Minimum Laboratory Testing Standards: Diagnosis of HIV Infection at the NRL. Presenter: Beverley Singh

Transcription

1 Minimum Laboratory Testing Standards: Diagnosis of HIV Infection at the NRL Presenter: Beverley Singh

2 Welcome Welcome to the Minimum Laboratory Testing Standards: Diagnosis of HIV Infections at the NRL Module

3 Module Objectives At the end of this module, participants will be able to: Describe the role of NRL in HIV laboratory testing; Discuss diagnosis of HIV infection using serological methods (ELISA testing) as the standard; Explain what advances have been done in diagnostic ELISA testing; Discuss Performance in detection for HIV infection; Describe the use of HIV Algorithms; Discuss Reporting Strategies; Discuss Test Method Verification; Describe new Algorithms/VISP.

4 Activity Group Discussion Duration: 5 minutes Enzyme-Linked Immunosorbent Assay! What assay does the above terminology relate to?

5 Diagnostic Tests for HIV Infection Serological methods for detection of antibody ELISA Rapid Tests Western blot Antigen detection methods P24 antigen capture test Polymerase Chain Reaction (also known as PCR) used in diagnosis: acute HIV infection, HIV infection in infants Recent Infection HIV-1 Limiting Antigen Avidity EIA

6 HIV Serological Diagnosis: Requirement at the NRL Tests are based on a reaction between HIV antigens and antibodies in the patient s serum Both a screening test and a confirmatory test should be used for diagnosis The NRL must have in place the most sensitive and specific test platforms Currently 3 rd generation assays are the norm but are being phased out and replaced by 4 th Generation Assays HIV Rapid tests are not routinely used for surveillance or diagnostics. Evaluation of RTD and Post Marketing Surveillance is a main function For Rapid Diagnostic testing RT can be used, but the NRL is expected to carry our routine surveillance/testing using 3rd/4th Generation testing.

7 Window Period Following HIV Infection Primary HIV infection Acute HIV syndrome Asymptomatic Antibody Viremia PCR P24 a.g ELISA a b (Weeks since infection) Years Source: S Conway and J.G Bartlett,

8 HIV Infection and Laboratory Markers

9 WHAT IS ELISA? Elisa- is enzyme linked immunosorbent assay sometimes called EIA enzyme immunoassay a technique used mainly in serology to detect the presence of an antibody or an antigen in a sample

10 False Negatives and Positives False negative results may result from: Window period Agammaglobulinemia Seroreversion (loss of HIV antibodies due to extreme immune system destruction) in late disease Technical error Type N or O strains, or HIV-2 False positive results may result from: Autoantibodies e.g., SLE HIV vaccines (VISP) Technical error

11 1 St and 2 nd Generation EIA

12 3 rd Generation Sandwich EIA

13 Fourth Generation ELISA Principle This ELISA principle is based on detecting both the antigen and/or antibodies associated with a virus in human serum or plasma, the antigen and antibody can be detected at the same time if both present. This assay is commonly used for serodiagnosis of HIV. Most 4 th generation assays are based on the sandwich" principle. Each manufacturer will have a slightly different design of the ELISA. Examples: Axsym HIV Ag/Ab combo, Genscreen Ultra HIV Ag/Ab, Vironostika HIV Ag/Ab.

14 Fourth Generation ELISA Microtitre wells are coated with HIV-1 and HIV-2 antigens and anti-hiv-1 P24, each microtitre well contains an HRP-labeled conjugate sphere of the same HIV- antibody/antigen mixture. Specimen diluent is added, patients samples and controls are added and incubated in the microtitre wells. If HIV- 1 and/or HIV-2 antibody is present in the sample a solid phase antigen/anti- HIV/enzyme labeled antigen complex is formed (Ag-Ab-Ag) If HIV-1 antigen is present in the patient s sample a solid phase antibody/hiv-antigen/enzyme labeled antibody complex is formed (Ab-Ag- Ab)

15 Fourth Generation ELISA (continued ) Microwells are then washed, substrate added (TMB) and incubate, color develops during this incubation, the color turns yellow when the stop( sulphuric acid) is added. If the patient s sample has anti-hiv-1, anti-hiv-2 and anti-hiv group O and/ or HIV antigen present an intense color develops in the microwells, however when the patient s sample does not have any anti-hiv and/or HIV antigen, no color forms or a low color forms with addition of substrate.

16 Western Blot Assay Solid-phase EIA with immobilised viral antigens (Ag) to detect antibodies (Ab) to specific HIV proteins. WB strips are commercially available; prepared using gel electrophoresis to separate HIV proteins by molecular weight. HIV proteins then transferred to a nitrocellulose membrane; the membrane cut into strips and used to detect specific protein antibodies

17 Western Blot Gold Standard WB should be viewed as a supplemental test which can be used to confirm positive/discordant results obtained from EIA. Advantages - 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

18 Sequence of Test Positivity Relative to WB

19 LAg (Limiting Antigen) Avidity Assay

20 LAg (Limiting Antigen) Avidity Assay (continued ) Developed by the CDC Estimates number of recent infections in a defined cohort Only HIV Positive samples Antibodies produced early in HIV Infection show a low avidity for the antigen Antibody Avidity increases progressively with time after exposure to an immunogen The avidity of the antibody can be measured in the presence or absence of a denaturing agent that will elute low-avidity antibody from the antigen-antibody complex Low avidity recent infection Higher avidity long term infection

21 Vaccine Induced Sero-Positivity/Reactivity (VISP) The phenomenon wherein a person who has received a vaccine against a disease would thereafter give a positive or reactive test result for having that disease when tested for it, despite not actually having the disease. This happens because many vaccines encourage the body to produce antibodies against a particular disease, and blood tests often determine whether a person has those antibodies, regardless of whether they came from an infection or just a vaccination A big concern in vaccine trials for HIV vaccine research People who give a positive result in an HIV test, even if that result is because of a vaccine and not because of an infection, may face discrimination because of the stigma attached to HIV.

22 Vaccine Induced Sero-Positivity/Reactivity When is a person classified as VISP: Reactive in one screening EIA test and positive in WB [CDC criteria] (Ackers et al., 2003, JID) Positive in at least one of the screening EIA test, regardless of WB-results (Silberman et al., 2008, AIDS Res Hum Retroviruses) Positive in 1 or more of the EIA tests, Western blot that is negative, indeterminate, or atypical-positive, negative test for HIV-1 by nucleic acid testing (Cooper et al, 2010, JAMA)

23 Examples of VISP EIA 1: Reactive EIA 2: Non-Reactive EIA 3: Reactive WB: Negative RNA PCR: Not Detected EIA 1: Reactive EIA 2: Reactive EIA 3: Reactive WB: Indeterminate RNA PCR: Not Detected

24 HIV Testing Algorithms and Reporting Strategies HIV EIA Algorithms HIV Reporting Strategies Test methods Test method validation Sensitivity and Specificity Results Interpretation

25 What is a strategy? Strategy is defined as the testing approach used to meet a specific need e.g. patient diagnosis. Parallel and serial testing is part of any testing strategy.

26 HIV Testing Strategies Parallel testing means samples are tested simultaneously by two different tests. Serial testing means samples are tested by a first test (screening test). The results of the first test determine whether additional testing is required. HIV EIA testing: the samples are screened on the first assay and the samples that are positive and confirmed on another assay or two other assays. If two or three assays are used, all assay principles and test methods must be different.

27 HIV Testing Strategies (continued ) Strategy 1: All samples are tested with one ELISA. All reactive results are considered infected and all non-reactive results are considered uninfected. Settings: transfusion/transplant services and prevalence/surveillance Strategy 2: All samples are tested first with one ELISA. Any sample found to be reactive with the first test must be tested by the second ELISA. Samples that are reactive on both ELISA tests are considered HIV Infected. Samples that are non-reactive on the first ELISA are not tested further.

28 HIV Testing Strategies (continued ) Strategy 3: similar to 2, except a third test is performed. All samples that are reactive on the first ELISA (Screen) is tested on the second (confirmatory 1) ELISA, samples reactive on the second ELISA is tested on the third ELISA (confirmatory 2). Samples that are positive on all 3 ELISA s is considered Infected. Samples that are non-reactive on the first ELISA are considered uninfected and not tested further.

29 What is an Algorithm? Combination and sequence of specific tests used in a given algorithm

30 HIV Testing Algorithms Identify appropriate tests Develop the algorithm Determine if tests are performing as expected Determine if any changes need to be made to the algorithm

31 Testing Algorithm (continued ) How do you know which tests to use in an algorithm? Test must be readily available in the country Test performance in country The tests must be validated prior to use Must be able to detect HIV 1 & HIV 2 The sensitivity and specificity of the assay determines which test will be used first

32 Testing Algorithm (continued ) Determine the testing strategy to be used, serial or parallel. ELISA testing is usually serial. NICD (NRL) : using a serial algorithm and testing strategy 1/2/3 dependent on the type of study e.g. surveillance versus a diagnostic algorithm For a diagnostic algorithm 3 rd /4 th generation testing may still be adequate but 4 th generation testing in diagnostic setting becoming a routine.

33 Testing Strategies and Algorithm Flow chart- Serial Algorithm EIA 1 Screening ELISA EIA 2 1 ST Confirmatory ELISA EIA 3 2 ND Confirmatory ELISA

34 Test Methods Used in Serology ELISA used routinely at NICD Western Blot used as a gold standard not done routinely P24 Antigen not done routinely, possible when using 4 th generation assays or to determine early sero-conversion. Rapid testing not used as routine test at the NICD. HIV RT Evaluations/Postmarketing surveillance.

35 Test Methods Used in Serology (continued ) Types of ELISA tests: Qualitative tests: A test that determines the presence or absence of a substance and is reported as either positive/negative or reactive/non reactive. Participants to give examples Quantitative tests: A test that determines the amount of a substance per unit volume or unit weight and is reported with a value or unit of measurement. Participants to give examples

36 Verification of test methods Before selecting test methods for use, these must be verified by the lab prior to placing In Use What is meant by verification? Verification is process of establishing evidence that provides high degree of assurance that a product or system accomplishes its intended requirements Verification is the process assessing: Precision Accuracy Sensitivity Specificity Linearity Analytical Measurement

37 Verification of test methods (continued ) Verification of the test method: test the kit using known reactive and non reactive samples. Compare the results of the new method with test method already in use. Use Proficiency testing panels to validate the test method. Test between samples if resources available. Determine sensitivity and specificity of the test Sensitivity and Specificity should be greater than 99% Perform precision and accuracy testing Write out report Approve report using special formula.

38 Verification of test methods (continued ) Precision testing Closeness of agreement between independent test/measurement results obtained under stipulated conditions. Test approx. 20 samples over a period of a few days (NICD tests over 5 days) using the same operator and the same equipment OR test the approx. 20 samples in the same day but in different test runs using same operator and same equipment. Analyze the results and calculate mean, Standard Deviation and %CV. The acceptable %CV can be obtained from package insert.

39 Verification of test methods (continued ) Accuracy Is the true value of a substance being measured. Verification of accuracy is the process of determining that the test system is producing true valid results Test approx. 20 samples over a period of a few days (NICD tests over 5 days) using the same operator and the same equipment OR test the approx. 20 samples in the same day but in different test runs using same operator and same equipment. Analyze the results. The final result should be the same for each sample tested on each day or in each test run.

40 Sensitivity and Specificity What is sensitivity? The ability of a test to accurately detect a true positive. What is specificity? The ability of a test to accurately detect a true negative

41 Sensitivity and Specificity (continued ) When choosing the test methods to be used in an algorithm, sensitivity and specificity of the test is key to selecting which assay will be used first, second and/or third The first test should be the most sensitive, all samples that are truly reactive should be accurately identified by the first test The second test should be more specific and also sensitive. Samples that are falsely reactive in the first test should be accurately identified as a true non reactive by the second test and samples that are truly reactive should be accurately identified by the second test The same principle applies to the third test as for the second. Sensitivity and specificity should be greater than 99%, 100% is optimal.

42 Sample types used in Serology Tests Serum Plasma Dried Blood Spot (DBS) requires overnight elution of sample prior to testing. Longer to process and test DBS samples than serum or plasma.

43 Result Reporting for HIV Serology tests The WB assay can be performed to determine final HIV results where results are discordant

44 LAg Testing Algorithm HIV-1 SEROPOSITIVE SPECIMENS TEST IN SINGLET (INITIAL) IF ODn > 2.0 LONG-TERM INFECTION IF ODn 2.0 REPEAT TEST IN TRIPLICATE (CONFIRMATORY) IF ODn > 1.5 LONG-TERM INFECTION IF ODn 1.5 PRELIMINARY RECENT INFECTION HIV WB and RNA can be done to confirm HIV status where repeat serology yields discordant results Transfer HIV-negatives to Negative Count Confirm HIV serology for specimens 0.4 ODn CONFIRMED HIV-1 POSITIVES FINAL RECENT INFECTION

45 New HIV Diagnostic Algorithm

46 Activity Write down the answers to the questions below. Duration: 5 minutes (3 minutes [preparation time] and 2 minutes [discussion]) What type of testing algorithm was displayed on slide 43? What testing strategy was used to report results?

47 Activity Group Discussion Duration: 20 minutes Discussions: Algorithms and Strategies used in NRL labs currently The place for HIV RTD in diagnostics at the NRL Acute HIV Infections Incidence Testing - why Incidence? Discuss Incidence Testing Algorithm

48 Summary Serological methods for HIV Diagnosis: ELISA s, Western Blot and HIV Rapid tests Principles of ELISA s assays: 1 st, 2 nd, 3 rd and 4 th generation ELISA s 1 st, 2 nd and 3 rd based on antibody detection 4 th generation based on antibody/antigen detection early diagnosis of infection Western Blot used to confirm positive/discordant results LAg assay determines number of recent and long term infections in cohorts Qualitative assays: ELISA, WB and HIV Rapid. Either Negative or Positive result. Quantitative assays: Viral Load and LAg. Results are quantified. VISP: Vaccine induced seropositivity whereby a person is reactive after being administered a vaccine against disease even though they did not have the disease Choose testing strategy suitable for your laboratory Select an algorithm based on assays available Test methods/assay must be verified prior to use. Determine Sensitivity, Specificity, Precision and Accuracy of all test methods selected for use Use most sensitive assay as screening and the confirmatory assays must be greater in specificity than sensitivity.

49 References & Links Ingrid V. Bassett, Senica Chetty, Janet Giddy, Shabashini Reddy, Karen Bishop, Zhigang Lu, Elena Losina, Kenneth A. Freedberg and Rochelle P. Walensky. Screening for acute HIV infection in South Africa: finding acute and chronic disease. HIV Med January ; 12(1): Celia Serna-Boleaa, Jose Muñoza,b, Jose M. Almeidab, Ariel Nhacolob, Emilio Letanga, Tacilta Nhampossab,c, Eliana Ferreirac,d, Pedro Alonsoa,b and Denise Nanichea High prevalence of symptomatic acute HIV infection in an outpatient ward in southern Mozambique: identification and follow-up AIDS 2010, 24: Evaluation of World Health Organisation Antibody Testing Strategy for individual patient diagnosis of HIV Infection (Strategy ΙΙΙ) written by: DJ. Martin, NK Blackburn, KF. O Connell, ET. Brant, EA. Goetsch Branson BM. The Future of HIV Testing. J Acquir Immune Defic Syndr 2010, 55:S102- S105 World Health Organization. Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy 2009.

50 Wrap Up Participants are now able to: Describe the role of NRL in HIV laboratory testing; Discuss diagnosis of HIV infection using serological methods (ELISA testing) as the standard; Explain what advances have been done in diagnostic ELISA testing; Discuss Performance in detection for HIV infection; Describe the use of HIV Algorithms; Discuss Reporting Strategies; Discuss Test Method Verification; Describe new Algorithms/VISP.

51 Questions