HIV TESTING TECHNOLOGIES ELISA / WESTERN BLOT

There are numbers of tests They should be used in combination (strategies) Combinations must be consistent

WHO Recommended Strategies Strategy I Test all samples with one EIA Strategy II Strategy I with all reactives retested in a more specific test with different principle and/or antigen. Strategy III Strategy II with reactives tested in a third test differing from the first two tests. WHO Recommended Strategies: Because of the cost of supplemental testing WHO has devised and recommended alternative testing strategies. These strategies are designed to avoid the use of the costly and poorly standardised Western Blot. The strategies rest entirely on assays that may be conducted by screening laboratories. It is emphasised that tests must be carefully selected. Each test used in the strategy should employ a different principle and different antigen preparation. The strategy selected also depends on the prevalence of HIV infection within the population being tested. The predictive values of the strategies will change with the prevalence.

Testing Strategies AIM: To develop the logic used in establishing the use of HIV tests (testing strategies) Developing Testing Strategies: To develop testing strategies effectively you should know:- the prevalence of the infection being detected the incidence of the infection the characteristics of tests used in testing your population that a supply of the tests to be used will be constant over time the interactive properties of the tests the requirements of the clinical staff that use the laboratory All these parameters need to be available and understood for a testing strategy to be developed so that the predictive values of the strategies will be understood.

Objectives of Testing Strategies To achieve the correct diagnosis in the most efficient manner To maintain consistency in testing To develop baseline data for assessing changes To deliver useful results Objectives Of Testing Strategies: Achieving the correct diagnosis In a fully developed testing strategy, the limitations of any tests and of the testing process are understood. Tests are used appropriately. There are supporting data against which ongoing performance may be measured. Consistency Laboratory performance can only be tracked if the testing process is consistent Laboratory staff and doctors alike understand results reported with consistent test usage. Laboratory records can be maintained with consistency. Strategies are based on data therefore the predictive value of the testing process is known. The limitations of the testing process may be defined. If any changes in tests or testing occur, the changes will be understood in the context of the strategy.

Screening Assays Are used to detect antibody-- specific or nonspecific Are designed to handle large numbers of samples with rapid throughput Must be high performance Should include a full range of HIV antigens Screening or First Line Tests: Screening assays usually detect antibody. Because the assays are geared to detecting any antibody that resembles the target antibody they demonstrate false reactivity that cannot be ignored. The most commonly used screening assays are enzyme immunoassays (EIAs) and particle agglutination(PA) assays. In many countries the blood service laboratories are using fully automated micro-particle immunoassays. The assays are designed so that large sample numbers can be processed in short time frames. The use of screening assays has almost eliminated the transmission of HIV, HCV and HBV through blood transfusion when the assays are used correctly. This has been possible because most screening assays are highly sensitive (designed to detect all positive sera). Screening assays are the assays used initially in diagnostic testing strategies.

Ab + Ag AbAg The Basis of Immunoassay reactions: All immunoassays are based on an equilibrium between the binding of an antibody with an antigen. Perfect Immunoassays: To create a perfect immunoassay there would be a single, totally specific interaction, which was 100% sensitive in detecting the particular antibody and 100% specific to the analyte in question

AbAg AbAg AbAg AbAg AbAg AbAg AbAg AbAg There Is No Perfect Assay! Because humans generate a repertoire of antibodies to many proteins there is always some degree of overlap and false reactivity can occurs in 0.1 to 2 or 3% of the population for any given assay. On the other hand sometimes antibody is of low level or low concentration and therefore is not detected. When 100% sensitivity and specificity are quoted for an assay - the reported evaluation has not incorporated sufficient samples (always look for Confidence Intervals). Therefore where a result is sensitive such as for the diagnosis of HIV, the result must be as close to a100% accurate as possible. Because this is not possible using a single test multiple tests must be used in sequence to arrive at a diagnosis that is as close to 100% as possible. The careful development of testing strategy will enable this. Ab +Ag AbAg Ab +Ag AbAg

Serological Testing Strategy NEG SCREENING TEST, highly sensitive POS SUPPLEMENTAL TEST, highly sensitive & higher specificity ADDITIONAL TESTS REACTIVE A Reference Laboratory Testing Strategy: In a reference laboratory tests additional to the normal screening and first supplemental test used in diagnostic laboratories, may be performed to decipher complex reactivity in samples that may be referred from other laboratories. “Supplemental tests” used could include other EIA’s, p24, immunoassays, commercial Western Blots and molecular methods. As a reference laboratory we aim to minimise the need for follow up of samples. It should be realised that most samples will never enter this complex type of strategy. Most samples will not go beyond the first screen.

HIV Testing Strategy HIV1/2 SCREEN SCREENING NEG REACTIVE HIV-1 WB POS Principles involved in a Testing Strategy: Testing strategies are set up according to the characteristics of the tests. The screening test is a highly sensitive test which identifies anything that resembles the target. Supplemental testing sorts out true from false reactivity - tests should therefore be: highly sensitive of different configuration use different capture targets NEG SUPPLEMENTAL ADDITIONAL TESTS POS POINT OF REPORTING

The Use of Screening Assays Define samples as negative for a given analyte Enable high throughput The Use Of Screening Assays: Screening assays are used in both blood bank and diagnostic programs to exclude negative samples from any further testing in the testing strategy. Because of the very high negative predictive value of screening tests, the first test in screening programs can use the specimen as a single sample and if it is negative no further testing need occur.

Why Follow a Strategy?

The Importance of Maintaining a Strategy Consistency of laboratory records Consistency of results Clarity of results to doctors Maintaining data base to assess performances Avoiding common false reactivity Avoiding technical errors Reducing costs The Importance of Maintaining a Strategy:. Consistency of Laboratory Records The records can be set up consistently and therefore data will be in better order If a strategy is carefully followed, laboratory records will be more easily maintained. Log books or computer records can be consistent. Consistency of results If a strategy is followed the results will be more consistent because technical staff will be more familiar with assays and procedures. The staff will become more familiar with interpretation procedures. Similarly, the clarity of reported results will be consistent and easier to understand. Therefore better diagnoses will be given to patients. Databases will be more useful if tests are used consistently. Any difficulties with the strategy may be assessed from data that are carefully maintained. If assays are changed over the course of time it may become evident that there is common false reactivity among samples. This will only be detected if a strategy is followed and a database maintained. Consistency of data and results will lead ultimately to the reduction in costs as will a reduction or avoidance of technical errors. In the previous example of developing HTLV strategies the reduction in the number of referrals and indeterminate results would only have been possible if the strategy had been maintained for a sufficient period of time to analyse the results.

WHO Recommended Strategies Strategy I Test all samples with one EIA Strategy II Strategy I with all reactives retested in a more specific test with different principle and/or antigen. Strategy III Strategy II with reactives tested in a third test differing from the first two tests. WHO Recommended Strategies: Because of the cost of supplemental testing WHO has devised and recommended alternative testing strategies. These strategies are designed to avoid the use of the costly and poorly standardised Western Blot. The strategies rest entirely on assays that may be conducted by screening laboratories. It is emphasised that tests must be carefully selected. Each test used in the strategy should employ a different principle and different antigen preparation. The strategy selected also depends on the prevalence of HIV infection within the population being tested. The predictive values of the strategies will change with the prevalence.

Objectives for HIV testing Diagnosis Surveillance Blood transfusion safety

Kinetics of Antibody Response to HIV KNOWLEDGE VIRAL STRUCTURE STRUCTURAL PROTEIN OF HIV1 AND HIV 2 HIV 1 ENV – gp41, 120, 160 core – p55, 18, 24 pol – p31, 51, 65 HIV 2 ENV – gp36, 140, core – p56, 26, 16 pol – 68, 53, 34 Viral entry, Target cell (CD4) Window period IgM. IgG

HIV STRUCTURE

Different Test for HIV DIRECT INDIRECT PRE-TEST COUNSELING, INFORMED CONSENT, CONFIDENTIALITY.

Challenges of HIV Testing Sensitivity - Early diagnostic ( window period) Specificity- Cross reactivity Easy to perform, low cost Détection of HIV-1 & HIV-2 and discrimination between the two viruses One test can not fulfill these requirements Need to perform a combination of HIV tests for screening and confirmation

Detection of antibodies Current HIV technologies Detection of antibodies Screening tests Enzyme immunosorbent assays (EIAs) Simple/rapid immuno-diagnostics assays Confirmatory or supplemental tests Western blot (WB) Alternatives to confirmatory tests Repetitive EIA or rapid assays

EIAs (Enzyme Immunosorbent Assays) This term describes a variety of assays that are based on the binding of antibodies with their antigens and the detection of this reaction using a component conjugated with an active enzyme. This enzyme acts on its substrate to produce a colour change.Test results are measured by measuring this colour. Four immunologic principles Indirect Competition Sandwich Immuno-capture

Indirect EIA

Competitive EIA A measured amount of known enzyme-labeled component (being measured) is added to the reaction at the same time patient sample is added. The labeled component therefore competes against the unlabeled component in the patient sample for binding sites. Results Negative Reaction has color change Positive Reaction no color change

Sandwich EIA(Ab-Ag-Ab)

IgG-Capture EIA Serum (1/100) Goat - anti - Human - IgG Capture Antibody Biotin - Labeled Ag Strepavidin - Substrate Peroxidase

Reason for EIA This test supplied as kit Easy to Perform Use to screen large number of sample Sensitive Specific Cost effective

Reason for EIA This test supplied as kit Easy to Perform Use to screen large number of sample Sensitive Specific Cost effective

Components of Commercially Available EIA Kits Solid–Phase Support Antigens bound to polystyrene microtiter plates (passive absorption) Blocking is necessary to reduce nonspecific binding (test accuracy) 96 microwell format Antigens The use of cloned antigens has reduced non- specific binding Antibodies Monoclonals of high titer, affinity and avidity

Components of Commercially Available EIA Kits Conjugates Ab conjugated with enzyme (without effecting binding site) Enzyme HRP (horseradish peroxidase) Substrate (chromogenic) Colorless chromogen reacts with enzyme ( color) Stop Solution Typically an acid, stabilizes the color for a limited time

Sources of Error for HIV EIA Tests Documented Sources of False Negative Results Operator Error FAIL TO ADD SERUM OR REAGENT TO THE CORRECT WELL REAGENT DILUTED IN WRONG DILUENT IN WRONG DILUTION

Equipment : 1- Pipettes Single and Multi channel Pipettes should be calibrated on a monthly basis. This can be done using a balance. Inaccurate pipetting

Equipment : 2- Microplate Washers Daily Prime the washer with wash solution before running sample plates Set the washer to wash the recommended number of times (with correct volume) Check for accurate dispensing and complete aspiration in each plate well, if not clean the washer head Listen for changes in the sound the washer makes, this can indicate a vacuum leak At the end of the day prime the washer with DI water

Equipment : Micro-plate Washers Weekly If a washer is not used during the week rinse it out with DI water to reduce microbial growth. Monthly Run a 10% solution of ethanol through the washer to disinfect. This can also be done if the washer exhibits signs of contamination (high background). Thoroughly rinse the washer after alcohol is used.

Equipment : Micro-plate Reader Daily Each time a reader is turned on it runs a self test, it will then report any errors. Weekly Run a control plate weekly. Variations in positive or negative specimens could be a sign of a bad diode or a spill on a diode.

Source of False Positive Results MULTIPLE PREGNANCY MULTIPLE TRANSFUSION AUTO IMMUNE DISORDER CHRONIC HEPATITIS, CHRONIC ALCOHOLIC HBV VACCINATION ANTIBODY TO POLYSTERENE

Cross contamination Can be caused by: Reusing pipette tips (contaminated with + plasma) Splashes from one well to another During removal of plate covers

Sample Quality Properly collected (no haemolysis) Transport conditions Storage conditions Number of freeze/thaw cycles Age of sample

Validation and Interpretation of Results Product inserts provide guidelines Positive and Negative controls must fall within a certain range. Controls are used to calculate a cut-off. Samples below cut-off are negative, those above are positive

Western Blot (Immunoblotting) Solid-phase EIA with immobilized viral antigens to detect antibodies to specific HIV proteins.

Principle AIDS is caused by at least 2 etiological agents HIV-1 & HIV-2 Inactivated and denatured protein of HIV-1 are fractioned by polyacrylamide gel electrophoresis Protein bands are transferred into nitrocellulose strips HIV-1 sample diluted with buffer are then incubated with the strip

Conjugate peroxidase labeled anti human IgG is added It will bind to the antibodies already bound to the strip Chromogen is then added forming color reaction Reaction is then stopped by aspiration and reaction

Sample requirement: Serum sample Maximum 8 days Stored 2o C – 8oC or frozen at – 25oC Lipemic sample must be centrifuged well Avoid heating

Creating Western Blot Strips HIV lysate proteins are separated by size using gel electrophoresis Proteins are transferred (blotted) onto the surface of a membrane Strips are incubated with patient serum and antihuman IgG conjugated with an enzyme (and chromagen) The membrane is cut into strips

HIV Western Blot Banding Pattern env gp160 gp120 gp 41 gag p55 p18 p24 pol p65 p51 p31

Interpretation of Results (General Consensus) Negative: No bands present Positive: 2 ENV band present (WHO Guidelines) Indeterminate Any bands present but do not meet criteria for positive

Western blot Banding * * *

When should WB be used? Western Blot assay should not be used as a screening test. WB should be viewed as a supplemental test which can be used to confirm positive results obtained from EIA. HOWEVER: Specificity is less than that of EIA A significant number of indeterminate blots are seen in low risk populations

Advantages Disadvantages Technically demanding Expensive Specific interaction of antibody and antigen can be directly visualized. Disadvantages Technically demanding Expensive Subject to interpretation Presence or absence of bands Intensity of those bands