01.04 laboratory diagnosis and monitoring of hiv infection


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  • Antigen test Not widely available in Kenya
  • Serial testing is the minimum that can be used. Parallel testing is effective but costly. Never give out results with one screening test, as there is the risk of giving out some false positive results. Initial (screening) tests must be sensitive, while the confirmatory test must be specific.
  • 01.04 laboratory diagnosis and monitoring of hiv infection

    1. 1. Module 1: Overview of HIV Infection Unit 01.04: Laboratory Diagnosis and Monitoring of HIV Infection
    2. 2. Objectives <ul><li>At the end of this session, the participant should be able to: </li></ul><ul><ul><ul><li>Describe the available laboratory testing strategies available and their disadvantages and advantages </li></ul></ul></ul><ul><ul><ul><li>Describe HIV Testing strategy used in Kenya </li></ul></ul></ul><ul><ul><ul><li>Understand about window period in HIV infection </li></ul></ul></ul><ul><ul><ul><li>Understand issues related to false positive and negative results and their implications </li></ul></ul></ul><ul><ul><ul><li>Describe tests used for monitoring HIV disease and response to ART </li></ul></ul></ul>
    3. 3. Introduction <ul><li>Importance of Laboratory tests in the management of HIV infection </li></ul><ul><ul><ul><li>Diagnosis (HIV test) </li></ul></ul></ul><ul><ul><ul><li>Staging of HIV disease (CD4 T cell analysis) </li></ul></ul></ul><ul><ul><ul><li>determining prognosis (CD4 T cell analysis/viral load) </li></ul></ul></ul><ul><ul><ul><li>Determine when to initiate preventive therapies and ART(CD4 T cell analysis) </li></ul></ul></ul><ul><ul><ul><li>To assess response to ART (CD4 T cell analysis/viral load) </li></ul></ul></ul><ul><ul><ul><li>To evaluate patients for toxicity to ARVs (RFT/LFT) </li></ul></ul></ul>
    4. 4. HIV diagnosis <ul><li>Choice of test used for diagnosis depends on: </li></ul><ul><ul><ul><li>Purpose of the test e.g. </li></ul></ul></ul><ul><ul><ul><ul><li>For Diagnosis of HIV infection </li></ul></ul></ul></ul><ul><ul><ul><ul><li>For Surveillance of HIV infection in the population </li></ul></ul></ul></ul><ul><ul><ul><ul><li>For Screening of blood and blood products </li></ul></ul></ul></ul><ul><ul><ul><li>Sensitivity and specificity of the test being used </li></ul></ul></ul><ul><ul><ul><li>Prevalence of HIV infection in the population where the test is being used </li></ul></ul></ul>
    5. 5. Tests that can be used <ul><li>Serological methods: antibody/antigen based test </li></ul><ul><ul><ul><li>Enzyme Immunosorbent Assay (ELISA) </li></ul></ul></ul><ul><ul><ul><li>Western Blot </li></ul></ul></ul><ul><li>Viral detection methods </li></ul><ul><ul><ul><li>PCR </li></ul></ul></ul><ul><ul><ul><li>Culture: Rarely used </li></ul></ul></ul>
    6. 6. Serological methods <ul><li>Common and widely used tests </li></ul><ul><li>Based on the principal of antigen antibody reaction </li></ul><ul><li>Antibody takes some time to be produced after an infection </li></ul><ul><li>Therefore all these tests have a problem of making a diagnosis during the period immediately after infection with HIV prior to the appearance of detectable antibodies </li></ul>
    7. 7. Window Period <ul><li>“ Period of time between the onset of infection with HIV and the appearance of detectable antibodies” </li></ul><ul><li>Depends on: </li></ul><ul><ul><li>Sensitivity of the test kit (ability to detect low levels of antibodies) </li></ul></ul><ul><ul><li>The duration the body takes to produces antibodies (5-7 days) after infection </li></ul></ul><ul><ul><li>The level of antibodies produced (low levels during early infection). </li></ul></ul>
    8. 8. ELISA <ul><li>Over 40 different kits available </li></ul><ul><li>Have relatively simple methodology </li></ul><ul><li>High sensitivity of 99.3-99.7% and high specificity of >99.7% </li></ul><ul><li>Suitable for testing large samples </li></ul><ul><li>Detect both HIV-1/2 antibody </li></ul>
    9. 9. Rapid Tests <ul><li>Developed in the late 1980s </li></ul><ul><li>Can be performed in less than 20 minutes </li></ul><ul><ul><li>therefore also referred as “Simple/Rapid” (S/R) assays </li></ul></ul><ul><li>Can be performed easily without instruments </li></ul><ul><li>A positive result is indicated by the appearance of a colored dot or line </li></ul><ul><li>Examples – Determine, Unigold and Oraquick. </li></ul>
    10. 10. Confirmatory Tests <ul><li>All positive tests should be confirmed with another test </li></ul><ul><li>Not usually necessary to confirm negative tests </li></ul><ul><li>There are very specific assays that are used to verify positive HIV results e.g. Western Blot </li></ul><ul><ul><li>Designed to identify individuals who are not infected but who have reactive screening test results </li></ul></ul><ul><ul><li>High specificity- therefore produce few false-negative results </li></ul></ul>
    11. 11. Alternative Confirmatory Strategies <ul><li>Developed by UNIADS/WHO for use in confirming initial HIV positive tests </li></ul><ul><li>maximizes accuracy while minimizing high cost associated with Western Blot but retaining similar predictive values to Western blot and others. </li></ul><ul><li>Three testing strategies recommended by UNAIDS/WHO </li></ul><ul><li>Strategy used depends on purpose of the test and the prevalence of HIV in the population </li></ul><ul><li>The strategy used works on the principle of using two screening tests either in parallel or sequentially </li></ul>
    12. 12. Report sensitive Consider +ve A +ve -ve specific B +ve -ve sensitive A +ve -ve Report Indeterminate Report sensitive C +ve -ve + - - Low risk A +ve -ve sensitive specific B +ve -ve Report Report TRANSFUSION TRANSFUSION SURVEILLANCE DIAGNOSIS DIAGNOSIS I II III WHO HIV antibody testing strategies + - + High risk Sensitivity >97% Specificity >95% SERIAL TESTING PARALLEL TESTING
    13. 13. False-Positive Result <ul><li>HIV tests are highly sensitive. </li></ul><ul><li>A positive HIV tests will be sometimes obtained in absence HIV infection (no HIV antibodies in the blood) referred as a “false-positive” </li></ul><ul><ul><li>Influenza immunization may temporarily cause a false-positive </li></ul></ul><ul><ul><li>Improper interpretation </li></ul></ul><ul><ul><li>Autoimmune diseases - RA/SLE </li></ul></ul><ul><ul><li>Identified by additional testing. </li></ul></ul><ul><li>All positive results must be confirmed by another test method. </li></ul><ul><li>A confirmed positive result from the second test method means that the individual is infected with HIV. </li></ul>
    14. 14. False-Negative Result <ul><li>This is a negative HIV test results in an HIV infected person ( where the HIV test should have been positive). </li></ul><ul><li>Causes </li></ul><ul><ul><li>newly infected patient at the window period </li></ul></ul><ul><ul><ul><li>No HIV antibodies are yet being produced </li></ul></ul></ul><ul><ul><li>Seroreversion </li></ul></ul><ul><ul><ul><ul><li>End stage of HIV </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Prolonged immune reconstitution with HAART </li></ul></ul></ul></ul><ul><ul><li>Atypical host response </li></ul></ul><ul><ul><li>Agammaglobulinemia </li></ul></ul><ul><ul><li>Type N or O strains or HIV-2 (ELISA 20-30% FN in HIV-2) </li></ul></ul><ul><ul><li>Technical error </li></ul></ul><ul><li>If it occurs in high-risk patients, repeat the test after a time before reassuring the person that they are not infected with HIV </li></ul>
    15. 15. Indeterminate Result <ul><li>Occurs when 2 different testing kits gives different results (-/+ or+/-) </li></ul><ul><li>Causes of Indeterminate results: </li></ul><ul><ul><ul><li>in people with clinical signs meeting WHO criteria 3, stages III or IV. </li></ul></ul></ul><ul><ul><ul><li>During sero-conversion phase </li></ul></ul></ul><ul><ul><ul><li>Autoantibody </li></ul></ul></ul><ul><ul><ul><li>Infection with O strain or HIV-2 </li></ul></ul></ul><ul><ul><ul><li>HIV vaccine recipients </li></ul></ul></ul><ul><ul><ul><li>Technical or clerical error </li></ul></ul></ul>
    16. 16. How to handle indeterminate results <ul><li>Obtain and test a second sample after a minimum of 2 weeks in asymptomatic individuals </li></ul><ul><li>test the second sample using the appropriate strategy and if indeterminate use confirmatory assay. </li></ul><ul><li>If confirmatory test result is also indeterminate, follow-up the person for a longer period (3 to 12 months). </li></ul><ul><li>If the results remain indeterminate after one year, the person is considered to be HIV-negative. </li></ul>
    17. 17. Unit 5B: Laboratory Monitoring and Follow-Up
    18. 18. Immunological Monitoring <ul><li>CD+4 absolute counts </li></ul><ul><li>CD4 Percentage </li></ul>
    19. 19. CD4+ T-Cell Counts <ul><li>CD4 cells have a central role in the function of immune system. </li></ul><ul><li>Reduced number and quality of CD4 cells causes a weakened immune system </li></ul><ul><li>The level of CD4 cells used to measures the strength of the immune system. </li></ul>
    20. 20. Monitoring ARV Therapy <ul><li>Normal values ranges from 500-1400 cells/ml of blood </li></ul><ul><li>With ART the median increase in CD4 count is 100 to 150 cells per year. </li></ul><ul><li>Magnitude of increase in CD4 cells depends on the baseline CD4 count and adherence to the ARV. </li></ul><ul><li>A reasonable frequency of CD4 count measurements in patients on antiretroviral therapy is every 6 months. </li></ul>
    21. 21. Factors Affecting CD4 Count <ul><li>Diurnal variation: low in the morning and high in the afternoon- stick to specific time appropriate for each patient </li></ul><ul><li>Active infections including Opportunistic Infections :therefore, treat OI’s before checking CD4 Count </li></ul>
    22. 22. Use of CD4 T cells in HIV management <ul><li>Prognostic indicator in HIV disease </li></ul><ul><li>Benchmark for initiating prophylactic medications and ART </li></ul><ul><li>Guidance on the management of asymptomatic patients </li></ul><ul><li>Information on the efficacy of ARVs </li></ul>
    23. 23. Virological Monitoring <ul><li>Use of Viral load and resistance testing </li></ul><ul><li>Not widely available </li></ul><ul><li>May become increasingly available in future </li></ul>
    24. 24. Viral load <ul><li>Assessment of circulating HIV RNA load </li></ul><ul><li>Has a prognostic value </li></ul><ul><ul><li>at any CD4 count the higher the viral load the faster the disease progression (decline in CD4 count) </li></ul></ul>
    25. 25. Current use of viral load <ul><li>Not currently recommended in general patient management </li></ul><ul><li>Current guidelines may change in future as the tests becomes more widely available and affordable </li></ul><ul><li>Potential uses: </li></ul><ul><ul><li>Early detection of new HIV infection </li></ul></ul><ul><ul><li>Diagnosis in children < 18 months </li></ul></ul><ul><ul><li>Determine response to therapy </li></ul></ul><ul><ul><li>Determine treatment failure </li></ul></ul><ul><ul><li>Deciding when to initiate therapy </li></ul></ul><ul><ul><ul><li>Initiation of HAART currently based on WHO Stage and CD4 count </li></ul></ul></ul>
    26. 26. Resistance testing <ul><li>Expensive and not generally available in many countries. </li></ul><ul><li>Can be used to identify drugs the HIV is resistant to. </li></ul><ul><li>Helps in the management of patients who have failed treatment, to select new ARVs where there is resistance </li></ul><ul><li>Recommendation: in research settings </li></ul>
    27. 27. Other tests: Hematological tests <ul><li>Hemoglobin </li></ul><ul><ul><li>Assess </li></ul></ul><ul><ul><ul><li>anemia before AZT </li></ul></ul></ul><ul><ul><ul><li>Bone marrow suppression during ARV </li></ul></ul></ul><ul><li>White blood cell counts and differentials </li></ul><ul><ul><li>High WBC level may signify infection </li></ul></ul><ul><ul><li>Low WBC may indicate disease progression or bone marrow suppression (cotrimoxazole) </li></ul></ul><ul><li>TLC </li></ul><ul><ul><li>TLC <1200/ml has been correlated with CD4 count<200 cells/ml </li></ul></ul><ul><ul><li>TLC should not be used make decisions on initiation of ARV in asymptomatic patients </li></ul></ul>
    28. 28. Others tests: Biochemistry tests <ul><li>Serum alanine or aspartate aminotransferases </li></ul><ul><ul><li>Assesses possible hepatitis co-infection </li></ul></ul><ul><ul><li>Monitors for liver toxicities from ARV </li></ul></ul><ul><li>Serum creatnine +/ – blood urea nitrogen </li></ul><ul><ul><li>Baseline renal function </li></ul></ul><ul><ul><li>Possible renal toxicity </li></ul></ul><ul><li>Serum glucose </li></ul><ul><ul><li>Hyperglycemia and diabetes in PI based regimen </li></ul></ul>
    29. 29. <ul><li>Pregnancy tests </li></ul><ul><ul><li>To rule out pregnancy and avoid drugs contraindicated in pregnant such as Efavirenz </li></ul></ul><ul><li>Serum bilirubin </li></ul><ul><ul><li>Elevation associated with PI regimen (Indinavir, Atazanavir ) </li></ul></ul><ul><li>Serum amylase </li></ul><ul><ul><li>Pancreatitis (ddI) </li></ul></ul><ul><li>Lipids (cholesterol, triglycerides) </li></ul><ul><ul><li>Elevation in PI based regimen, d4T </li></ul></ul>Others tests: Biochemistry tests
    30. 30. Others <ul><li>RPR </li></ul><ul><ul><li>For screening of syphilis </li></ul></ul><ul><li>CXR </li></ul><ul><ul><li>Evaluation of possible active TB </li></ul></ul><ul><li>Pap smear/VIA or VILI </li></ul><ul><ul><li>Screening cervical cancer </li></ul></ul><ul><ul><ul><li>cervical cancer an AIDS indicator </li></ul></ul></ul><ul><ul><ul><li>Commonest cancer in women </li></ul></ul></ul><ul><ul><ul><li>Regular screening recommended in HIV+ women </li></ul></ul></ul>
    31. 31. Summary <ul><li>HIV infections cause production of antibodies and viral products which can be used to make a diagnosis </li></ul><ul><li>EIA long and rapid tests are commonly used for diagnosis of HIV </li></ul><ul><li>CD4 counts can be used as a guide for initiating prophylactic medications and ART, to provide information on the efficacy of ARVs and to monitor HIV progression </li></ul><ul><li>Viral load tests can be used for early detection of new HIV infection, for determination of response to therapy and sometimes for when to initiate therapy </li></ul>