Laboratory Diagnosis of AIDS Notes

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Notes on Laboratory diagnosis of AIDS.. By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com... Facebook: www.facebook.com/pathologybasics

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OVERVIEW
1. Structure of HIV
2. Understanding immune response to HIV
a. Humoral response
 Binding antibodies
 Neutralising antibodies
b. Cellular response
 CD4 cells
 CD8 cells
3. Diagnosis of AIDS
Antibody detection
a. Screening tests
 Particle agglutination tests
 Specialized rapid spot test
b. Confirmatory tests
 Western blot
Antigen detection
 p24 antigen capture
 RT – PCR
 bDNA
 NASBA
4. Laboratory monitoring of anti retroviral therapy
a. CD4+ T cell counts
b. HIV RNA determinants
c. HIV resistance testing


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* STRUCTURE OF HUMAN IMMUNODEFICIENCY VIRUS


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* IMMUNE RESPONSE TO HIV INFECTION
(I) HUMORAL RESPONSE
Binding antibodies
1. appear within 6-12 weeks of viral infection
2. forms basis of most screening tests
3. First antibodies detected are those against
structural proteins like p24 –
This correlates with plasma leves of p24
antigen.
4. This is followed by appearance of
antibodies to env (gp41) proteins

Neutralizing antibodies
1. these appear following decrease in viral
load
2. co relate with appearance of CD8+
lymphocytes
3. They are –
Type specific – directed against V3 loop
region – neutralizes viruses of specific strain
Group specific – neutralizes wide variety of
HIV isolates

only antigen can be detected in this period

(II) CELLULAR RESPONSE
CD 4+ cells
1. These cells though first to be infected and
get destroyed, have high affinity for HIV
infected cells
2. They can undergo clonal expansion in
response to HIV antigens
In response to high viral loads, the response
shifts from proliferation to one of IFN-γ
production

CD 8 + cells
1. CTCLs have been identified within weeks
of HIV infection
2. They bind to infected cells and cause lysis
through class I MHC molecules
3. Patients who mount a good CTCL response
have a favourable prognosis than those who
don’t.


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* DIAGNOSIS
Introduction
1. An arsenal of laboratory methods is available to screen blood, diagnose infection, and
monitor disease progression in individuals infected by HIV.
2. These tests can be classified into those that: 1) detect antibody, 2) identify antigen, 3)
detect or monitor viral nucleic acids, and 4) provide an estimate of T-lymphocyte
numbers (cell phenotyping).

General Considerations
Tests to detect antibody to HIV can be further classified as:
1) screening assays, which are designed to detect all infected individuals, or
2) confirmatory (supplemental) assays, which are designed to identify individuals who
are not infected but who have reactive screening test results.
Accordingly, screening tests possess a high degree of sensitivity, whereas confirmatory
assays have a high specificity.

Early Detection and the Window Period
1. Antibody may be present at low levels during early infection but not at the detection
limit of some assays.
2. Using the early-generation tests, antibody could be detected in most individuals by 6 to
12 weeks after infection. Newer-generation assays, including the third-generation antigen
sandwich assays, can detect antibody at about 3-4 weeks after infection.
3. This window period before the detection of antibody can be shortened by several days
using antigen tests, and by several more days using nucleic acid detection methods.
Therefore, in most individuals, the window period may be only 2-3 weeks if an allinclusive testing strategy is used.


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Tests to Screen for HIV Infection

Considerations in Choosing a Screening Test Methodology
Regardless of the particular screening test used, serum or plasma samples first are tested
(screened) using a test with high sensitivity, most often an enzyme-linked immunosorbent
assay (ELISA), "rapid test," or "simple method" (described below).
More recently, tests have been developed using fluids that can be obtained conveniently
outside the clinical laboratory. Whole blood from fingerstick and oral fluid (saliva) has
been shown to be as effective as serum or plasma for detecting antibodies to HIV.
Reactive Results
Regardless of the screening method, a sample producing a reactive result must be
screened again in duplicate, with at least 2 of the 3 results being repeatedly reactive
before verifying infection with confirmatory assays. The most common reason for
nonrepeatable results by screening tests is technical error.
Samples that produce repeatedly reactive results by screening tests must be further tested
using confirmatory tests, or other confirmatory strategies (see below).

HIV Screening Assays

Enzyme-Linked Immunosorbent Assays/Enzyme Immunoassays
ELISA is the most commonly used type of test to screen for HIV infection because of its
relatively simple methodology, inherent high sensitivity, and suitability for testing large
numbers of samples, particularly in blood testing centers.
A common feature of all varieties of ELISA is the use of enzyme conjugates that bind to
specific HIV antibody, and substrates/chromogens that produce color in a reaction
catalyzed by the bound enzyme conjugate.
The most popular ELISA involves an indirect method in which HIV antigen is attached to
a well of a 96-well microtiter plate. Antibody in the sample is allowed to react with the
antigen-coated solid support, usually for 30 minutes at 37º C or 40º C.
After a wash step to remove unbound serum components, addition of a conjugate (an
antihuman immunoglobulin with a bound enzyme) binds to the specific antibody that is
attached to the antigens on the solid phase.


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Following another wash, addition of an appropriate substrate results in color development
that is detected by a spectrophotometer and is proportional to specific HIV antibody
concentration in the sample.
Alternate ELISA methodologies include a competitive format in which specific HIV
antibody in the sample competes with an enzyme-bound antibody reagent for antigen
sites on the solid phase. In this method, color development is inversely proportional to
specific HIV antibody concentration.
A more recent addition to ELISA technology is the antigen sandwich method in which an
enzyme (alkaline phosphatase or horseradish peroxidase) is conjugated to an HIV antigen
(similar to the immobilized antigen on the solid phase). The antibody in the sample is
"sandwiched" between 2 antigen molecules, 1 immobilized on the solid phase and 1
containing the enzyme. Subsequently, the addition of substrate results in color
development in proportion to antibody concentration. The antigen sandwich ELISA is
considered the most sensitive screening method, given its ability to detect all isotypes of
antibody (including IgM).(2) One disadvantage of this method is the relatively large
volume (150 µL) of sample required, which may make repeat testing and testing of
samples from infants difficult.


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Rapid tests
1. Rapid assays for detecting specific HIV antibody were developed in the late 1980s,
and are defined as tests that can yield results in <30 minutes.
2. Technical errors are common with these assays, however, because users become
careless with these simple procedures. For example, pipettes are not always held in a
vertical position as recommended, resulting in an incorrect delivery of reagent volumes.
In addition, many laboratory workers attempt to test multiple samples simultaneously,
resulting in inaccuracies in the timing of steps.
3. When performed correctly, rapid HIV assays are accurate and have wide utility in a
number of testing situations. Application includes emergency rooms, physicians' offices,
point-of-care testing, autopsy rooms, funeral homes, small blood banks, and situations
involving stat HIV testing (where immediate treatment is recommended for exposures).
4. Rapid HIV assays have proven particularly useful for testing pregnant women in labor
who have not received prenatal care (ie, of unknown HIV status).
5. Importantly, these rapid assays are easy to perform and have utility in developing
countries, where facilities may not be optimal, stable electricity may be unavailable, and
formal education programs for laboratorians are absent.
Dot blot/immuno blot test
1. One class of rapid tests is the "dot blot" or "immunoblot"; they produce a wellcircumscribed colored dot on the solid phase surface if the test is positive.
2. Most of these rapid assays now incorporate a built-in control to indicate that the test
was performed correctly. This control is an antihuman immunoglobulin that binds any
immunoglobulin in the sample and produces a separate indicator when all reagents are
added appropriately.
3. In addition, several varieties are available that include 2 "dots," which allows the
differentiation of HIV-1 and HIV-2 infection.
40 The procedures for the dot-blot assays are similar regardless of the exact format of the
test. Most require drop-wise additions of reagents in the following sequence: buffer,
sample, wash buffer, conjugate, wash buffer, substrate, and stop solution. Some assays
substitute an IgG binding dye (protein A gold reagent) for the antiimmunoglobulin
conjugate, thereby decreasing the procedure by a step.


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immunochromatographic assays
1. The newer 1-step rapid assays, also known as immunochromatographic assays, are
convenient, self-contained tools for HIV serologic testing, consisting of a flat cartridge
device, usually plastic or paper.
2. Whole blood, oral fluid, or serum is placed at the tip of the device and allowed to
diffuse along a strip that is impregnated with reagents (often protein A colloidal gold)
that bind and permit visual detection of HIV antibodies; some use third-generation
(antigen sandwich) technology.
3. These tests can be completed in <10 minutes (some within 2 minutes), require little or
no addition of reagents, and contain a built-in quality-control reagent to control for
technical errors.
4. Some tests can be stored at a wide range of temperatures (from 15º C to 30º C), and are
transported easily. For example, one type (Determine; Abbott) comes in "cards" .The
cards require no reagents, just addition of serum or plasma.
5. Other rapid test formats include dipsticks, in which antigen is attached on the "teeth"
of comblike devices; several of these rapid tests have the ability to differentiate HIV-1
and HIV-2.


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HIV cassette device


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Comb aids device.
Simple Tests (Agglutination assays)
1. This type of HIV test requires longer than 30 minutes for results, but consists of
procedures that can be performed easily without instrumentation.
2. Within this class of tests are agglutination assays in which antigen-coated particles
(red blood cells, latex particles, or gelatin particles) are allowed to react with serum
antibodies to form visible clumping (agglutination).
3. If red blood cells are used, the technique is termed passive hemagglutination; with the
use of latex particles, it is known as latex agglutination.


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Tests to Confirm HIV Infection
1. Most testing algorithms require the use of very specific assays, such as the Western
blot, indirect fluorescent antibody (IFA) assay, or the radioimmunoprecipitation assay
(RIPA), to verify reactive screening test results.
2. If performed and interpreted correctly, these extremely specific tests should not
produce biologic false-positive results. They are, however, more laborious and more
expensive than screening assays.

Western Blot Test
(gold standard)
Methodology
It is based on using an electrophoretic technique to separate HIV antigens derived from a
lysate of virus grown in culture.
This technique denatures the viral components, imparts a negative charge to the antigens,
and separates them primarily on the basis of their molecular weights.
The separation of antigens in the technique allows for the identification of specific
antibodies to each of the viral antigens in a subsequent set of steps similar to the ELISA
methodology.
1. A purified HIV antigen mixture is layered onto a sodium dodecyl sulphate (SDS)
polyacrylamide gel slab and then electrophoresed.
2. The viral proteins (HIV antigens) migrate through the molecular pores of the gel at
rates determined by electrical charge and molecular weight.
3. The proteins with higher molecular weight migrate less and form bands closer to the
starting point. The proteins on the gel are then transferred ("blotted") to nitrocellulose
paper by another electrophoretic procedure.
4. This paper is cut into thin strips, each with the full distribution of viral protein antigen
bands. A single test strip is incubated with a 1:50 or 1:100 dilution of a test sample or a
control and then washed and incubated with a labeled (tagged) antihuman globulin. At
this point, the procedure is similar to any other indirect immunoassay.
5. The label usually is an enzyme (horseradish peroxidase or alkaline phosphatase) that
will react with a specific colorless substrate to produce an insoluble colored band on the
strip wherever there is an antigen-antibody complex.
6. Reaction with a positive serum sample produces a pattern of bands on the strip that is

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characteristic of HIV. Many of these bands have been identified as specific viral gene
products.
7. The HIV-1 viral antigens are separated as follows (from top to bottom): gp160, gp120,
p66, p55, p51, gp41, p31, p24, p17, and p15. The "gp" designation refers to
glycoproteins; "p" indicates proteins. The numeric values (x100) indicate molecular
weights.

Interpretation of Results
1. Depending on the particular antibodies in the sample, reactivities with the separated
antigenic components result in band profiles. The type of profile (the combination and
intensity of bands that are present) determines whether the individual is considered
positive for antibodies to HIV.
2. The classification of Western blot results is determined by certain criteria. Most
institutions now follow the CDC guidelines, which require reactivity to at least 2 of the
following antigens: p24, gp41, gp120/160 for a positive classification. It is now
universally accepted that a negative result is the absence of all bands.


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Indirect Immunofluorescent Antibody Assay
In this technique, cells (usually lymphocytes) are infected with HIV and are fixed to a
microscope slide.
Serum containing HIV antibodies is added and reacts with the intracellular HIV.
The slide is washed and then allowed to react with antiimmunoglobulin antibodies with a
covalently bound fluorescence label attached.
The reaction is visualized using a fluorescent microscope.
This technique has the advantage of sometimes providing definitive diagnosis of samples
that have yielded indeterminate results by Western blot analysis.
Disadvantages to its use include the requirement of an expensive microscope and a
subjective interpretation, thus necessitating well-trained individuals.
Modified Western Blot
Western blot assays that have the ability to identify and differentiate infections by HIV-1
and HIV-2 have been developed.
Most incorporate the use of viral lysates from HIV-1 and synthetic peptides artificially
applied from HIV-2 on the same nitrocellulose strip (a modified or augmented Western
blot).
In this case, multiple HIV-1 antigens and 1 HIV-2-specific band (gp36 or gp41) are
present on the strip.
Criteria established by manufacturers include reactions to 1 gene product from each of
the 3 major groups (Gag, Pol, and Env) for positivity for HIV-1.
To be considered positive for HIV-2, the test must show reactions to the HIV-2-specific
antigen plus a reaction to HIV-1-specific antigens, which alone do not meet the criteria
for positivity for HIV-1.
Line Immunoassay
Another alternative to the classic Western blot and IFA confirmatory tests is the line
immunoassay (LIA).


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In this assay, recombinant or synthetic peptide antigens are applied on a nitrocellulose
strip, rather than electrophoresed as in the Western blot.
This use of "artificial" antigens decreases the presence of contaminating substances
derived from cell culture that can cause interference and sometimes false reactions.

Alternatives to Classic Tests and Testing Strategies
As technology evolves, alternatives to the classic tests and testing strategies arise. Each
offers 1 or more attractive features that may simplify collection, testing, or interpretation
of results.

Oral Fluid ("Saliva") HIV Tests
Noninvasively collected specimens, such as oral fluids, have been used for HIV testing as
a more convenient alternative to blood samples. Although generally referred to as
"saliva," the fluid used for testing is actually crevicular fluid from capillaries beneath the
tooth-gum margin, which is a transudate of blood and therefore similar to the samples
used in serum-based tests. The concentration of antibodies in oral fluids is about 1/400 of
that in plasma, however, because of the dilutional effect of fluids from the salivary glands
(true saliva),(36) necessitating extremely sensitive tests that are able to detect small
quantities of antibody. The testing technology to detect these low quantities is now
available, and oral fluid tests, both ELISA and rapid tests, are accurate.(37,38)

Urine Tests
Intact IgG antibodies are found in urine, but their exact origin is unknown. The collection
of urine is simple, noninvasive, and inexpensive, and the sample can be stored at room
temperature for extended periods of time. The use of urine for testing is appropriate for
physicians' offices, health clinics, and in developing countries where health care
personnel may not be trained professionally or where clean needles for drawing blood
may not be available. The major disadvantage is that there is not an approved urine-based
confirmatory assay, necessitating the collection of blood when results are reactive. The
FDA has approved an ELISA and Western blot for use to test urine for antibodies to
HIV-1.


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Tests to detect HIV antigens
1. p24 antigen capture assay
1. ELISA type assay in which solid phase consists of antibodies to p24 antigen
2. During first few weeks, before antibody response develops to HIV, p24 antigen can be
detected in serum.
3. After that there is an equilibrium between p24 antigen and antibodies.
2. Measuring HIV RNA in plasma by




RT-PCR
bDNA
NASBA


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* Laboratory monitoring of antiretroviral therapy

CD4+ T cell
Counts
Provides information on
Current immunological status

HIV RNA
determinants
predicts what will
happen to CD4 counts
In near future

HIV resistance
testing
helps in selecting
drugs and changing
therapy

(i) CD4+ T cell counts
1. Best indicator of immediate state of immunological competence – should be
performed at time of diagnosis and every 3 to 6 months thereafter
2. calculated as
% of CD4 cells (by flow) x Total lymphocyte count

3.

If CD4 count is
<200
High risk for P. Carinii infection
<50
High risk for MAC infection

4. If CD4 count
<350
Decreases by 25%
<200
<50

Start anti retroviral treatment
Start anti retroviral treatment
Place on P. carinii prophylaxis
Place on MAC prophylaxis

(ii) HIV RNA determinants
1. Method used is RTPCR and bDNA
2. should be done at time of diagnosis and every 3-4 months therafter
3. Target is to get RNA copies to <50/ml – known as steady state – achieved usually
within 6 months of therapy
4. Treatment to be started when copies are >50,000/ml


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(iii)HIV resistance testing
Genotypic measurements
Sequence analysis of HIV RNA genome is
compared with known HIV RNA genome
sequences having known resistance to
drugs

Phenotypic measurements
Growth of viral isolates of the patient is
compared with growth of reference strains.

In hands of an expert, resistance testing enhances short term ability to decrease viral load
by 0.5 log compared to changing drugs merely on the basis of drug history.


Laboratory Diagnosis of AIDS Notes

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