HIV Flashcards by Arriane Cyrel (MTI)

Lymphadenopathy Associated Virus (LAV)

Luc Montagnier at the Pasteur Institute in France

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1983:

Luc Montagnier at the Pasteur Institute in France

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Human T Lymphotropic Virus Ill

Robert Gallo of US confirmed the discovery of the virus

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1984

Robert Gallo of US confirmed the discovery of the virus

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HUMAN IMMUNODEFICIENCY VIRUS (HIV)

President Mitterand of France and President Reagan of US resolved the issue

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TWO TYPES OF HIV VIRUS

HIV-1

HIV-2

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: HIV virus common worldwide

HIV-1

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: HIV virus isolated in Africa

HIV-2

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3 Groups of HIV-1

Group M
Group N
Group O

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– major group

Group M

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– non m/non

Group N

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– outlier group

Group O

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HIV Genus

Lentivirinae

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HIV Family

Retroviridae

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HIV Contains 2 positive single stranded RNA

SSrNA

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HIV Structure

[?] in diameter

100nm

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You can get HIV from:

 Sex without condom
 Passed from mother to baby (Vertical and perinatal)
 Sharing of equipment
 Contaminated transfusion transplants and blood organ

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You cannot get HIV from:

 Kissing
 Hugging
 Sharing food
 Insect bites
 Toilet seats
 Bathing together
 Sneezes and cough
 Heat

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HIV main MOT

Sex without condom

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Target cell of HIV:

CD4 T cells (T helper cells)

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Important Proteins

Gp41/Gp120 comples

p24

p17

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• Large glycoprotein that traverses the bilipid layer

Gp41/Gp120 comples

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• Gp120 and CD4 complex will interact with CCR5 and CXCR4 receptors of CD4 T cells p24

Gp41/Gp120 comples

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• Nucleocapsid core protein

p24

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One of the earliest marker to HIV infection

p24

The presence of [?] does not mean (+) for HIV

p24

• Matrix shell protein

p17

Important Enzymes

Reverse transcriptase Integrase Protease

• Enables the virus to convert viral RNA to DNA

Reverse transcriptase

• (Normal process: [?])

DNA to RNA

• Inserts viral DNA into host DNA

Integrase

Making it impossible to kill the virus; ability to persist in the host

Integrase

• Cleaves other enzymes and structural proteins from their polyproteins; important in formation of new virion

Protease

• Progressive deterioration of the immune system due to the destruction of CD4 cells

HIV INFECTION

Normal CD4:CD8 ratio =

2:1

CD4:CD8 ratio With HIV =

0.5:1

can occur in HIV INFECTION

Opportunistic infections

STAGES OF HIV INFECTION

Primary Stage Intermediate Stage Final Stage

Acute HIV

Primary Stage

Clinical Latency

Intermediate Stage

Opportunistic infections

Final Stage

• May be asymptomatic

Primary Stage

• Development of flu-like symptoms

Primary Stage

• Lymphadenopathy

Intermediate Stage

• Fever

Intermediate Stage

• Weight loss

Intermediate Stage

• Diarrhea

Intermediate Stage

• Fatigue, night sweats

Intermediate Stage

Intermediate Stage Labs:

T4 cells < 400/mm2 CD4:CD8 ratio is <1 (vs. normal ratio of 2:1) Thrombocytopenia, Leukopenia, Anemia

Aka HIV dormancy

Clinical Latency

Some may show s/s, while others may show little to none

Clinical Latency

Lasts 10 years or longer for some

Clinical Latency

rapid multiplication of the virus

Acute HIV

high viral load = high risk of transmission

Acute HIV

(inflamed/swollen lymph nodes)

Lymphadenopathy

Acquired Immune Deficiency Syndrome - AIDS (after 2-10 years)

Final Stage

Final Stage Labs:

T4 cells < 200/mm2

Opportunistic infections in Final Stage:

Pneumocystis jiroveci (formerly carinii) Candidiasis CMV Herpes simplex M. tb Kaposi’s sarcoma Hairy leukoplakia

(formerly carinii)

Pneumocystis jiroveci

HIV IMMUNOLOGIC FEATURES

1. Progressive depletion of T4 cells 2. Reversing the normal CD4:CD8 ratio (normal 2:1) to as low as 0.5:1 3. First detectable serologic marker is the core protein p24

HIV Screening tests

ELISA

HIV Confirmatory tests

Western blot assay

Solid phase assay

ELISA

>99.5% sensitivity

ELISA

Gold standard

Western blot assay

Most sensitive and specific for HIV-1

Western blot assay

Detects IgG Ab specific to HIV Ag (Ab against core protein p24)

Western blot assay

Positive results are bands on nitrocellulose membrane strip

Western blot assay

Why is antibody detection more commonly used?

1. Small size of pathogen especially HIV 2. Antibodies are widely distributed 3. Longer detection time of antibodies 4. Antibody testing is more practical

Assay Generations

1st Generation 2nd Generation 3rd Generation 4th Generation

• viral lysate

1st Generation

• recombinant disrupted virion

1st Generation

• use of biological vehicle

2nd Generation

• Synthetic peptide - mimics CHON

3rd Generation

• Peptide synthesizer

3rd Generation

• Recombinant Ag, Synthetic Peptide, and Monoclonal antibodies

4th Generation

1st Generation

western blot

2nd Generation

recombinant Ag - placed in fungi

3rd Generation

most of the test (test kits)

4th Generation

ELISA

"lateral flow" assay

Immunochromatography

Most widely used test for HIV screening

Immunochromatography

Easy to carry; used in outreaches; HIV tests in Session Road and lab exp in the lab

Immunochromatography

Advantages storage at room temperature, ease of use, and fast results, field testing

Immunochromatography

Disadvantages less sensitive and not recommended in blood banks

Immunochromatography

One of the first serological test developed for HIV

Enzyme-linked Immunosorbent assay (ELISA)

worldwide use

Enzyme-linked Immunosorbent assay (ELISA)

Advantages high sensitivity and specificity

Enzyme-linked Immunosorbent assay (ELISA)

Disadvantages false positives and machine maintenance; due to high sensitivity

Enzyme-linked Immunosorbent assay (ELISA)

Used by big hospitals and laboratories

Luminescence Assays

Relative light unit (luminometer)

Luminescence Assays

Advantages high sensitivity and specificity

Luminescence Assays

Disadvantages false positives, expensive maintenance issues

Luminescence Assays

Widely used in blood banks before

Agglutination

Results are difficult to distinguish; Need expertise

Agglutination

gelatin particles or RBC as carriers (TPHA)

Agglutination

Advantages differentiating test, high sensitivity and specificity

Agglutination

Disadvantages subjective reading, prozone phenomenon or Ab excess

Agglutination

obsolete test

Immunoconcentration Immunodot Assay

Aka "flow through" assay

Immunoconcentration

Same as Lateral flow or Immunochromatography

Immunoconcentration

Advantages storage at room temperature, ease of use, and fast results, field testing

Immunoconcentration

Synthetic test

Line Immunoassay

Disadvantages less sensitive, less specific, subjective reading

Immunoconcentration

Principle: Solid-phase ELISA

Immunodot Assay

Aka "dipstick" ELISA

Immunodot Assay

Same as Lateral flow but observation on dots instead of band or line

Immunodot Assay

Advantages highly specific, ease of use

Immunodot Assay

Disadvantages need ref for storage, subjective

Immunodot Assay

gold standard for HIV

Western blotting

Protein detection

Western blotting

Confirmatory test

Western blotting

Performed by the National Reference Laboratories

Western blotting

: followed to confirm truly (+) results

CDC/ASPHL criteria

Advantages very specific

Western blotting

Disadvantages tedious procedure, trained personnel

Western blotting

Requires electrophoresis after testing

Western blotting

According to these criteria, a result should be reported as positive if at least two of the following three bands are present:

o p24 o gp41 o gp120/gp160

A [?] test result is reported if either no bands are present.

negative

Specimens that have some of the characteristic bands present but do not meet the criteria for a positive test result are considered to be [?]

Neither positive nor negative; in between

indeterminate

Repeat result after 3 months

indeterminate

Only 1 of the bands mentioned in the criteria for (+) result is present

indeterminate

Ex. Not diagnostic if only p24 is present

indeterminate

Monitoring test (for effectiveness of treatment)

CD4 and Viral Load

To check for viral load

CD4 and Viral Load

Low/high count = ineffective treatment

CD4 and Viral Load

Hallmark feature of HIV: presence of reverse transcriptase and integrase

CD4 and Viral Load

Laser counting

CD4 count

FACS (fluorescence activated cell sorter)

CD4 count

Detects RNA/DNA ; HIV (RNA)

PCR/NAT/Viral load

Viral load (quanti)

PCR/NAT/Viral load

Qualitative or quantitative

PCR/NAT/Viral load

old name of HIV

Lymphadenopathy Associated Virus (LAV)

Common signs and symptoms: swollen lymph nodes

Lymphadenopathy Associated Virus (LAV)

Theories for HIV:

An individual made a sexual contact with a monkey Most accepted: due to Bush meat

Butchering different types of meat in Africa; Animal blood enters the body; High conc in blood, saliva, and sweat

Bush meat

Body fluids containing high viral load:

blood and sexual fluids (semen and vaginal)

vertical transmission or perinatal - Treatment:

Antiretroviral therapy

- Decreases viral load in the blood to prevent transmission

Antiretroviral therapy

Contaminated blood transfusion and organ transplants other viruses

Hepa B, Hepa C, HIV, Syphilis

1. HIV attaches to [?]

host CD4 cell

2. DNA is made from HIV’s RNA via [?]

reverse transcriptase

3. HIV DNA is [?] into host DNA (integrase)

integrated

4. Viral components are [?]

reproduced

5. HIV virus is [?]

assembled

6. HIV virus is [?]

distributed

1st thing to happen once infected with HIV

Progressive depletion of T4 cells

Ab rises during the [?] of infection (Ab test kits)

6th week

Ag is present as early as the [?] of infection (expensive machines)

0 week

CD4 T cells is high during the [?]; declines during the acute phase, but eventually rises with Ab as Ag declines

0 week

Normal RBC size: 7-8 micrometers; HIV is like a dot compared to RBC

Small size of pathogen especially HIV

Not only detected in serum but also in other body fluids

Antibodies are widely distributed

Once increased, there will be a constant production

Longer detection time of antibodies

Ag is expensive (PCR, NAT, RIBA; Ab is cheaper (rapid test kits)

Antibody testing is more practical

Ab-testing Principle:

Target and Capture

(reagent)

Capture analyte

Target and Capture:

Antigen (capture analyte) + Antibody (target analyte) = antigen-antibody complex

Western blotting HCV:

- Srip Immunoblot Assay (SIA) - Recombinant Immunoblot Assay(RIBA)

PRINCIPLE OF WESTERN BLOT Look for [?] 1st reagent: [?] Add [?] to stop the reaction Detection signal (?)

target protein Primary Antibody enzyme-conjugated secondary antibody and enzyme substrate colorimetric or chemiluminescent

Criteria for determining a positive test result have been published by the [?].(Stevens)

Association of State and Territorial Public Health Laboratory Directors and CDC, the Consortium for Retrovirus Serology Standardization, the American Red Cross, and the FDA