LABORATORY DIAGNOSIS OF HIV - AIDS

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Notes on Laboratory diagnosis of AIDS.. By Dr. Ashish V. Jawarkar Contact: [email protected]... 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 Neutralizing 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

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 CD 8 + 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

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 all-inclusive 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 well-circumscribed 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 HIV RNA HIV resistance Counts determinants testing Provides information on predicts what will helps in selecting Current immunological status happen to CD4 counts drugs and changing In near future 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 Start anti retroviral treatment Decreases by 25% Start anti retroviral treatment <200 Place on P. carinii prophylaxis <50 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 Phenotypic measurements Sequence analysis of HIV RNA genome is compared with known HIV RNA genome sequences having known resistance to drugs

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 HIV - AIDS

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