Immunologic Methods in Diagnosis of HIV Infection

M P i Ph DM Parsania, Ph.D.Tehran Medical Sciences Branch, Islamic Azad 

UniversityUniversity

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.

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

• 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

HIV Structure

surface transmembrane

matrix protein

capsid protein

nucleocapsid protein

RTIntegraseprotease

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

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.

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.

HIV ‐ Life Historyh

HIV ‐ Life HistoryhCCR5 ‐ the co receptorCCR5 ‐ the co receptor

HIVHIV

chemokineMutant

CD4

CD4CD4

CCR5CCR5

CCR5

macrophage

Virion interaction with CD4 receptor and CXCR4 co‐receptorreceptor

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 

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. 

Retroviruslife cycle:

HIV and AIDSThe cellular and immunological picture - The course of the diseaseThe cellular and immunological picture The course of the disease

HIV Diagnostic Testing

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

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

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

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

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

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

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

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

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Common HIV‐1 or HIV‐1/2 DiagnosticAlgorithm

APHL/CDC HIV‐1/2 DiagnosticAlgorithm Template

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

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

Body Fluids Used for HIV Rapid Testing

• Serum                              • Plasma• Whole bloodWhole blood                      • Oral fluids

Reading Results

Reactive Control

Positive

P iti N ti

HIV-1/2

Positive Negative

Read results in 20 – 40 minutes

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

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)

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

How Particle Agglutination WorksAnti‐HIV antibodies bind to the antigen‐coated latex 

How Particle Agglutination Works

particles..

Antigen

Antibody

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

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

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)

HIV-1 Western Blot AntigensHIV 1 Western Blot Antigens

p = protein

gp = glycoprotein

Number = molecular weightNumber = molecular weight

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)

HIV-1 and HIV-2 Gene Products &W BlWestern Blot

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

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

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

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.

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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

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

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

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

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

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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