![Update on HIV
Testing
[A Very Fast]](https://image.slidesharecdn.com/3-updateonhivtesting-130417015103-phpapp01/85/3-update-onhivtesting-1-320.jpg)
3 update onhivtesting
- 1. Update on HIV Testing [A Very Fast]
- 2. Message There are numbers of tests They should be used in combination (strategies) Combinations must be consistent
- 3. Laboratory Tests diagnosis of infection acute, recent, established or late stage disease prognostic markers monitoring of ARV therapies immunological and virological markers toxicities diagnosis of opportunistic infections drug resistance testing
- 4. ‘typical’ primary HIV-1 infection symptoms HIV-1 p24 antigen 0 1 2 3 4 5 6 / 2 4 6 8 10 weeks years HIV antibodies Time following infection HIV viral load HIV proviral DNA symptoms ‘window’ period 1° infection
- 5. DIAGNOSIS Virus Detection & Quantification Antibody Antigen Detection RNA modified Ag Viral Culture, phenotyping CD4+ ARV Resistance – genotyping ARV Sensitivity HIV Assays: Methodologies FOR THE DIAGNOSIS (DETECTION) & MANAGEMENT OF HIV EIA Simple, rapid tests Immunoblots Incident assays MANAGEMENT DNA (RNA)
- 6. DNA PCRDNA PCR RNA PCRRNA PCR p24 Agp24 Ag 3rd gen ELISA 1st gen ELISA Detuned ELISA 1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yr gp160 gp120 p68 p55 p53 gp41-45 p40 p34 p24 p18 p12 gp160 gp120 p68 p55 p53 gp41-45 p40 p34 p24 p18 p12 gp160 gp120 p68 p55 p53 gp41-45 p40 p34 p24 p18 p12 early recent / established advanced SpectrumSpectrum of anti-HIVof anti-HIV testingtesting
- 7. HIV Testing - Direct Detection of Virus HIV p24 antigen– serology - In isolation or Ab/Ag Combo test - Diagnosis of primary infection viraemia Virus culture / isolation Nucleic acid detection - (NAT) Clinical uses Proviral DNA vs. plasma RNA(viral load) resolution of inconclusive serology / neonatal subtyping drug resistance monitoring
- 8. Principle of Immunoassays SOLID PHASESOLID PHASESOLID PHASESOLID PHASE ANTIGEN SAMPLE ANTIBODY ANTI-HUMAN IMMUNOGLOBULIN WITH DETECTOR
- 9. Available Assays EIAs including rapid, simple particle agglutination, dot/blot Western blot Antigen & Ab/Ag Incidence assays Direct Virus Detection
- 14. Western Blot Expensive – $ 80 - 100 technically more difficult visual interpretation lack standardisation - performance - interpretation - indeterminate reactions – resolution of ?? ‘Gold Standard’ for confirmation
- 15. Antibody testing limitations Difficulties in interpretation Limitations - ‘window period’ antibodies appear within 3-4 weeks Direct detection – HIV p24 antigen or DNA/RNA (NAT) – pre-antibody Combo test = earlier detection Primary infection + therapy = delayed antibody response
- 16. Ag/Ab Combo tests Detection of Ag & Ab in a single test utility in primary infection – pre- seroconversion ‘window period’ Incident populations – ‘at risk’ Blood bank Automated platforms availableAg & AbAg & AbAg & AbAg & Ab Ab & Ag
- 17. Issues with Combo Assays Testing strategies False reactivity rates Confirmation strategies Replacement of other assays (especially in the USA) Cost Legal issues
- 18. Conclusions HIV-1/2 Ag/Ab combo assays perform well Differences in limit of detection of Ag (140 - <25 pg/mL) May shorten window period by 3-5 days PHI detection without indication Issues associated with introduction Strategies and confirmatory algorithms ARV therapy effects seroconversion events
- 19. What about simple assays?
- 20. HIV Determine test Detect HIV-1 & HIV-2 Cannot differentiate Procedural control – anti Hu IgG Whole blood or serum/plasma Widely available No additional reagents required Room temperature storage 15 minutes to result
- 21. BioRad HIV-1/2 Multispot Detects HIV-1 and HIV-2 Will differentiate 1 and 2 Procedural control – anti-Hu IgG Serum / plasma only Additional reagents (included) Requires refrigerated storage ‘Immunoconcentration’ principle 15 minutes to result
- 23. 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.
- 24. WHO Recommended Testing Strategies Transfusion safety Surveillance Diagnosis Risk factors No risk factors Strategy I >10% I <10% II Strategy II >10% II <10% III
- 25. Testing Strategies AIM: To develop the logic used in establishing the use of HIV tests (testing strategies)
- 26. Objectives of Testing Strategies To achieve the correct diagnosis in the most efficient manner To maintain consistency in testing To know the predictive value of the testing process To develop baseline data for assessing changes To deliver useful results
- 27. Aims in Developing HIV Testing Strategies To arrive at the correct sero-diagnosisTo arrive at the correct sero-diagnosis To minimise total testing; thus costTo minimise total testing; thus cost Minimise samples classed as indeterminateMinimise samples classed as indeterminate or dual reactorsor dual reactors Detect HIV-1 negative but HIV-2 positiveDetect HIV-1 negative but HIV-2 positive Follow likely seroconverters (HIV-1 or -2)Follow likely seroconverters (HIV-1 or -2)
- 28. 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
- 29. Ab + Ag AbAg
- 30. Ab +AgAb +Ag Ab +AgAb +Ag AbAgAbAg Ab +AgAb +Ag Ab +AgAb +Ag Ab +AgAb +Ag Ab +AgAb +Ag Ab +AgAb +Ag Ab +AgAb +Ag AbAgAbAg AbAgAbAg AbAgAbAg AbAgAbAg AbAgAbAg AbAgAbAg AbAgAbAg
- 31. Serological Testing Strategy NEG SCREENING TEST, highly sensitiveSCREENING TEST, highly sensitive POS SUPPLEMENTAL TEST,SUPPLEMENTAL TEST, highly sensitive & higherhighly sensitive & higher specificityspecificity ADDITIONALADDITIONAL TESTSTESTS REACTIVE NEGNEG POS IND NEG IND
- 32. POINT OF REPORTING NEG HIV1/2 SCREEN POS HIV-1 WB ADDITIONAL TESTS REACTIVE NEG NEG IND POS IND HIV Testing Strategy SC R EEN ING SU PPLEM EN TA L
- 33. Supplemental Assays Range of assays that further define sero-status High Performance (higher specificity)
- 34. The Use of Screening Assays Define samples as negative for a given analyte Enable high throughput
- 35. Assay Selection depends on: laboratory infrastructure access to reference laboratory desired characteristics of the test equipment performance time shelf life and stability of reagents price technical skills of personnel support (technical, kit supply, etc)
- 36. Predictive Values Positive Predictive ValuesPositive Predictive Values:: The likelihood of a sample identified as aThe likelihood of a sample identified as a reactive by a test being truly POSITIVE forreactive by a test being truly POSITIVE for the analyte used as the basis of the test.the analyte used as the basis of the test. PPV = True Positives True Positives + False Reactives X 100%
- 37. Predictive Values Negative Predictive ValuesNegative Predictive Values:: The likelihood that a sample identified as a non-The likelihood that a sample identified as a non- reactive by a test is truly NEGATIVE for thereactive by a test is truly NEGATIVE for the analyte used as the basis of the test.analyte used as the basis of the test. NPV = True Negatives True Negatives + False Negatives X 100%
- 38. Negative Predictive Value Non-reactive test, prevalence 2% 99.17 99.38 99.59 99.79 99.90 99.96 99.9899.17 99.38 99.59 99.79 99.90 99.96 99.98 99.17 99.38 99.59 99.79 99.90 99.96 99.9899.17 99.38 99.59 99.79 99.90 99.96 99.98 99.17 99.38 99.59 99.79 99.90 99.96 99.9899.17 99.38 99.59 99.79 99.90 99.96 99.98 Specificity Sensitivity 60 70 90 60 70 80 90 95 98 99%
- 39. Positive Predictive Value Reactive test, prevalence 2% 4.95 4.954.95 4.95 6.49 6.496.49 6.49 9.43 9.43 9.439.43 9.43 9.43 17.24 17.24 17.24 17.24 17.24 17.24 17.2417.24 17.24 17.24 17.24 17.24 17.24 17.24 29.41 29.41 29.41 29.41 29.41 29.41 29.4129.41 29.41 29.41 29.41 29.41 29.41 29.41 51.02 51.02 51.02 51.02 51.02 51.02 51.0251.02 51.02 51.02 51.02 51.02 51.02 51.02 67.57 67.57 67.57 67.57 67.57 67.5767.57 67.57 67.57 67.57 67.57 67.57 Sensitivity Specificity 60 70 80 90 95 98 99 60 70 80 90 95 98 99%
- 40. Cut-off valueCut-off value XX 11 FrequencyFrequency 3SD 2SD 1SD 22 33 Cross reactivity
- 41. Positive Predictive Value Assays used separately Assay 1 Sens 99.0% Spec 99.5% PPV 28.4% 80.2% 98.0% Assay 2 Sens 99.0% Spec 99.9% PPV 66.4% 95.3% 99.6% Prevalence 0.2% 2.0% 20.0%
- 42. Positive Predictive Value Assays used sequentially Assay 1 Sens 99.0% Spec 99.5% PPV 28.4% 80.2% 98.0% Assay 2 Sens 99.0% Spec 99.9% PPV 99.75% 99.97% 99.99% Prevalence 0.2% 2.0% 20.0%
- 43. Assay 1 Assay 2 Assays 1+2Assay 1 Assay 2 Assays 1+2 PPV vs Prevalence (in sequence) Sensitivity 99.0% 99.0% Specificity 99.5% 99.9% Prevalence 0.2% 2.0% 20.0% 28.4% 80.2% 66.4% 99.75% 98.0% 80.2% 99.97% 99.6% 99.99% PPV PPV PPV
- 45. 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
- 46. 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.
- 47. WHO Recommended Testing Strategies Transfusion safety Surveillance Diagnosis Risk factors No risk factors Strategy I >10% I <10% II Strategy II >10% II <10% III
- 48. WHO Guidelines Other possibilities strategy for confirmation combination of affordable & simple assays different test principles different antigen preparations two or three ELISAs or rapid tests diagnosis confirmed by second sample detection of virus (PCR) antigen detection (limited lab.facilities) Always use a QC sample
- 49. Cost of HIV Testing comparative costs (AUD$) ELISA (Ab only) - $2 per test EIA (Ab/Ag combo) - $3.50 rapid test - $10-20 per test Western blot $80 - 100 p24 antigen $30 PCR - qualitative $80 - 100 PCR - quantitative (viral load) $90 – 150* DNA sequencing (resistance) $400 – 700
- 50. Summary of Testing Strategies -Screening test x1 Screening test x2 Supplemental test Other tests NEG R - NEG - - NEG + POS Eliminates laboratory error RR or R-
- 51. Message There are numbers of tests They should be used in combination (strategies) Combinations must be consistent
Editor's Notes
- #6: HIV Assays - Methodologies and Utilisation: The diagnosis of HIV infection usually rests on the identification of specific antibody to HIV in plasma or serum. To identify positively the presence of anti-HIV, two assays using different technology and different antigen preparations, should be used. The detection of HIV infection by antibody tests is an indirect confirmation of infection. There has been a change in test usage with the introduction of molecular-based tests. The emphasis in using anti-HIV tests has been somewhat eclipsed by the news around tests that quantify viral load. Viral load estimations are used to gauge prognosis and to monitor therapy. Other tests monitor the status of immune function indirectly but these are used widely in HIV management.
- #9: Principles of Immunoassay: There is a general basic construction format of immunoassays which include: An antigen which is used to capture any sample antibody, attached to a solid phase, be it a well on a microtitre plate or a gelatin particle, bead or membrane. The anchored antigen is incubated with the sample so that an antigen-antibody reaction occurs. In PA assays bound antibody causes particle agglutination. In EIAs a wash step removes the serum or plasma and leaves only bound antibody. A conjugate which is usually anti-human immunoglobulin labelled with enzymes or fluorochromes which will subsequently bring about a reaction that will result in a colour change. After a wash step to remove excessive conjugate a substrate on which the enzyme reacts or by which the fluorochrome is activated, the colour change is read in a spectrophotometer. Newer machine-based assays may use variations on the system diagrammed above.
- #10: Available Assays: There is a large range of assays available, especially for anti-HIV screening and HIV-diagnosis. In considering the most suitable assay for a given testing strategy there will be a number of considerations including: suitability of assay format antigens used characteristics of the assay skills of the technical staff, etc.
- #24: 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.
- #25: WHO Recommended Testing Strategy 2 The strategies are recommended by their prospective use and depending on the prevalence of the population being tested. Furthermore if the test strategy is being used to achieve diagnoses, risk factors will be taken into account. These alternative testing strategies have been used to advantage in numbers of countries.
- #26: 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.
- #27: 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.
- #28: Aims in Developing HIV Testing Strategies: To arrive at a correct sero-diagnosis Characteristics of the tests need to be understood. The sequence in which the tests are to be used have to be understood by laboratory personnel and health care workers alike. The deficiencies of the strategies must be understood. Minimising total testing Developing strategies generates data that will enable laboratories to achieve the correct diagnosis in the most efficient manner. Therefore, total testing is minimised. If different tests are continually introduced into testing there is confusion about what aberrant test results might mean, and no consistent means of resolving these. Therefore further testing and confusion is generated. This can be costly. CONTINUED ON NEXT PAGE
- #29: 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.
- #30: 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
- #31: 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.
- #32: 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.
- #33: 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
- #34: Supplemental assays - Definition: Supplemental assays are those that further define the sero-status. To distinguish true from falser reactivity supplemental assays are necessarily of high specificity.
- #35: 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.
- #36: Assay Selection: The assay selected in strategy development will depend on a large number of factors. These factors will be different for every country or region. An advantage of collaborations between regions is that each may utilise aspects of others testing strategies for unusual situations.
- #37: Predictive Values: Understanding predictive values is the key to understanding the predictability of a strategy’s effectiveness. The predictive values of the same strategy in different areas will be different because of factors such as the prevalence of HIV in the community the characteristics of the tests being used the rate of false reactivity. Low Prevalence : True Positives < False Reactives High Prevalence: True Positives > False Reactives Therefore the same tests and strategies used in different areas will not give the same predictive values. Thus it is important that each testing network understand what its individual predictive values are.
- #38: Predictive Values Continued: Negative predictive values are much higher than positive predictive values because of the way of tests are constructed. Antibody tests are geared to recognise antibody . (The tests can be thought of as hypersensitive therefore, the number of false negatives is usually very low.)
- #39: Negative Predictive Values: Because assays are constructed to pick up antibody that even vaguely resembles anti-HIV antibody, the negative predictive value of tests is very high, even for tests with relatively low sensitivity and specificity. This explains why it is possible to screen using a single sample. It is notable that most samples screened will fall into the negative category. This means that if samples are screened appropriately with a high sensitivity test the chances of a false negative are very low.
- #40: Positive Predictive Value: The positive predictive value depends on the prevalence of the measured analyte in the community being tested as well as the false reactivity of the tests i.e. test specificity. The positive predictive value is the ratio of true reactivity to the sum of and true false reactivity and therefore predicts the likelihood of a reactive test being a true positive. It is notable that for tests with high sensitivity that if the specificity is low the predictive value falls precipitously. This is particularly true of populations where the true prevalence is low. In developing a testing strategy, laboratories need to have a clear understanding of what the positive predictive value of tests are. Furthermore it is extremely important to be able to communicate to the public and to the health care workers and the public the importance of predictive values. How many times have we all heard that the test is 100% sensitive therefore the performance is perfect!
- #41: The Cutoff Value: Cutoff values are set by manufacturers by examining wide populations - both negative and positive for the analyte. If the cutoff value is move the predictive value of the test will change. Cutoff values should never be altered unless a full evaluation has been undertaken.
- #42: Positive Predictive Values Of Strategies: If an assay of high sensitivity and specificity is used in a low prevalence population its predictive value will be low but in a high prevalence population it will be much higher. Even if the sensitivity and specificity of an assay approach 100% (as is the case in assay 2 above) the predictive value may still be quite low for low prevalence populations.
- #43: Positive Predictive Values (2): If assays are used in sequence as in assay 1 and assay 2 in the example above, the predictive value, even at low prevalence, is enhanced considerably. It is the use of assays in sequence that makes strategy development essential in populations of low prevalence.
- #46: 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.
- #47: 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.
- #48: WHO Recommended Testing Strategy 2 The strategies are recommended by their prospective use and depending on the prevalence of the population being tested. Furthermore if the test strategy is being used to achieve diagnoses, risk factors will be taken into account. These alternative testing strategies have been used to advantage in numbers of countries.