1. HIV
TESTING TECHNOLOGIES
ELISA / WESTERN BLOT

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

3. 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.

4. 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.

5. 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.

6. 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.

7. 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

8. 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

9. 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.

10. 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

11. 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.

12. Why Follow a Strategy?

13. 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.

14. 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.

15. Objectives for HIV testing
Diagnosis
Surveillance
Blood transfusion safety

16. 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

17. HIV STRUCTURE

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

20. 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

21. 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

22. 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

23. Indirect EIA

24. 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

25. Sandwich EIA(Ab-Ag-Ab)

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

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

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

29. 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

30. 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

31. 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

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

33. 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

34. 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.

35. 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.

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

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

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

39. 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

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

41. 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

42. 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

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

44. 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

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

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

47. Western blot Banding * * *

48. 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

49. 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