HIV Flashcards
Lymphadenopathy Associated Virus (LAV)
Luc Montagnier at the Pasteur Institute in France
1983:
Luc Montagnier at the Pasteur Institute in France
Human T Lymphotropic Virus Ill
Robert Gallo of US confirmed the discovery of the virus
1984
Robert Gallo of US confirmed the discovery of the virus
HUMAN IMMUNODEFICIENCY VIRUS (HIV)
President Mitterand of France and President Reagan of US resolved the issue
TWO TYPES OF HIV VIRUS
HIV-1
HIV-2
: HIV virus common worldwide
HIV-1
: HIV virus isolated in Africa
HIV-2
3 Groups of HIV-1
Group M
Group N
Group O
– major group
Group M
– non m/non
Group N
– outlier group
Group O
HIV Genus
Lentivirinae
HIV Family
Retroviridae
HIV Contains 2 positive single stranded RNA
SSrNA
HIV Structure
[?] in diameter
100nm
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
You cannot get HIV from:
Kissing
Hugging
Sharing food
Insect bites
Toilet seats
Bathing together
Sneezes and cough
Heat
HIV main MOT
Sex without condom
Target cell of HIV:
CD4 T cells (T helper cells)
Important Proteins
Gp41/Gp120 comples
p24
p17
• Large glycoprotein that traverses the bilipid layer
Gp41/Gp120 comples
• Gp120 and CD4 complex will interact with CCR5 and CXCR4 receptors of CD4 T cells p24
Gp41/Gp120 comples
• Nucleocapsid core protein
p24
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
- Progressive depletion of T4 cells
- Reversing the normal CD4:CD8 ratio (normal 2:1) to as low as 0.5:1
- 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?
- Small size of pathogen especially HIV
- Antibodies are widely distributed
- Longer detection time of antibodies
- 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
- HIV attaches to [?]
host CD4 cell
- DNA is made from HIV’s RNA via [?]
reverse transcriptase
- HIV DNA is [?] into host DNA (integrase)
integrated
- Viral components are [?]
reproduced
- HIV virus is [?]
assembled
- 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
