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

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Presentation on theme: "Laboratory Testing Unit 6 Diagnosis & Follow-up of HIV Infection."— Presentation transcript:

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

2 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 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 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 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 6 “Window Period” Following HIV Infection P24 a.g ELISA 0 234 (Weeks since infection) ab Viremia Antibody Asymptomatic Acute HIV syndrome Primary HIV infection Source: S Conway and J.G Bartlett, 2003 Years ------------------------------------ PCR

7 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 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 9  T1: Screening test If (+) go to T2  T2: Confirmatory test If (-) go to T3  T3: Tie breaker HIV Serial Rapid Test Algorithm Neg Pos NegPos SAMPLE Neg Pos T1 T3 T2

10 10 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 Neg SAMPLE T1 NegPos T3 T2 PosNegPos

11 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 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 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 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 15 Direct Detection Methods  Uses: Window period Indeterminate WB results Monitor HIV infected individuals  Two methods P24 antigen capture test Polymerase Chain Reaction

16 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 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 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 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 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 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 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 23 RBCs plateletsWBCs PMNs Eos Lymphs Grans Macroph. CD4 (T helper)CD8 (T helper) others Cellular Blood Elements & CD4 Count

24 24 ART Baseline Labs  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

25 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 26 Laboratory Indicators for Initiating ART  Total lymphocyte count <1200  CD4 count <200  Viral load

27 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/mm 3 CD4 count is 380 and 23%

28 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 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 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 31 Recovery of CD4 Cells Continues Years After Starting HAART Source: Binquet C, et al. Am J Epidem, 2000.

32 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 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 34 Pediatric Age-Specific HIV Classification: CD4+ Immunologic Categories

35 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 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 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 38 Detection of Treatment Failure  CD4 cell count  Viral load testing  Viral resistance testing Genotype Phenotype

39 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 40 Types of Resistance Testing  Genotype  Phenotype  Virtual phenotype

41 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 42 Ethiopian Guidelines for Laboratory Monitoring of Patients Taking ART

43 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

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