HIV Testing

Group 4
Introduction
 The human immunodeficiency virus (HIV)
infection and acquired immunodeficiency syndrome
(AIDS) continue to present a major challenge to
healthcare professionals and society through out much
of the world.
 Testing for AIDS virus became a cornerstone for
surveillance and prevention programs and for provision
of appropriate medical care for those who are infected.
 The controversy surrounding the tests now stems
not from a lack of confidence in their reliability but from
the perceived social consequences of an individual
being identified as infected with the AIDS virus. With
better understanding by the public of HIV infection/AIDS
and a greater acceptance of those who are infected,
this attitude can be hoped to change.

 1981
 The isolation of human T-lymphotropic virus type 1 (HTLV-I)
ended the skepticism that no retrovirus will be found to
infect humans.


 1983
 Researchers at the Pasteur Institute in Paris isolated a
retrovirus from a homosexual man with lymphadenopathy
and named it lymphadenopathy-related virus (LAV)


 1984
 The human T-lymphotropic retrovirus (HTLV-III) was isolated
by an American research team led by Robert Gallo.

 1985
 The 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.

1996
 Blood banks in the United States added the p24
antigen capture assay to the screening process
to help identify the rare infected individuals who
were donating blood in the time (up to 3 months)
between infection and the development of
antibodies.
 Window period was 16 days.


2002
 The 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.



Viral Characteristics
Structure
 HIV has a cylindrical, eccentric nucleoid or core.
The nucleoid contains the HIV genome, which is
diploid. Encoded in the genome are the entire
complement of genes of the virus. These genes
code for the structural proteins, which are used
to assemble the virus particles, and the
regulatory proteins involved in the regulation of
viral gene expression. The surface of HIV
manifests external knoblike structures formed by
the envelope glycoprotein.
Life Cycle of HIV
1.The virus attaches to the CD4 membrane receptor and sheds
its 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 host
cell.
6.
7.New virus particles are produced as the result of normal
cellular activities of transcription and translation.
8.
9.New particles bud from the cell membrane.
Epidemiology
Incidence
 Africa has been the continent hardest hit by HIV
infection and AIDS.

 In the USA, 80% of AIDS cases are reported in gay
or bisexual men, and 16% are intravenous drug
users, the majority of whom live in major
metropolitan areas.


Modes of Transmission
 The modes of transmission of HIV are similar to
those of hepatitis B, in particular with respect to
sexual, parenteral, and vertical transmission. The
risk of sexual transmission varies with particular
sexual practices.
 HIV has been isolated from blood, semen, vaginal
secretions, saliva, tears, breast milk, CSF,
amniotic fluid, and urine.
 HIV may be indirectly transmitted.
 Children born to women with HIV have a 20% to
30% risk of HIV infection.


Signs and Symptoms
 Many individuals with HIV infection remain
asymptomatic for years, with a mean time of
approximately 10 years between exposure and
development of AIDS. When symptoms occur
they may be remarkably protean and nonspecific.
Physical examination may be entirely normal.
Abnormal findings may range from completely
nonspecific to highly specific for HIV infection .
 Kaposi’s sarcoma

 HIV ScreeningTest

ENZYME
IMMUNOASSAY
Diagnosis of HIV Infection
 The CDC has recommended that screening for HIV
infection be performed as a matter of routine health
care. The diagnosis of HIV infection depends upon
the demonstration of antibodies to HIV and/or the
direct detection of HIV or one of its components. As
noted above, antibodies to HIV generally appear in
the circulation 2-12 weeks following infection.
ELISA
 The standard blood screening test for HIV infection is
ELISA, also referred to as an enzyme immunoassay
(EIA). The solid-phase assay is an extremely good
screening test with a sensitivity of >99.5%.

 Most diagnostic laboratories use a commercial EIA kit that
contains antigens from both HIV-1 and HIV-2 and thus
are able to detect either.

 EIA tests are generally scored as positive (highly reactive)
negative (nonreactive), or intermediate (partially active).
 While the EIA is an extremely sensitive test, it is not optimal
with regard to specificity. Among the factors associated with
false-positive EIA tests are antibodies to class II antigens,
autoantibodies, hepatic disease, recent influenza
vaccination, and acute viral infection. For these reactions,
anyone suspected of having HIV infection based upon a
positive or inconclusive EIA result must have the result
confirmed with a more specific assay such as the Western
blot.



 Commercial enzyme immunoassay for HIV-1
and HIV-2 use several types of anitgens, that
is, whole virus lysate, recombinant proteins,
and chemically synthesized peptides. The
first FDA-approved assays using a whole
virus lysate antigen remain the most
commonly used antigen preparation in the
United States. This is because whole virus
lysate antigens contain all the viral proteins
present in the virus.

EIA Procedures – Indirect
Assay
 The EIA screening tests for HIV-1 antibody currently
licensed in the United States use microwells or
beads coated with antigenic preparations of
inactivated and lysed whole virus.

 Diluted patient serum is added to the microwell, and
HIV-1 specific antibody, if present, is allowed to
react with the immunosorbent.

 Thorough washing is done after incubation to
remove extraneous serum proteins and unbound
antibody.
 An enzyme-conjugated second antibody specific for
human immunoglobulin is added and allowed to react
with antibody bound to the absorbed viral lysate.

 An appropriate substrate is added to the microwell, and
the color change resulting from hydrolysis of the
substrate by the enzyme is measures
spectrophotometrically.

 This is compared to a reference standard of known
concentration, this color change is directly proportional
to the amount of HIV-1-specific antibody present in the
patient specimen.
EIA Procedures – Competitive
Assay
 Competitive assay use the same types of antigens
as the indirect EIA. These antigens are bound to
a solid support, usually a microplate well.

 The patient’s sample (serum or plasma) is added to
the well simultaneously with enzyme-labeled HIV
antibody.

 During incubation, the patient’s antibodies compete
with the enzyme-labeled HIV antibody for the
limited number of antigen binding sites available
on the walls of the microplate.
 As the concentration of antibody increases in the
patient’s sample, the enzyme-labeled antibody
is displaced from the antigen binding site and
the color development is less than that
observed with antibody negative specimens.

 In contrast to the indirect assays, the intensity of
the color change observed in the competitive
EIA is inversely related to the amount of HIV
antibody present in the test sample.
PARTICLE
AGGLUTINATION ASSAYS
HIV Screening Test
Particle Agglutination Assay
 The most widely used test for HIV.
 The assay is simpler to perform than the
standard EIA and requires practically no
equipment.
 Well suited for use in large seroprevalence
surveys.
 Has been found to be slightly less sensitive
than EIA.
Procedure
 Mix 25 uL of diluted serum with a suspension
of gelatin particles coated with purified HIV
antigen.
 The assay is performed in a microplate and
can be read after incubating for two hours
at room temperature.
Results
 (+) positive = sera that agglutinate coated
particles alone.
 (-) negative = sera that produce no
agglutination.
 Sera that agglutinate both coated and
uncoated particles are retested.
 Positive specimens should be confirmed by
the use of a supplemental assay such as
Western Blot.
HEMAGGLUTINATION
ASSAY
HIV Screening Test
Hemagglutination Assay
 A quantification of viruses or bacteria by
hemagglutination.
 It is an easy, simple and rapid method and
can be applied to large amounts of
samples.



LATEX AGGLUTINATION
ASSAYS
HIV Screening Test
Latex Agglutination Assay
 Has not gained wide acceptance because of
its cost, difficulty in reading and lack of
sensitivity 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 are
used as recommended.
 When performed by well-trained, experienced
technologists, the results have been found
to be reliable.
SOLID-PHASE
IMMUNOASSAY
HIV Screening Test
Solid-Phase ImmunoAssay
 Often referred to as “rapid tests” since results
can be obtained in 10-15 minutes.
 Antigens are reacted with the patient’s sera
and 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
 Read as positive or negative based on the
presence of the color reaction as compared
to 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 test
results are required.
Western blot
HIV Screening Test
More informative followup test for EIA.
Standard method for confirming HIV-1
seropositivity.

 Procedure
HIV viral protiens are seperated according
to their molecular weight.
Nitrocellulose is applied to the surface of
the gel.
To allow the proteins to migrate from the
gel to the surface of the nitrocellulose
an electric current will be applied.
Block and wash
Cut the nitrocellulose into strips.
Serum will be incubated in the strip
If present, Ab to HIV proteins will bind to
the viral Ag on the surface of the
nitrocellulose.

Proteins:
Env
Pol
Gag

Most reports indicate that Ab to gag
proteins appear first.



Disadvantages


Time consuming and expensive

Many source of error




Immunoflourescence assay

Commonly used in the lab
Less expenssive

 Infected cells and uninfected cells are
mixed or placed on the separate area of
the slide. UNINFECTED cells serves as
negative controls.
 Incubated with the serum
 Wash
 Incubate with flourescencein
isothiocyanate-labeled anti-human IgG.
 Observe the flourescence pattern using
flourescent microscope.



Radioimmunoassay

2 Techniques


Solid-phase
Liquid-phase
 Solid phase
 Microtiter wells are coated with
monoclonal or polyclonal antibody to
HIV-1 proteins
 Add the sample and incubate
 Wash
 Add the I-labeled IgG (anti-HIV) solution
 Wash
 Cut the wells
 Radioactivity is measured with gamma
counter
 Liquid phase
Serum or cultured medium containing
HIV-1 are mixed with I-labeled IgG in a
small volume liquid
Incubate
Centrifuge
Wash
Measure the radio activity
References
 AIDS Testing Methodology and Management Issues by Gerald
Schochetman and J. Richard George
 Blood, 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 Turgeon
 2006 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.
 Harrison’s 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)
