1. HIV Diagnosis Jose-Luis Burgos, MD, AAHIVM Prevenmed-UCSD International Heath & Cross Cultural Medicine

2. “Appearances to the mind are of four kinds. Things either are what the appear to be; or they neither are, nor appear to be; or they are, and do not appear to be; or they are not, yet appear to be. Rightly to aim in all these cases is the wise man’s task.” Epicteus, 2 nd Century A.D.

3. Objectives  Demonstrate knowledge about current HIV testing methods and interpretation of results  Ability to understand how a VCT program can prevent HIV transmission  Distinguish what tests are useful in infants  Identify rapid tests and their use in high risk and vulnerable population

4. 1 mil 100,000 10,000 1,000 100 10 + _ HIV RNA HIV-1 Antibodies Exposure P24 + 020304050 Symptoms Days HIV RNA Ab Typical Course of Primary HIV

5. Basic Principles of HIV testing  All persons receiving an HIV test should receive counseling for risk reduction before and after an HIV testing  Positive results need to be referred for initial evaluation.  HIV tests should be voluntary and with informed consent

6.  Many patients at highest risk for HIV have not been tested  Up to one third of patients receive their first HIV test within two months of an AIDS defining illness  40% of patients undergoing HIV testing do not return for results  ARV, Prevention of OI, effective interventions

7. Risk Assessment  Tuberculosis  Sexually transmitted disease  Behavioral risk factors –Sexual contact with commercial sex workers, injection drug users, multiple partners, MSM –Unprotected sex involving exchange of body fluids –Injection drug abuse –Blood product receipt (before reliable screening introduced)  In countries with generalized epidemics (e.g. South Africa), screening for HIV should be part of routine primary care

8.  Testing should be recommended for all pregnant women as soon as pregnancy is detected  Women with high risk for HIV infection, should have a repeat test during the third trimester  During labor, all women with unknown serologic status for HIV should be tested with the use of rapid tests  All exposed new born children should be tested between within 48 hours, 1 - 2 months and 3-6 months. Risk Assessment

9. Anonymous vs Confidential  Anonymous –Identifying information not provided –Results not linked to identifying information –Allows reporting of HIV infection without breaching confidentiality –Disadvantage: may not be able to locate clients for test results  Confidential –Clients linked to test result by identifying information –Results remain confidential  Informed consent

10. Pre-Test Counseling  Goal: reduce HIV acquisition and transmission  Accurate and current information about HIV  Obtain informed consent  Transmission and acquisition  HIV test info: risk, benefits, meaning of potential test results  Assessment of individuals risks and appropriate risk reduction activities  Capacity to comprehend HIV testing and consent

11. Post-Test Counseling  Accurate and current information about HIV  Local resources  Risk reduction education  Referrals for ongoing care and support  Healthy living strategies  Meaning of test results and state reporting guidelines  Mental health support / counseling

12. Patient centered counseling  Talk with rather then to the patient  Individualize sessions (patients needs)  Neutral non-judgmental  Use open ended questions  Aim for a realistic risk-reduction plan  Offer options not directives  Recognize the counselor's limited role.

13. Diagnosis of HIV Infection  Viral antibodies  Viral antigens  Viral RNA/DNA  Culture Lancet, 1996; 348: 176.

14. Sensibility & Specificity Sensibility: Sensibility: Ability to identify infection in individuals that are truly infected. Ability to identify infection in individuals that are truly infected. Specificity: Specificity: Ability of the test to identify as negative individuals that are NOT infected Ability of the test to identify as negative individuals that are NOT infected

15. HIV Diagnostics  Approximate times to positivity* –1 st generation ELISA 42 days –3 rd generation ELISA23 days –p24 antigen assays16 days –RNA PCR11 days  95% seroconvert by 6 months  False positives –autoimmune disease, renal failure, cystic fibrosis, multiple pregnancies/transfusions, liver disease, post immunization  False negatives –window period, agammaglobulinemia, Group O, N, ± HIV-2 *Busch et al., Transfusion 1995;35:91-97

16. Negative Antibody Test Results  HIV negative  Recent infection: too early for seroconversion  CDC: follow-up testing at 6 weeks, 12 weeks, 6 months

17. Confirmation Process  Non-negative screenings should be confirmed –Western Blot (WB) –Immunofluorescent Antibody Assay (IFA)  Higher specificity than EIA  Interpretation can be subjective

18. IFA (Immunofluorescent Antibody Assay  Fluorognost HIV-1 IFA uses human material from infected T cells that express HIV-1 antigens. The cells are fixed to the surface of a glass slide, non infected cells are used as controls. When HIV antibodies are present in serum or plasma they attach to the infected cells but not to the non- infected cells.  Uses microscope, faster results

19. Western Blot  Detects antibodies to HIV-1 proteins –Core: p17, p24, p55 –Polymerase: p31, p51, p66 –Envelope: gp41, gp120, gp160  Negative: no bands  Positive: –Reactivity to gp41 + gp120/160 or –Reactivity to p24+gp120/160  Indeterminate: –EIA repeatedly reactive –Presence of any band pattern not meeting criteria for positive results

20. Predictive Value: HIV Ab Tests  Importance of pre-test probability in determining false + rate (Bayes theorem)  Depends on the prevalence of HIV infection in the population  Low HIV prevalence: predictive value of a positive test is low  HIV Ab testing of low prevalence populations likely to produce more false- positive than true-positive results

21. False Positive Results in Low vs. High Prevalence of HIV infection Prevalence in tested group Per 100,000 tests Number of false positives assuming 0.1 % prevalence Number of True Positives Ratio of False Positive/True Positives 1 in 100,000 1001100:1 1 in 10,000 1001010:1 1 in 1000 1001001:1 1 in 100 1001,0001:10 1 in 10 10010,0001:100

22. Window Period  Time delay from infection to positive EIA –Average: 10-22 days –Most seroconvert within six months Am J Med 2000; 109

23. False Negative Results  High-prevalence population: 0.3%  Low-prevalence: <0.001%  Usually due to testing during window period  Rare patients seroconvert in late- stage disease  Technical or clerical error  Type N or O  HIV-2

24. False Positive Test Results  Much less common than in earlier times –Frequency: 0.0004% to 0.0007%  Causes –Autoantibodies (single case, Lupus, ESRD) –HIV vaccines  EIA+: 68%  WB+: 0-44% –Technical / clerical error NEJM 1988;319:961 Ann Intern Med 1989;110:617

25. Indeterminate Results  4-20% of WB assays with positive bands  Testing during seroconversion –p24 usually appears first  Late stage HIV: loss of core antibody  HIV vaccine recipients  Technical / clerical error  Infection with O strain or HIV-2

26. Indeterminate Results (continued)  Cross-reacting nonspecific antibodies –Collagen-vascular disease –Autoimmune disease –Pregnancy –Organ transplantation –Lymphoma, other malignancies –Liver disease –Multiple sclerosis –Recent immunization

27. Indeterminate Results  Evaluate HIV risk  Low risk: almost never infected with HIV-1 or HIV-2 –Repeat testing: often continued indeterminate –Cause: frequently not established –HIV unlikely –Follow-up serology in 3 months  Seroconversion: usually WB+ in 1 month –Repeat testing at 1, 2, 6 months  Counseling to reduce potential transmission

28. Frequency of HIV Testing  High risk behavior: every 6-12 months  Annual seroconversion –General population: 0.02% –Military recruits: 0.04% –MSM: 0.5 - 2% –IDU in high prevalence area: 0.7-6%

29. Alternative Testing  Rapid Testing  Alternative body fluids –Saliva –Urine –Vaginal secretions  Viral detection  Home test kits

30. Rapid HIV Antibody Detection  Results in 15-20 minutes  Occupational exposure  Women in labor with unknown HIV status  Clients unlikely to return for visits  Outreach  ERs

31. Rapid HIV Antibody Detection  OraQuick HIV-1 Antibody Test (OraSure) –Results read by provider in 20 minutes –Sensitivity: 99.6% / Specificity: 100% –$20-30  Fingerstick sample of blood  Negative test: definitive  Positive test: needs standard serology confirmation  Not recommended for HIV-2 screening

32. OraQuick Advance HIV-1/2  CLIA (Clinical Laboratory Improvement Amendment) waived for whole blood, saliva, plasma  Room temperature  Detects VIH-1/2  Results in 20 minutes

33. HIV 1/2 HIV 1/2

34. Stored Serum Phase II, Whole blood Phase II, plasma Phase III, whole blood Rapid test Se ns. Spec. Sens. Spe c. Sens.Spec.Sens.Spec. Determine 10 0% 98.0 % 100 % 100%100%100%99.9% MedMira 99. 0 10097.0 98. 5 97.999.893.199.0 Multispot 10 0 100--10099.1100*100* OraQuick 100--10099.898.699.9 OraQuick oral ------98.6**98.9** SUDS 97. 5 94.5--99.199.698.6*99.5* Unigold 99. 0 96.095.5 99. 8 99.199.889.299.8 *Plasma specimens **Oral fluid specimens

35. Rapid Test Results  Reactive (preliminary positive) rapid test –Screening test is positive –Preliminary result –Confirmatory testing required –Precautions to avoid viral transmission  Negative rapid test –No recent exposure: definitive negative –Possible recent exposure:  Recommend re-test  Counseling to prevent transmission

36. P24 Antigen  Part of blood bank algorithms since 1996  Uncommon in clinical practice  Detects free, non-complex HIV antigens in peripheral blood

37. Indications for HIV Viral Detection  Confusing / indeterminate serologic test results  Acute retroviral infection  Neonatal infection  Window period following exposure  Not FDA approved for diagnosis of HIV  Expensive

38. Viral Detection  p24 Antigen  HIV-1 DNA PCR –Most sensitive: able to detect 1-10 copies of proviral DNA –S/S: 99% / 98%  HIV-1 RNA (RT-PCR, bDNA) –S/S: 95-98%  Viral culture of PBMC: expensive, labor intensive, reliability variable

39.  Recommendations for HIV Screening of Pregnant Women Universal testing for all pregnant women as a routine part of prenatal care using an “opt out” approach –Labor and Delivery: routine rapid testing if HIV status unknown –Postnatal: rapid testing for all infants whose mother’s status is unknown  Regulations, laws, and policies about HIV screening of pregnant women vary from state to state National Recommendations For HIV Testing of Pregnant Women

40. Diagnosis in Children < 18 months

41. DNA PCR DNA PCR (detects HIV proviral DNA within PBMC) Qualitative  Reactive  Non Reactive Sensitivity increases from 38% at birth to93% at 14 days at one mont S-S between 90-100% (Dunn 1995)

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43. Detuned Antibody Testing  Less sensitive ELISA test  May help distinguish between recent seroconverters and those with long- standing HIV infection  Current ELISAs can detect relatively low levels of Ab  HIV Ab levels increase over first few months –Recent infection: standard ELISA positive –Detuned assay: negative  Able to diagnose individuals who have already seroconverted on a standard ELISA but are still early in infection

44. Recommended Websites  AIDSInfo website (www.aidsinfo.nih.gov) www.aidsinfo.nih.gov  AETC National Resource Center  http://www.aidset.org http://www.aidset.org  http://www.cdc.gov/spanish/enfermedade s.htm http://www.cdc.gov/spanish/enfermedade s.htm http://www.cdc.gov/spanish/enfermedade s.htm  http://www.womenchildrenhiv.org/ http://www.womenchildrenhiv.org/  www.aahivm.org

45.   “If we begin with certainties, we shall end in doubts; but if we begin with doubts, and are patient with them, we shall end with certainties” Sir Francis Bacon