Laboratory Testing

1. Laboratory Testing Unit 6 Diagnosis & Follow-up of HIV Infection

2. Learning Objectives • Describe the role of laboratory tests in: • The diagnosis of HIV infection • Ongoing monitoring of HIV’s effect on the body • Initiating ART and prophylaxis of OIs • Monitoring response to treatment • Monitoring toxicity of treatments

3. Patient Management Tasks That Depend on Laboratory Support • Diagnosis of HIV • Staging of HIV’s effects • Baseline lab assessment • ARV regimen selection • Monitoring response to ART • Monitoring adverse drug effects • Detection of treatment failure • Diagnosis of OIs • Resistance profile

4. Diagnostic Tests for HIV Infection • Serological methods for detection of antibody • Rapid tests • ELISA • Western blot • Antigen detection methods • P24 antigen capture test • Polymerase Chain Reaction (also known as PCR or viral load)

5. HIV Serological Diagnosis • Tests are based on a reaction between HIV antigens and antibodies in the patient’s serum • Tests use viral antigen extracts as a testing material • Both a screening test and a confirmatory test should be used for diagnosis

6. “Window Period” Following HIV Infection Acute HIV syndrome Antibody Primary HIV infection Asymptomatic Viremia ------------------------------------ PCR WINDOW P24 a.g ELISA a b Years 0 2 3 4 (Weeks since infection) Source: S Conway and J.G Bartlett, 2003

7. 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 • Technical error

8. Rapid Test • Use two consecutive kits • Initial test kit is very sensitive • Repeat test is very specific • Visual tests that do not require the ELISA Reader • Technically simpler to perform • Most have sensitivity and specificity comparable to ELISA

9. SAMPLE T1 Neg Pos T2 Neg Pos T3 Neg Pos HIV Serial Rapid Test Algorithm • T1: Screening test • If (+) go to T2 • T2: Confirmatory test • If (-) go to T3 • T3: Tie breaker

10. SAMPLE T2 T1 Neg Pos Neg Pos T3 Neg Pos HIV Parallel Rapid Test Algorithm • T1 and T2: Screening and Confirmatory tests • If T1 and T2 do not agree go to T3 • T3: Tie breaker

11. ELISA Test • Very sensitive • Results: • Negative means patient does not have evidence of HIV • Repeatedly reactive indicates need for confirmatory test (by Western blot) • Ideally, all tests performed would screen for HIV 1 and 2

12. Western Blot Test • Confirmatory test for all positive ELISA assays • Two or more “key” bands indicate a positive test • Should not be used alone for HIV diagnosis

13. Western Blot Result Interpretation • Results are interpreted as follows: • Negative: no bands • Positive: reactivity to gp120/160, plus either gp41 or p24 • Indeterminate: one reactive band (or anything other than a positive test) should be repeated at a later time, e.g., 1-3 months later • Repeatedly indeterminate: no HIV infection

14. Case Study: Tewabech • Tewabech is a 27 year-old female who began a new relationship with an older man last month • Yesterday she learned that this man had been married previously and that his wife died from AIDS • Tewabech is concerned that she might have HIV and wants to be tested

15. Direct Detection Methods • Uses: • Window period • Indeterminate WB results • Monitor HIV infected individuals • Two methods • P24 antigen capture test • Polymerase Chain Reaction

16. P24 Antigen Capture Test • Detected during acute phase of primary HIV infection • Sensitivity in adults: 50-75% • Sensitivity in children: 20% • Specificity: 95% • Following seroconversion, antigen is less detectable (sensitivity declines)

17. Polymerase Chain Reaction • PCR or “viral load” • Very expensive • Not routinely used for diagnosis except in children <18 months • Also used for: • Diagnosis in special situations • Evaluation of response to treatment (success) • Prognosis (Viral set point) • Detection of virological treatment failure

18. Case Study: Tewabech (cont.) • Tewabech requests an HIV test • The test is performed and comes back negative • Tewabech remains concerned • Although she is counseled to return in one month for repeat testing, she returns one week later with fever, rash, pharyngitis and adenopathy • HIV PCR is positive

19. PCR for Viral Load • Commercial methods/kits in use: • Amplicor • Amplification: RT-PCR detection of DNA • Detection: labeled probe • B-DNA • Amplification: RT-PCR detection of DNA • Detection: labeled antibodies • EasyQ • Amplification: NASBA detection of RNA • Detection: labeled probe

20. Viral Load Monitoring • Where available, PCR or NASBA monitoring provide valuable information about ART effectiveness • Viral load should be checked: • Every 3-6 months when not on ART • One month after starting or changing ART • Every 3-4 months in stable ART patients

21. CD4 Determination • CD4+ cells • Subset of T lymphocytes • CD4 receptor serves as a target for HIV • Normal Values • T helper (CD4+) cell count = 400-1200 • T suppressor (CD8+) cell count = 400-800

22. Uses of CD4 Cell Count • Decision to initiate ARV • Guide in initiating OI prophylaxis • Assess progression of disease • Measure response to treatment (prognostication) • Detect immunologic treatment failure

23. Cellular Blood Elements & CD4 Count RBCs WBCs platelets PMNs Eos Lymphs Grans Macroph. CD4 (T helper) CD8 (T helper) others

24. HIV antibody test Full blood count and differential AST or ALT Serum creatinine or blood urea nitrogen Serum glucose Pregnancy tests for women Urine dipstick Hep B surface antigen Serum RPR Serum lipids (for pts. with other cardiac risk factors or to receive PIs or EFV) CD4 lymphocyte count Viral load ART Baseline Labs

25. ART Baseline Labs (2) • Minimum required in Ethiopian context • HIV antibody test • CBC and differential • Liver function test – ALT • Pregnancy test • BUN and creatinine

26. Laboratory Indicators for Initiating ART • Total lymphocyte count <1200 • CD4 count <200 • Viral load

27. Case Study: Tewabech (cont.) • Tewabech is referred to her local hospital clinic for ongoing care • Baseline laboratory tests performed all return with normal results, except: • White blood cell count is 4200 cells/mm3 • CD4 count is 380 and 23%

28. Total Lymphocyte Count (TLC) • May be used as a substitute marker for CD4 count • TLC value <1200 cells/ul corresponds roughly to CD4 cell count <200 cells/ml • TLC used to initiate ART but not for monitoring response to therapy

29. CD4 Count and TLC Thresholds for OI Prophylaxis • Pneumocystis pneumonia • CD4 <200 • TLC <1200 • Toxoplasmosis • CD4 <100 and positive Toxoplasma serology • Cryptococcal meningitis • CD4 <100

30. Case Study: Tewabech (cont.) • Tewabech establishes ongoing care in the HIV clinic, with repeat CD4 counts done every 6 months • Ultimately, her CD4 count declines to 180 and 13%; she begins zidovudine, lamivudine and nevirapine

31. Recovery of CD4 Cells Continues Years After Starting HAART Source: Binquet C, et al. Am J Epidem, 2000.

32. Interpretation of CD4 Cell Count • CD4 count and interpretation are affected by many factors • Patient age • Time blood is drawn (diurnal variation ) • Seasonal variation • Presence of intercurrent infections, e.g., HTLV-1, flu • Steroid administration • Stress (psychological, physical, pregnancy) • Splenectomy • Results may fluctuate up to 30% in same individual

33. Interpreting CD4 Counts in Pediatric Patients • Level varies with age • Absolute CD4 cell count is high in children <6 years of age • CD4 percentage varies less than CD4 number • Because of this, CD4 percentage (rather than count) is often used in pediatric ART initiation and follow-up

34. Pediatric Age-Specific HIV Classification: CD4+ Immunologic Categories

35. Case Study: Tewabech (cont.) • Viral load monitoring becomes available after Tewabech has started on ART, and her first level checked 8 months after starting ART is undetectable (<50 copies/mcl) • One year after starting ART, her viral load is 255; the next viral load is undetectable • Eighteen months later, her viral load again rises to 330, though the next viral load is undetectable

36. Drug Resistance • Definition: growth of HIV despite presence of therapeutic levels of drug concentration • Arises from genetic mutations of HIV • As the number of mutations increases, this leads to an increasing degree of resistance

37. Treatment Failure • Detected by laboratory evaluation before it becomes obvious clinically • Detected by a few different methods • Treatment failure due to resistance is detected early by rise in virological means

38. Detection of Treatment Failure • CD4 cell count • Viral load testing • Viral resistance testing • Genotype • Phenotype

39. Indications for Resistance Testing • Recently acquired infection (primary HIV infection) • Chronically infected treatment-naïve patients • Patients receiving ART who show evidence of virologic failure (rising viral load)

40. Types of Resistance Testing • Genotype • Phenotype • Virtual phenotype

41. Toxicity Monitoring Goals • Assure safety of ARVs • Detect specific organ dysfunctions: • Known to be associated with specific ARVs • That can be detected before they become clinically apparent • To prevent or limit adverse health effects

42. Ethiopian Guidelines for Laboratory Monitoring of Patients Taking ART

43. Key Points • Laboratory tests determine: • The diagnosis of HIV infection • Timing for initiating ART and OI prophylaxis • Laboratory tests measure: • HIV’s effect on the immune and other body systems • Response to and toxicity of treatments • Laboratory testing is key to determine HIV diagnosis, optimize ART effect, and minimize ART toxicity