hiv testing cao.pptx

8/14/2019 HIV Testing CAO.pptx

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Group 4

HIV Testing


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The human immunodeficiency virus (HIV)infection and acquired immunodeficiency syndrome(AIDS) continue to present a major challenge tohealthcare professionals and society through out muchof the world.

Testing for AIDS virus became a cornerstone for

surveillance and prevention programs and for provisionof appropriate medical care for those who are infected.

The controversy surrounding the tests now stemsnot from a lack of confidence in their reliability but fromthe perceived social consequences of an individualbeing identified as infected with the AIDS virus. With

better understanding by the public of HIV infection/AIDSand a greater acceptance of those who are infected,this attitude can be hoped to change.

Introduction


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1981The isolation of human T-lymphotropic virus type 1 (HTLV-I)

ended the skepticism that no retrovirus will be found toinfect humans.

1983 Researchers at the Pasteur Institute in Paris isolated a

retrovirus from a homosexual man with lymphadenopathyand named it lymphadenopathy-related virus (LAV)

1984The human T-lymphotropic retrovirus (HTLV-III) was isolated

by an American research team led by Robert Gallo.

1985The first tests became available to identify antibody to a

retrovirus, the human immunodeficiency virus (HIV). Interval between infection and detection (window period) was

22 days.


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1996Blood banks in the United States added the p24

antigen capture assay to the screening process

to help identify the rare infected individuals whowere donating blood in the time (up to 3 months)between infection and the development ofantibodies.

Window period was 16 days.

2002The licensure of nucleic acid testing (NAT) as a

routine part of blood donor screening allowed the

early detection of HIV infection.Window periods was 12 days.


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StructureHIV has a cylindrical, eccentric nucleoid or core.

The nucleoid contains the HIV genome, which isdiploid. Encoded in the genome are the entirecomplement of genes of the virus. These genes

code for the structural proteins, which are usedto assemble the virus particles, and theregulatory proteins involved in the regulation ofviral gene expression. The surface of HIVmanifests external knoblike structures formed by

the envelope glycoprotein.

Viral Characteristics


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1.The virus attaches to the CD4 membrane receptor and shedsits protein coat, exposing its RNA core.

2.

3.Reverse transcriptase converts viral RNA into proviral DNA.

4.

5.The proviral DNA is integrated into the genome of the hostcell.

6.

7.New virus particles are produced as the result of normalcellular activities of transcription and translation.

8.

9.New particles bud from the cell membrane.

Life Cycle of HIV


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IncidenceAfrica has been the continent hardest hit by HIV

infection and AIDS.

In the USA, 80% of AIDS cases are reported in gayor bisexual men, and 16% are intravenous drugusers, the majority of whom live in majormetropolitan areas.

Epidemiology


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Modes of TransmissionThe modes of transmission of HIV are similar to

those of hepatitis B, in particular with respect tosexual, parenteral, and vertical transmission. Therisk of sexual transmission varies with particularsexual practices.

HIV has been isolated from blood, semen, vaginalsecretions, saliva, tears, breast milk, CSF,amniotic fluid, and urine.

HIV may be indirectlytransmitted.Children born to women with HIV have a 20% to

30% risk of HIV infection.


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Signs and SymptomsMany individuals with HIV infection remain

asymptomatic for years, with a mean time ofapproximately 10 years between exposure anddevelopment of AIDS. When symptoms occur

they may be remarkably protean and nonspecific.Physical examination may be entirely normal.Abnormal findings may range from completelynonspecific to highly specific for HIV infection .

Kaposis sarcoma


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ENZYMEENZYME

IMMUNOASSAYIMMUNOASSAY

HIV ScreeningTest


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Diagnosis of HIV Infection

The CDC has recommended that screening for HIVinfection be performed as a matter of routine healthcare. The diagnosis of HIV infection depends upon

the demonstration of antibodies to HIV and/or thedirect detection of HIV or one of its components. Asnoted above, antibodies to HIV generally appear inthe circulation 2-12 weeks following infection.


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ELISA

The standard blood screening test for HIV infection isELISA, also referred to as an enzyme immunoassay(EIA). The solid-phase assay is an extremely goodscreening test with a sensitivity of >99.5%.

Most diagnostic laboratories use a commercial EIA kit thatcontains antigens from both HIV-1 and HIV-2 and thusare able to detect either.

EIA tests are generally scored as positive (highly reactive)negative (nonreactive), or intermediate (partially active).


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While the EIA is an extremely sensitive test, it is not optimalwith regard to specificity. Among the factors associated withfalse-positive EIA tests are antibodies to class II antigens,autoantibodies, hepatic disease, recent influenzavaccination, and acute viral infection. For these reactions,

anyone suspected of having HIV infection based upon apositive or inconclusive EIA result must have the resultconfirmed with a more specific assay such as the Westernblot.


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Commercial enzyme immunoassay for HIV-1and HIV-2 use several types of anitgens, thatis, whole virus lysate, recombinant proteins,and chemically synthesized peptides. Thefirst FDA-approved assays using a whole

virus lysate antigen remain the mostcommonly used antigen preparation in theUnited States. This is because whole viruslysate antigens contain all the viral proteins

present in the virus.


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EIA Procedures IndirectAssay The EIA screening tests for HIV-1 antibody currentlylicensed in the United States use microwells or

beads coated with antigenic preparations ofinactivated and lysed whole virus.

Diluted patient serum is added to the microwell, andHIV-1 specific antibody, if present, is allowed toreact with the immunosorbent.

Thorough washing is done after incubation to

remove extraneous serum proteins and unboundantibody.


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An enzyme-conjugated second antibody specific forhuman immunoglobulin is added and allowed to react

with antibody bound to the absorbed viral lysate.

An appropriate substrate is added to the microwell, andthe color change resulting from hydrolysis of the

substrate by the enzyme is measuresspectrophotometrically.

This is compared to a reference standard of knownconcentration, this color change is directly proportional

to the amount of HIV-1-specific antibody present in thepatient specimen.


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EIA Procedures CompetitiveAssay

Competitive assay use the same types of antigensas the indirect EIA. These antigens are bound toa solid support, usually a microplate well.

The patients sample (serum or plasma) is added to

the well simultaneously with enzyme-labeled HIVantibody.

During incubation, the patients antibodies competewith the enzyme-labeled HIV antibody for the

limited number of antigen binding sites availableon the walls of the microplate.


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As the concentration of antibody increases in the

patients sample, the enzyme-labeled antibodyis displaced from the antigen binding site andthe color development is less than thatobserved with antibody negative specimens.

In contrast to the indirect assays, the intensity ofthe color change observed in the competitiveEIA is inversely related to the amount of HIV

antibody present in the test sample.


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PARTICLEAGGLUTINATION ASSAYS

HIV Screening Test


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Particle Agglutination Assay

The most widely used test for HIV.The assay is simpler to perform than the

standard EIA and requires practically noequipment.

Well suited for use in large seroprevalencesurveys.

Has been found to be slightly less sensitive

than EIA.


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Procedure

Mix 25 uL of diluted serum with a suspensionof gelatin particles coated with purified HIVantigen.

The assay is performed in a microplate and

can be read after incubating for two hoursat room temperature.


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Results

(+) positive = sera that agglutinate coatedparticles alone.

(-) negative = sera that produce noagglutination.

Sera that agglutinate both coated anduncoated particles are retested.

Positive specimens should be confirmed bythe use of a supplemental assay such asWestern Blot.


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HEMAGGLUTINATIONASSAY

HIV Screening Test


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Hemagglutination Assay

A quantification of viruses or bacteria byhemagglutination.

It is an easy, simple and rapid method andcan be applied to large amounts of

samples.


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LATEX AGGLUTINATIONASSAYS

HIV Screening Test


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Latex Agglutination Assay

Has not gained wide acceptance because ofits cost, difficulty in reading and lack ofsensitivity and specificity compared to EIA.

Very easy to perform and produces results in

less than 10 minutes. Difficult to read even when the appropriate

high-intensity light and magnification areused as recommended.

When performed by well-trained, experiencedtechnologists, the results have been foundto be reliable.


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SOLID-PHASEIMMUNOASSAY

HIV Screening Test


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Solid-Phase ImmunoAssay

Often referred to as rapid tests since resultscan be obtained in 10-15 minutes.

Antigens are reacted with the patients seraand the resulting immune complexes are

trapped on to filters. Detection of immune complexes is by the

sequential reaction with enzyme-anti-human IgG conjugates and then a

precipitating substrate. Color reaction occurs where immune

complexes are present and stains the


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Read as positive or negative based on thepresence of the color reaction as comparedto positive and negative control spots.

Gaining popularity due to their simplicity and

rapid results. Used in situations where standard EIAs

cannot be performed or immediate testresults are required.


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Western blotWestern blot

HIV Screening Test


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More informative followup test for EIA.Standard method for confirming HIV-1

seropositivity.


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ProcedureProcedure

HIV viral protiens are seperated accordingto their molecular weight.

Nitrocellulose is applied to the surface ofthe gel.

To allow the proteins to migrate from thegel to the surface of the nitrocellulosean electric current will be applied.


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Block and washCut the nitrocellulose into strips.Serum will be incubated in the stripIf present, Ab to HIV proteins will bind to

the viral Ag on the surface of thenitrocellulose.


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Proteins:EnvPolGag

Most reports indicate that Ab to gagproteins appear first.


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Disadvantages

Time consuming and expensiveMany source of error


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Immunoflourescence assay


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Commonly used in the labLess expenssive


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Infected cells and uninfected cells aremixed or placed on the separate area ofthe slide. UNINFECTED cells serves asnegative controls.

Incubated with the serum Wash Incubate with flourescencein

isothiocyanate-labeled anti-human IgG.

Observe the flourescence pattern usingflourescent microscope.


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Radioimmunoassay


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2 Techniques

Solid-phaseLiquid-phase


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Solid phaseSolid phase

Microtiter wells are coated withmonoclonal or polyclonal antibody toHIV-1 proteins

Add the sample and incubate

Wash Add the I-labeled IgG (anti-HIV) solution Wash

Cut the wells Radioactivity is measured with gammacounter


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Liquid phaseLiquid phase

Serum or cultured medium containingHIV-1 are mixed with I-labeled IgG in asmall volume liquid

IncubateCentrifugeWashMeasure the radio activity


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AIDS Testing Methodology and Management Issues by Gerald

Schochetman and J. Richard GeorgeBlood, Blood Products and HIV, 2nd Edition by R. Madhok, C.D.

Forbes and B.L. Evatt Immunology and Serology in Laboratory Medicine, 2nd Edition

by Mary Louise Turgeon2006 Current Medical Diagnosis and Treatment, 45th Edition

by Lawrence M. Tierney, Jr. et. al.1991 Medical Diagnosis and Treatment, 30th Edition by

Steven A. Schroeder et. al.Harrisons Principles of Internal Medicine, 17th Edition by

Fauci, Braunwald, Kasper, Hauser, Longo, Jameson,Loscalzo

The Jounal of Infectious Diseases, Volume 162 number 6(December 1990 issue)

References

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