Human Immunodeficiency Virus

1. Human Immunodeficiency Virus The Virus & The Laboratory Diagnosis Dr. SudheerKher

2. Lab workers Health workers Counselors Strategies and Algorithms • Strategies – Testing approach used to meet a specific need, such as: • Blood Safety • Surveillance • Diagnosis • Algorithms – The combination and sequence of specific tests used in a given strategy

4. HIV Genes • Three basic genes • gag – encodes matrix, capsid, nucleic acid binding proteins • pol – encodes reverse transcriptase • env - encodes surface glycoproteins, transmembrane proteins

5. Regulatory genes • tat ( transactivator of transcription) • rev (regulator of expression of viral protein) • nef ( negative expression factor) • vif (virion infectivity factor) • vpr (stimulating promoter of virus) • vpu (HIV-1) & vpx (HIV-2) small viral proteins promoting maturation and release of progeny virus from cells.

6. HIV Antigens • Major surface/envelope glycoproteins • gp120 • gp41 anchors gp120 to virus • Major capsid proteins • p24 • P18 • Other minor surface & structural proteins • p55

7. Lab workers Health workers Counselors HIV Testing Strategies • Parallel testing • Samples are tested simultaneously by two different tests • Serial testing • Samples tested by a first test • Result of first test determines whether additional testing is required

8. Lab workers Health workers Counselors Advantages of National Testing Strategies and Algorithms • Facilitates: • Country-level standardization • Procurement and supply management • Training • Quality assurance

9. Lab workers Health workers Counselors Key Factors in Determining a Country’s Algorithm • Test performance in country • Test availability in country • Program needs • Ease of use • Type of specimen • Cost • Potential need to differentiate between HIV 1 & HIV 2

10. Lab workers Health workers Evaluating Test Performance: Basic Terms • Sensitivity (Se) of a test is its capacity to correctly identify people that are infected with HIV. • Specificity (Sp) of a test is its capacity to correctly identify people that are not infected with HIV. • Positive Predictive Value (PPV) is the probability that a person who tests reactive is indeed infected with HIV. • Negative Predictive Value (NPV) is the probability that a person who tests negative is not infected with HIV.

11. Lab workers Health workers Calculating Sensitivity, Specificity, PPV, & NPV Actual HIV status (Gold Standard) Test result HIV infected HIV -uninfected Total A+B Positive B A C+D Negative C D A+C B+D Total Sensitivity = A ÷ (A+C) Specificity = D ÷ (B+D) Positive Predictive Value = A ÷ (A+B) Negative Predictive Value= D ÷ (C+D)

12. Lab workers Health workers Calculating Sensitivity, Specificity, PPV, & NPV (Cont’d) Actual HIV status (Gold Standard) Test result HIV infected HIV -uninfected Total A+B(372) Positive B (2) A (370) C+D(628) Negative C (4) D (624) A+C (374) B+D (626) 1000 Total Sensitivity = A ÷ (A+C) = 370 ÷ 374 = 98.9% Specificity = D ÷ (B+D) = 624 ÷ 626 = 99.7% PPV = A ÷ (A+B) = 370 ÷ 372 = 99.5% NPV = D ÷ (C+D) = 624 ÷ 628 = 99.4%

13. Lab workers Health workers Counselors HIV Rapid Test Performance • No test is 100 % sensitive • No test is 100 % specific Note: Performance of tests and subsequent algorithm must be determined in context of population

14. Lab workers Health workers How Prevalence Affects PPV & NPV (Prevalence) (Se) PPV= (Prevalence) (Se) + (1- Prevalence) (1- Sp) (1-Prevalence) (Sp) NPV= (1-Prevalence) (Sp + (Prevalence) (1- Se)

15. Lab workers Health workers How Prevalence Affects PPV & NPV (Cont’d) PPV for 10 % prevalence population: (10/100) (98.9/100) = ----------------------------------------------------------------------- = 97.3% (10/100) (98.9/100) + (1- 10/100) (1- 99.7/100) PPV for 1% prevalence population: (1/100) (98.9/100) = --------------------------------------------------------------------------- = 76.9% (1/100) (98.9/100) + (1- 1/100) (1- 99.7/100)

16. Consent Issues • Opt in strategy – • Pretest & Post test counseling a must • Opt out strategy – • Consent taken as a part of general consent for investigations & treatment • Consent is assumed • Patient given option to opt out any time • Pre test counseling not done • Post test counseling done.

17. Testing in Developing countries • To cater for the cost of testing • Infrastructure • Technical manpower • Magnitude of workload • Without compromise on quality

18. Lab workers Health workers Counselors Testing Algorithm Describes the Sequence of Tests to be Performed • An HIV Positive Status should be based upon the outcome of 2 or more tests • When two test results disagree (one is reactive, the other non-reactive), the finding is called “discordant.” In this case, a third test must be performed. Always follow the sequence of the tests in the algorithm

19. Lab workers Health workers Counselors Ideal Algorithm • Tests need to be : • Highly sensitive • Highly specific • Tests should not share the same false negatives and false positives • 3rd test (if needed)

20. Blood Sample Test 2 Test 1 Both Non-reactive Report Negative DiscordantResult Both Reactive Report Positive Test 3 Reactive Result Report Positive Non-reactive Result Report Negative Lab workers Health workers Counselors Testing Algorithm* * Develop appropriate algorithm diagram (parallel or serial) and insert names of HIV tests that represent test 1, 2 or 3

21. Lab workers Health workers Counselors Possible HIV Test Outcomes: Parallel Algorithm

22. Test Serology ELISA Latex Agglutination Western Blot Immunofluorescence Virion RNA, RT-PCR P24 antigen Isolation of virus Lymphocyte CD4:CD8 T cell ratio Objective Initial Screening Initial Screening Confirmatory test Confirmatory test Detection of virus in blood Early marker of infection Test not readily available Correlate of HIV Disease Laboratory Diagnosis

23. Classical way of Lab diagnosis • Initial Screening test • Repeat test using different technique / Ag • Confirm by • IFA – Popular in US • Western Blot ( Rest of the world) • Used for diagnosis of a patient

24. Laboratory tests for diagnosis of HIV infection

25. Are you asking for the test because you feel it is mandatory? Are you asking for the test out of fear? Have you acquired basic knowledge of HIV/AIDS? Do you value human rights? Are you aware that your patient too has rights? Are you prepared to do counseling before asking for the test? Are you prepared to counsel the patient after the test? Are you prepared to take informed written consent of the patient before sending the sample? Are you aware that HIV test is no substitute for your precautions? Before you ask for HIV test

26. Strategies for HIV testing in India • Strategy-I • Serum tested by one of the E/R/S. • If reactive, sample considered positive • If not reactive, sample considered negative • Use – Transfusion safety • Highly sensitive & reliable kits used

27. Strategies for HIV testing in India • Strategy-II • Serum reactive with one E/R/S retested with second E/R/S using different Ag/test principle. If reactive, reported positive. • Use – Unlinked anonymous HIV Surveillance

28. Strategies for HIV testing in India • Strategy –III • Serum reactive with two E/R/S tests is retested with a third E/R/S. • Or two of the ERS followed by confirmatory test. • Use – Diagnosis of HIV infection

29. Blood counts Leucopenia Lymphocyte count less than 400/cmm Thrombocytopenia T-cell subset assay CD4 count below 200/cmm Normal CD4:CD8 ratio 2:1. Reversed in AIDS to 0.5:1. Hypergammaglobulinaemia Diminished CMI – Candidial, tuberculin tests. Lab diagnosis of opportunistic infections Malignancies -Non-specific tests for HIV