bl t (WB)Western blot (WB)
Early diagnosis in infantsp24p24
Initiation and monitoring of ARTCD4 countCD4 count
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Enzyme Immunoassays (EIAs)Enzyme Immunoassays (EIAs)• Quantitative assay to measure HIV antibodiesQ y
Most detect antibodies to HIV‐1 and HIV‐2
Antigens coated in microwells
/ b d d d b lHIV Antigen / Antibody reaction is detected by color change
I t it f l fl t t f tib d tIntensity of color reflects amount of antibody present serum
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After several incubation and wash steps a color reaction occurs if HIV
An automated reader gives a measurement of optical densitysteps, a color reaction occurs if HIV
antibody is presentmeasurement of optical density (presence of color) for each well
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Generation of EIA Tests
First Second Third *Fourth
U d i lU d i l D t t I M d I GD t t I M d I GU bi t HIVU bi t HIV D t t HIV tib diD t t HIV tib di
*Not US FDA‐approved as of 10/1/09
Uses crude viral lysate
Uses crude viral lysate
Detects IgM and IgG in “Sandwich” EIA
Detects IgM and IgG in “Sandwich” EIA
Uses recombinant HIV antigens or peptides
Uses recombinant HIV antigens or peptides
Detects HIV antibodies and p24 antigen
Detects HIV antibodies and p24 antigen
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False Negatives and PositivesFalse Negatives and Positives• False negative results may result from:
Window period– Window period– Agammaglobulinemia– Seroreversion (loss of HIV antibodies due to (extreme immune system destruction) in late diseaseTechnical error– Technical error
– Type N or O strains, or HIV‐2 • False positive results may result from:False positive results may result from:
– Autoantibodies e.g., SLE– vaccines– Technical error
24
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Common HIV‐1 or HIV‐1/2 DiagnosticAlgorithm
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APHL/CDC HIV‐1/2 DiagnosticAlgorithm Template
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Rapid tests• Qualitative assay to detect HIV antibodies• Most detect HIV 1 and HIV 2
Advantages:• As reliable as EIAs
1. Quick 2. Easy to perform3. No sophisticated instruments are required4. Can be done on single sample
Di d tDisadvantages:1. Costly2 Tedious if large no samples have to be tested at one time2. Tedious if large no. samples have to be tested at one time
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Tests Based on I h t hImmunochromatography
Lateral Flow Devices– Determine– Hema‐Strip– OraQuick
HIV Antigen
Control
OraQuick– Unigold
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Sample pad
Specimen Flow
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Body Fluids Used for HIV Rapid Testing
• Serum • Plasma• Whole bloodWhole blood • Oral fluids
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Reading Results
Reactive Control
Positive
P iti N ti
HIV-1/2
Positive Negative
Read results in 20 – 40 minutes
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4th Generation Immunochromatograpict t f d t ti f HIV A /Abtest for detection of HIV Ag/Ab
Control bar (anti-HIV)
p24 bar (avidin)
Ab bar (rHIV Ag + synt peptides)
Conjugate pad(Selenium colloid-HIV Ag/
BBiotinylated anti-p24+Selenium colloid anti-p24)
B
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4th Generation Immunochromatograpict t f d t ti f HIV A /Abtest for detection of HIV Ag/Ab
B
Control bar (anti-HIV)
Bp24 bar (avidin)
Ab bar
B
(rHIV Ag + synt peptides)
B
Conjugate pad(Selenium colloid-HIV Ag/
B
Patient sample
Biotinylated anti-p24+Selenium colloid anti-p24)
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Determine HIV-1/2 Ag/Ab ComboDetermine HIV 1/2 Ag/Ab Combo
p24 Antigen barAntibody bar
Control bar
Antibody bar
Selenium colloid
Conjugate padConjugate pad
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How Particle Agglutination WorksAnti‐HIV antibodies bind to the antigen‐coated latex
How Particle Agglutination Works
particles..
Antigen
Antibody
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Proposed HIV Point of Care AlgorithmTwo Rapid Tests (A1/A2) Performed in Sequence on Blood or
HIV Diagnostic Algorithm (Rapid Test)
A1[HIV-1 or HIV-1/2 rapid test (Blood or oral fluid)]
Oral Fluid (A1 and A2 must be different rapid tests)
A1-Negative for HIV-1 d HIV 2 tib di *
A1+
A2 [HIV-1 or HIV 1/2 rapid test from a
different manufacturer (blood)]
and HIV-2 antibodies*
A1+ A2+Presumptive positive for
HIV 1 or HIV 2
A1+ A2-Inconclusive rapid test
result;HIV-1 or HIV-2 antibodies; requires medical follow-up for further evaluation and
testing
result;requires additional testing
g
*If using an HIV-1 only rapid test, Negative for HIV-1 antibodies only
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Western Blot / Line ImmunoassaysWestern Blot / Line Immunoassays
• Used as supplemental test for confirmation (only difficult ( ycases)
• Detects antibodies to specific HIV antigens on cellulose strip
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Confirmatory Tests
Western Blot, Line immunoassay
• WB use antigens from whole virus lysates electrophoretically transferred to a membrane
• LIA use recombinant or synthetic HIV antigens mechanically applied on to support membrane
• Presence or absence of bands is scored
• Highly specific, Labor intensive, expensive WHO criteria- presence of at least 2 envelope bands (gp120,
gp160, gp41)gp160, gp41)
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HIV-1 Western Blot AntigensHIV 1 Western Blot Antigens
p = protein
gp = glycoprotein
Number = molecular weightNumber = molecular weight
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Components Used in HIV-1 Western BlotComponents Used in HIV 1 Western Blot
HIV Western blot StripHIV Western blot Strip
Color Reagent
Human HIV AntibodiesY YY YYYYY Y
Antihuman IgG AntibodiesEnzyme Detector
Human HIV Antibodies(from patient serum)
Y YY YYY
HIV Antigens( W bl )(on Western blot)
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HIV-1 and HIV-2 Gene Products &W BlWestern Blot
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Interpretive Criteria for HIV-1 Western Blot
Positive ControlPositive Control PositivePositiveNegativeNegative IndeterminateIndeterminateAt least two of the Following bands: p24, gp41, gp120/160
No bands:One or more bands presentNot meeting positive criteria
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b d d b h d b dIndirect immunofluorescence
• Can be used to detect both virus and antibody to it.• Antibody detected by testing patient serum against
ti li d t lid i b t d h d dantigen applied to a slide, incubated, washed and a fluorescent antibody added.
• Virus is detected by fixing patient cells to slide• Virus is detected by fixing patient cells to slide, incubating with antibody
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Direct Methods of HIV diagnosis:24 i d ip24 antigen detection
• EIA for detection of p24 antigen in serum, plasma, CSF or cell culture
• Can detect infection in window period, in late stage of disease ,and in newborns
• To monitor response to anti-retroviral therapy
• To monitor disease progression
• Sensitivity is limited (only 69% in patients with AIDS and low in 1 h ld)neonates < 1 month old)
• Negative test does not rule out HIV infection
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Window Period
• Period between infection and first reliable• Period between infection and first reliable detection of HIV by lab test.
• Window period varies by test and by individual.
• The majority of infected individuals are positiveThe majority of infected individuals are positive by ELISA, antigen, and/or DNA/RNA tests by 6‐8 weeks after infection.weeks after infection.
44
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‘typical’ primary HIV‐1 infection
symptoms
HIV proviral DNA
symptoms
HIV antibodies
HIV proviral DNA
‘window’
HIV i l l d
windowperiod
HIV-1 p24 antigen
HIV viral load
HIV-1 p24 antigen
0 1 2 3 4 5 6 / 2 4 6 8 101° infection weeks yearsTime following infection1° infection
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Minimum Time from Infection to First D t ti f HIV 1 M kDetection of HIV‐1 Markers
KEY:
TIME WHEN HIV DETECTION BY TEST IS POSSIBLE
TIME PERIOD BEFORE DETECTION
ELISA
IS POSSIBLE
p24
DNA PCR
RNA (VL)
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5 10 15 20 25 30 35
Detection of HIV: time (in days) after infectionInfection
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Detection of HIV by Diagnostic Tests
Symptoms
p24 Antigen
HIV RNAHIV RNA
HIV Enzyme Immune Assay (EIA)*
0 1 2 3 4 5 6 7 8 9 10Western blot
Weeks Since Infection
*3rd generation, IgM-sensitive EIA*2nd generation EIA
*4th generation, Ag/Ab Combo EIA
*2nd generation EIA*viral lysate EIA
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Management of HIVManagement of HIV
• Goal is “undetectable” (<48 copies/mL) HIVGoal is undetectable (<48 copies/mL) HIV viral load (Assays vary on limit of detection from <75 to <20 copies/mL)from <75 to <20 copies/mL)
• Monitor the HIV viral load : 3 months once goal is achievedgoal is achieved
• CD4 counts can be repeated : 6‐12 months if i l l d ll dviral load controlled
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CD4 T LymphocyteCD4 T‐Lymphocyte• CD4 T‐lymphocyte countsCD4 T lymphocyte counts
used for:– Determining clinical prognosis
– Assessing criteria for antiretroviral therapy
– Monitoring therapyM l d t t d• Manual and automated methods
• Issues:Requires high level of– Requires high level of technical skill for test performance and interpretation
– Properly maintained equipment
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CD4 Cell CountsCD4 Cell Counts• CD4 cell count measures the number of CD4 cells per cubic milliliter of blood.
• The CD4 count is a measure of the degree of immuno‐compromise and stage of HIV disease progression.
• The CD4 count is an important test for deciding whether ARV therapy is required and for monitoring the recovery py q g yof the immune system under treatment.
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