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HIV tests in adults and children


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HIV tests in adults and children

  1. 1. Testing Related to HIV in Adults & Children and ART MonitoringTesting Related to HIV
  2. 2. Session Objectives At the end of the session, the participant should be able to: • understand the general principles of HIV testing • classify the testing procedures • elaborate on the policy of three strategies of testing and its applications • learn the tests for diagnosis of HIV-infection in children • discuss the tests for monitoring disease progression in adults and childrenTesting Related to HIV 2
  3. 3. Objectives of Testing • Diagnosis – Adults – Children • Test blood/tissue/organ for transplantation safety • Monitor trend of HIV infection and surveillance • ResearchTesting Related to HIV 3
  4. 4. General Principles of Testing • Voluntary and part of overall comprehensive prevention and treatment programme • Use of quality testing kits, standardised techniques, and QA/QC procedures • Test kit and procedure must be appropriate to the field situation • Cost-effectiveTesting Related to HIV 4
  5. 5. Uses of Laboratory Testing of HIV/AIDS • Establishing diagnosis – Adults – Children • <18 months • >18 months • Monitoring progression of HIV disease and response to ART – CD4 T-cell estimation (Indirect Marker) – Viral load estimation (Direct Marker)Testing Related to HIV 5
  6. 6. Types of HIV Diagnostic Tests • HIV antibody test: Serology • Viral antigen test: p24 • Viral isolation and culture • Viral DNA PCRTesting Related to HIV 6
  7. 7. Typical Course of Untreated HIV Infection 800 Acute HIV 10^6 HIV antibodiesCD4 Asymptomaticcount HIVcells/µl RNA Minor HIV-related Copies /ml symptoms Virologic set-point 200 Varies from patient to Opportunistic patient infections 10^2 Death 1 3 Wk about 6mths // 5yrs 10 yrs Time DurationTesting Related to HIV 7
  8. 8. Typical’ HIV-1 infection: Lab Markers symptoms symptoms HIV proviral DNA HIV antibodies ‘window’ period HIV viral load HIV-1 p24 antigen 0 1 2 3 4 5 6 / 2 4 6 8 101° infection weeks years Time following infectionTesting Related to HIV 8
  9. 9. HIV Structure p66 & 31 Image Courtesy GHTM , Tambaram, HIV Fellowship ProgrammeTesting Related to HIV 9
  10. 10. Tests for Diagnosing HIV • Antibody Tests • Screening Tests – Rapid tests – Enzyme linked immunosorbent assays (ELISA) • Confirmatory/Supplemental Tests – 2nd/3 rd R/E to confirm 1st R/E – Western blot assay • Same blood sample is utilised for performing all the tests for identifying HIV antibodiesTesting Related to HIV 10
  11. 11. Antibody Tests: HIV EIA/ELISA (Microwell Format) Advantages: Disadvantages: • Used as a screening test – Technical expertise since 1985 – Special equipment • Easy for mass screening – More stringent • Easy to automate standardisation • Accurate • Less costly than other testsTesting Related to HIV 11
  12. 12. ELISA Test: Microwell Format After several An automated reader incubation and wash gives a measurement of steps, a colour optical density reaction occurs if (presence of colour) HIV antibody is for each well present Images Courtesy: GHTM, Tambaram, ChennaiTesting Related to HIV 12
  13. 13. ELISA Screening Tests Generation Antigen / Antibody First Generation Viral Lysates as Ag (Not in use anywhere) Second Generation Synthetic peptides/Recombinant Ag Synthetic peptides/Recombinant Ag, Third Generation Double Ag binding, Enables IgM, IgA Ab detection Fourth Generation Detects Ag and Ab to HIVTesting Related to HIV 13
  14. 14. Concept of Window Period • Window Period (WP) is present between time of infection and the point at which evidence of infection is detected. Relative shortening of Window Period with different ELISA Kits 4 GENERATIONS OF HIV Ab TESTS Image Courtesy Abbott PublicationTesting Related to HIV 14
  15. 15. HIV Rapid Tests • Rapid tests to detect antibodies to HIV-1 and HIV-2 are in-vitro qualitative tests • Can be performed on whole blood, plasma, serum and saliva. In India whole blood and serum are used • Most rapid test kits come with all materials required to perform the testTesting Related to HIV 15
  16. 16. Overview of workflow at ICTC Step Personnel Time Pre test counselling and 1. Counsellor 15-20 min informed consent Blood (2-5ml) collection in 2. Lab Technician 5-10 min sterile vial 3. Serum separation Lab Technician 30-60 min 4. HIV testing on serum sample Lab Technician 30-60 min 5. Report preparation Lab technician 30-60 min Report dispensing with Post 6. Counsellor 15-20 min test counselling All times excluding waiting times; Waiting time depends on client load; Reports will be available on the same dayTesting Related to HIV 16
  17. 17. HIV Rapid Tests Positive Result Immunological Principles (Visual) Particle agglutination Clumping Immunofiltration/dot Dot Immunochromatography Line ELISA based rapid tests Dot Most rapid tests detect Antibodies to both HIV-1 & HIV-2Testing Related to HIV 17
  18. 18. Rapid Test: Line Test (Immunochromatography) Lateral Flow Devices Non- Reactive Control HIV Antigen Reactive Specimen Flow Sample pad Sample Pad Test line Control lineTesting Related to HIV 18
  19. 19. Rapid Test: Dot Test (Immunoconcentration) HIV antibody links to bound HIV peptide antigens forming the colour spot Internal Control HIV-1 peptide HIV-2 peptideTesting Related to HIV 19
  20. 20. False Positive Test Reasons for False Positive Test  Antibody based tests: • Lab error • Cross reacting antibodies (Autoimmunity) • IV drug abuse • Multiple pregnancies • Recent immunisation • Chronic alcoholics • Cirrhosis of LiverTesting Related to HIV 20
  21. 21. False Negative Test Reasons for False Negative Test  Antibody based test: • Window period • Lab/Clerical error • Other immunodeficiency statesTesting Related to HIV 21
  22. 22. HIV Testing Strategies • Strategy I: – All samples tested with one ELISA/Rapid • Strategy II: – All samples tested with one ELISA/Rapid (E/R) – Reactive samples tested again on different system (different antigen or principle) • Strategy III: – All samples tested with one ELISA/Rapid (E/R) – Reactive samples from the first test tested with different antigen or preparation – Reactive samples from the second test again tested with third system of different antigen or principleTesting Related to HIV 22
  23. 23. HIV Testing Strategies Testing Objective of Place of Type of Testing Strategy Testing Testing Transfusion/ I Donation Mandatory Blood Bank safety Unlinked Designated II Surveillance Anonymous laboratories Voluntary Diagnosis of  Counselling III patients at  Informed ICTC ICTC Consent  ConfidentialTesting Related to HIV 23
  24. 24. HIV Testing Strategy III A1 Report: A2 A1+ A1- Non-Reactive A2+ A2- Report: A3- Non-Reactive A3 A3 A1+, A2-,A3+ A3+ (indeterminate) A3+ A3- A1+, A2+, A3- (indeterminate) A1+, A2+, A3+ Report: ReactiveTesting Related to HIV 24
  25. 25. Tests of Choice for Infants & Children <18 Months of Age Name the tests used for the diagnosis of HIV in infants and children aged <18 Months and give their relevance Test Recommendation ReasonHIV antibodyHIV p 24 AntigenHIV viral cultureHIV DNA PCRTesting Related to HIV 25
  26. 26. Tests of Choice for Infants & Children <18 Months of Age Name the tests used for the diagnosis of HIV in infants and children aged <18 Months and give their relevance Test Recommendation Reason False +ve due to persistentHIV antibody No maternal antibodies Lower sensitivity than PCRHIV p 24 Antigen Yes, but (27% at 6 weeks) Costly, result takes 2-4 wks,HIV viral culture Yes, but not readily available 98% sensitive from 6 weeksHIV DNA PCR Yes of ageTesting Related to HIV 26
  27. 27. Diagnosis of HIV Exposed Infants & Children • Positive HIV antibody (Ab) alone indicates there has been exposure to HIV: DOES NOT mean child is infected, can be circulating maternal antibodies • Test to diagnose HIV in this population: HIV DNA PCR (Qualitative) • Ideal: First positive virological test (HIV DNA PCR) should be confirmed by a repeat positive test on a separate specimenTesting Related to HIV 27
  28. 28. Diagnosis of HIV Exposed Infants & Children Schedule of visits at ICTC 6 weeks 14 weeks 9 months 18 months 10 weeks 6 months 12 months Birth DNA PCR HIV Antibody test followed by DNA PCR if HIV+ DNA PCR for all HIV exposed infants Final confirmatory Antibody Test for all HIV exposed infants irrespective of earlier testing results / treatment status All HIV infected and/ or symptomatic infants/children are to be referred to ART centreTesting Related to HIV 28
  29. 29. Diagnosis of HIV Exposed Infants & Children National Guidelines: General Principles• Follow two different diagnostic algorithms A. Infants <6 months old and born to HIV positive mother B. Child of age 6-18 months born to HIV positive mother• First HIV DNA PCR test sample at ICTC (Dry blood spot)• If DBS is positive, Second sample for DNA PCR test (whole blood) will be collected at linked ART Centre – A positive DNA PCR test reveals the infant/child as HIV-1 infected – A negative DNA PCR test reveals discordance between the first and second tests; needs a tie breaking third DNA PCR test from the whole blood sample to be taken at ART centreTesting Related to HIV 29
  30. 30. Diagnosis of HIV Exposed Infants & Children National Guidelines: General Principles • Sample for DNA PCR test (whole blood) taken at ART Centre for the tie-break – A positive DNA PCR test reveals the infant/child as HIV-1 infected – A negative DNA PCR test reveals the infant/child as HIV-1 uninfected • Final confirmatory Antibody Test for all HIV exposed infants irrespective of earlier testing results / treatment status at 18 monthsTesting Related to HIV 30
  31. 31. Children <18 months with unknown HIV status of Mother • In Children (<18months) with signs and symptoms of HIV, whose exposure status is unknown, perform rapid test for HIV antibodies • If negative, label child as uninfected • If positive, follow algorithm A or B, depending on age of the childTesting Related to HIV 31
  32. 32. Tests for Monitoring • Tests are required to monitor - Disease progression - Staging of disease - Response to ART • Include - CD4 T-cell Assay - Viral load assayTesting Related to HIV 32
  33. 33. Standard Technologies for Monitoring ART • CD4 by Flow Cytometry advised by NACO – Variations across methods can occur • Nucleic acid Amplification Technologies (NAT) – Cannot be widely used in countries with limited resources because: • Lack of adequate infrastructure • Requires highly skilled lab personnel • NAT testing is too expensiveTesting Related to HIV 33
  34. 34. FACS Machines for CD4 Estimation Available Through NACOFACS Count FACS Calibur Partec Images Courtesy: GHTM, Tambaram, ChennaiTesting Related to HIV 34
  35. 35. When to Perform CD4 Test • All HIV patients accessing Hospitals - immediately after their HIV status known • Pre-ART: once in 6 months till they are being initiated on ART • During ART: – Once in 6 months for monitoring – As and when their clinical conditions demandTesting Related to HIV 35
  36. 36. CD4 counts and CD4 % • Used to assess immunological status of the HIV-infected child • CD4 counts are higher in infants as compared to adults and fall to adult values by age 5 – Varies due to diurnal change, undercurrent illness, steroid treatment, splenectomy, after immunisations – Test variability: repeated measurements are more informative than single value • CD4% varies less than CD4 counts, hence considered more valuable in children <5 years of ageTesting Related to HIV 36
  37. 37. Key Points • Encourage voluntary testing for HIV with pre-test and post-test counselling • HIV testing should follow recommended strategies I/II/III depending on the situation • HIV Ab test significance varies in adults and children <18 months; DNA PCR test is advised • A CD4 test for monitoring must be done with a fresh sample and at the same lab • Paediatric HIV monitoring and staging relies on CD4%Testing Related to HIV 37