Presentation on theme: "Unit 6 Diagnosis & Follow-up of HIV Infection"— Presentation transcript:
1Unit 6 Diagnosis & Follow-up of HIV Infection Laboratory TestingUnit 6Diagnosis & Follow-upof HIV InfectionThis unit should take 1 hour and 10 minutes to complete.Step 1: Overview of Unit Learning Objectives (Slide 2) – 5 minutesStep 2: Laboratory Monitoring for HIV/AIDS (Slides 3-42) – 60 minutesStep 3: Key Points, Questions (Slide 43) – 5 minutes
2Learning Objectives Describe the role of laboratory tests in: The diagnosis of HIV infectionOngoing monitoring of HIV’s effect on the bodyInitiating ART and prophylaxis of OIsMonitoring response to treatmentMonitoring toxicity of treatmentsStep 1: Overview of Unit Learning Objectives (Slide 2) – 5 minutes
3Patient Management Tasks That Depend on Laboratory Support Diagnosis of HIVStaging of HIV’s effectsBaseline lab assessmentARV regimen selectionMonitoring response to ARTMonitoring adverse drug effectsDetection of treatment failureDiagnosis of OIsResistance profileStep 2: Laboratory Monitoring for HIV/AIDS (Slides 3-42) – 60 minutesUse this slide to elaborate on the objectives described in the previous slide, and to create an interactive environment with the participants.Select a few of the items on the slide and ask the audience to describe how lab tests are needed for that particular task.For example, “How do we use laboratory support in the diagnosis of OIs?” Correct responses might be: 1) staining of BAL specimen for Pneumocystis 2) detection of cryptococcal antigen in a patient’s serum or CSF, etc.Don’t make the group go through this exercise for all the items on this page – that would be tedious. Just do this enough to “activate” and engage the group, e.g., 3-4 items
4Diagnostic Tests for HIV Infection Serological methods for detection of antibodyRapid testsELISAWestern blotAntigen detection methodsP24 antigen capture testPolymerase Chain Reaction (also known as PCR or viral load)HIV infection is in almost all instances diagnosed and confirmed by detecting anti-HIV antibodiesDiagnostic Methods:Serological assays for HIV infection have been refined over the past years to unprecedented levels of sensitivity and specificity.However, diagnostic problems, which cannot be resolved with serological methods, are usually cleared using methods for direct detection of HIV infection.In addition, molecular amplification methods are currently the methods of choice for prediction of HIV progression and for monitoring antiretroviral treatment.
5HIV Serological Diagnosis Tests are based on a reaction between HIV antigens and antibodies in the patient’s serumTests use viral antigen extracts as a testing materialBoth a screening test and a confirmatory test should be used for diagnosisSerological methods detect antibodies to HIV.These methods are used solely for diagnosis of HIV infection, not for treatment monitoring.Tests are more than 99% sensitive and 99% effectiveBecause antibodies develop within 2-8 weeks after infection, testing shortly after a potential exposure may not be reliable.
6“Window Period” Following HIV Infection Acute HIV syndromeAntibodyPrimary HIV infectionAsymptomaticViremiaWINDOWPCRP24 a.gELISAStandard HIV antibody tests do not detect HIV in the “window period.”PCR and p24 antigen tests can detect HIV during the latter part of the “window period” but there is not presently a test that reliably identifies early HIV infection within the first two weeks following acute infection.Source: S Conway and J.G Bartlett, 2003abYears234(Weeks since infection)Source: S Conway and J.G Bartlett, 2003
7False Negatives and Positives False negative results may result from:Window periodAgammaglobulinemiaSeroreversion (loss of HIV antibodies due to extreme immune system destruction) in late diseaseTechnical errorType N or O strains, or HIV-2False positive results may result from:Autoantibodies e.g., SLEHIV vaccines
8Rapid Test Use two consecutive kits Initial test kit is very sensitiveRepeat test is very specificVisual tests that do not require the ELISA ReaderTechnically simpler to performMost have sensitivity and specificity comparable to ELISABased on the principles of dot immunoassay, or particle agglutination (e.g. gelatin or latex)Requires a second test only if the first test is positive:A single negative result is valid and need not be repeated, due to the very high sensitivity (> 99.7%) of the test.First test is relatively less specific (may have false positives) but the second test is very specific and differentiates the true positives from false positives.These tests enable health providers to supply definitive negative and preliminary positive results to clients at the time of testing.The method of determination is projected in subsequent slides.
9HIV Serial Rapid Test Algorithm NegPosSAMPLET1T3T2T1: Screening testIf (+) go to T2T2: Confirmatory testIf (-) go to T3T3: Tie breakerThis testing algorithm and method of interpretation is used in rapid tests, because a single positive result is not reliable due to the high sensitivity of T1.It is a cost effective algorithm, because tests are only done when needed, i.e., when indicated based on the previous test’s results.Results that appear in bold text on the flow diagram represent final results.
10HIV Parallel Rapid Test Algorithm T1 and T2: Screening and Confirmatory testsIf T1 and T2 do not agree go to T3T3: Tie breakerNegSAMPLET1PosT3T2This mode of testing is more expensive than the serial method, because two test kits are used for even a negative test. However, it provides results more quickly because it requires only two rounds of testingResults that appear in bold text on the flow diagram represent final results.
11ELISA Test Very sensitive Results: Negative means patient does not have evidence of HIVRepeatedly reactive indicates need for confirmatory test (by Western blot)Ideally, all tests performed would screen for HIV 1 and 2Uses the same principles of interpretation of results as in the Rapid testMany of the current HIV ELISA assays also contain HIV-2 antigens. Specimens failing to demonstrate HIV-1 should be examined for the presence of HIV-2 specific proteins in patients where the suspicion of HIV infection is high.
12Western Blot Test Confirmatory test for all positive ELISA assays Two or more “key” bands indicate a positive testShould not be used alone for HIV diagnosisThe Western Blot (WB)Detects antibodies to HIV-1 proteins, including core (p17, p24, p55), polymerase (p31, p51, p66) and envelope (gp41, gp 120, gp160).Should not be used alone; should always be coupled with a screening test due to a 2% rate of false positives.
13Western Blot Result Interpretation Results are interpreted as follows:Negative: no bandsPositive: reactivity to gp120/160, plus either gp41 or p24Indeterminate: one reactive band (or anything other than a positive test) should be repeated at a later time, e.g., 1-3 months laterRepeatedly indeterminate: no HIV infectionWestern blot techniques involves placing specimen at one end of a gel strip and subjecting the specimen to electrophoresis, where an electrical current causes migration of antibodies in the specimen.Antibodies to HIV proteins of specific molecular weights can be detected, and these correlate to particular HIV proteins.
14Case Study: TewabechTewabech is a 27 year-old female who began a new relationship with an older man last monthYesterday she learned that this man had been married previously and that his wife died from AIDSTewabech is concerned that she might have HIV and wants to be testedSUGGESTED DISCUSSIONDiscuss the following questions regarding the case study with the participants. Read each question and ask participants to volunteer answersShould she be tested today? If not, when?If she tests positive, does that mean her new partner infected her?If she tests negative, does that mean she does not have HIV?What tests are available to diagnose HIV infection?
15Direct Detection Methods Uses:Window periodIndeterminate WB resultsMonitor HIV infected individualsTwo methodsP24 antigen capture testPolymerase Chain ReactionIn certain situations serologic tests are not reliable and direct detection of HIV is required. Such as:Newborns born to HIV-infected mothers, individuals with indeterminate WB results, to determine the HIV status during “window period”, and to monitor HIV infected individuals
16P24 Antigen Capture TestDetected during acute phase of primary HIV infectionSensitivity in adults: 50-75%Sensitivity in children: 20%Specificity: 95%Following seroconversion, antigen is less detectable (sensitivity declines)p24 antigenCan be detected in serum or plasmaAssociated with intact or disrupted virions or released by infected cells.Detected using EIA, utilizing immobilized antibodies to p24, which bind to p24 antigen present in the patient's serum.TestUsed during primary HIV infection (“window period”), during very late symptomatic stages of infection, and in newborns to HIV-infected mothers.Negative result does not rule out HIVFor confirmation of a reactive result, the sample must be subjected to an additional confirmatory neutralization assayEstimated average time from detection of p24 antigen to detection of HIV antibody by routine screening examinations is approximately six days (not all recently infected individuals exhibit p24 antigenemia)
17Polymerase Chain Reaction PCR or “viral load”Very expensiveNot routinely used for diagnosis except in children <18 monthsAlso used for:Diagnosis in special situationsEvaluation of response to treatment (success)Prognosis (Viral set point)Detection of virological treatment failureA significant drop in viral load is expected three to four weeks after initiation of treatment.Diagnosis in special situationsConfusing serological testExamples: indeterminate WB or serologic positive result in children less than 18 months (and particularly less than 12 months of age) due to maternal antibodiesNeonatal HIV infectionsWindow periodEvaluation of response to treatment (success)Drop of >1 log within 4-8 weeks of starting ARTGoal: undetectable viral level at 4-6 monthsUndetectable viral load is defined as < 50 copies virus per mcl of serumPrognosis (Viral set point)Viral load at 6-12 month after acquisition of HIVDetection of virological treatment failureFailure to achieve complete viral suppressionDetectable virus after complete suppression
18Case Study: Tewabech (cont.) Tewabech requests an HIV testThe test is performed and comes back negativeTewabech remains concernedAlthough she is counseled to return in one month for repeat testing, she returns one week later with fever, rash, pharyngitis and adenopathyHIV PCR is positiveDISCUSSION SUGGESTIONSWhat do her symptoms represent? (Acute HIV Syndrome)What other symptoms are associated with this syndrome? (see other unit)Was the test defective?
19PCR for Viral Load Commercial methods/kits in use: Amplicor B-DNA Amplification: RT-PCR detection of DNADetection: labeled probeB-DNADetection: labeled antibodiesEasyQAmplification: NASBA detection of RNAMethodologies used to determine viral load.
20Viral Load MonitoringWhere available, PCR or NASBA monitoring provide valuable information about ART effectivenessViral load should be checked:Every 3-6 months when not on ARTOne month after starting or changing ARTEvery 3-4 months in stable ART patients
21CD4 Determination CD4+ cells CD4 receptor serves as a target for HIV Subset of T lymphocytesCD4 receptor serves as a target for HIVNormal ValuesT helper (CD4+) cell count =T suppressor (CD8+) cell count =CD4+ cellsIdentified by functional proteins (receptors) on the cell surfacePreferentially depleted during the course of the diseaseHIV binds to the receptor in the process of entering/infecting the cell
22Uses of CD4 Cell Count Decision to initiate ARV Guide in initiating OI prophylaxisAssess progression of diseaseMeasure response to treatment (prognostication)Detect immunologic treatment failureAccurate and reliable enumeration of CD4 T cell counts is crucial for monitoring the rate of progression to AIDS, both for initiating prophylaxis for opportunistic infections as well as monitoring the impact of antiretroviral therapy (ART).
23Cellular Blood Elements & CD4 Count WBCsRBCsplateletsPMNsEosLymphsGransMacroph.This slide puts “CD4 cells” in context of all cellular blood elements.CD4 cells are identified in the lab by a process called “flow cytometry.”CD4 (T helper)CD8 (T helper)others
24ART Baseline Labs HIV antibody test Full blood count and differential AST or ALTSerum creatinine or blood urea nitrogenSerum glucosePregnancy tests for womenUrine dipstickHep B surface antigenSerum RPRSerum lipids (for pts. with other cardiac risk factors or to receive PIs or EFV)CD4 lymphocyte countViral loadIn the absence of resource constraints, this list provides a thorough baseline assessment which can help you to determine baseline health status, and assist in deciding which medications the patient can use and when they should be started.These tests might not be available in all Ethiopian settings.
25ART Baseline Labs (2) Minimum required in Ethiopian context HIV antibody testCBC and differentialLiver function test – ALTPregnancy testBUN and creatinine
26Laboratory Indicators for Initiating ART Total lymphocyte count <1200CD4 count <200Viral loadEthiopian guidelines for initiation of ART also include clinical indicators, such as Stage IV regardless of CD4 count, and Stage III with CD4 <350.
27Case Study: Tewabech (cont.) Tewabech is referred to her local hospital clinic for ongoing careBaseline laboratory tests performed all return with normal results, except:White blood cell count is 4200 cells/mm3CD4 count is 380 and 23%DISCUSSION SUGGESTIONSWhat ART is needed presently? (nothing; ART is not initiated until CD4<200)What OI prophylaxis is needed? (nothing; no significant risk for serious OIs. Generally, none started until CD4<200)
28Total Lymphocyte Count (TLC) May be used as a substitute marker for CD4 countTLC value <1200 cells/ul corresponds roughly to CD4 cell count <200 cells/mlTLC used to initiate ART but not for monitoring response to therapyWhile the total lymphocyte count correlates poorly with the CD4 T cell count in asymptomatic patients, in combination with clinical staging, it is still a useful marker of prognosis and survival.Reference Laboratory Support to HIV Diagnosis and Monitoring of Antiretroviral Therapy Report of a Regional WorkshopPune, India, July 2004
29CD4 Count and TLC Thresholds for OI Prophylaxis Pneumocystis pneumoniaCD4 <200TLC <1200ToxoplasmosisCD4 <100 and positive Toxoplasma serologyCryptococcal meningitisCD4 <100
30Case Study: Tewabech (cont.) Tewabech establishes ongoing care in the HIV clinic, with repeat CD4 counts done every 6 monthsUltimately, her CD4 count declines to 180 and 13%; she begins zidovudine, lamivudine and nevirapineDISCUSSION SUGGESTIONSAsk the group if they agree with her care and treatment (they should, since it is in accord with national guidelines).How will they follow-up with (what laboratory tests will they order for) Tewabech now that she is on ART? How often will they be repeated?
31Recovery of CD4 Cells Continues Years After Starting HAART The CD4 count typically increases > 50 cells/mm3 at 4-8 weeks after viral suppression with HAART and then increases an additional 50 – 100 cells / mm3 per year thereafter. (Ref. GID 2000;185:471 JAMA 2002;288:222)Image source: Binquet C, et al. Modeling changes in CD4-positive T-lymphocyte counts after the start of highly active antiretroviral therapy and the relation with risk of opportunistic infections: the Aquitaine Cohort, Am J Epidem Feb 15;153(4):Source: Binquet C, et al. Am J Epidem, 2000.
32Interpretation of CD4 Cell Count CD4 count and interpretation are affected by many factorsPatient ageTime blood is drawn (diurnal variation )Seasonal variationPresence of intercurrent infections, e.g., HTLV-1, fluSteroid administrationStress (psychological, physical, pregnancy)SplenectomyResults may fluctuate up to 30% in same individual
33Interpreting CD4 Counts in Pediatric Patients Level varies with ageAbsolute CD4 cell count is high in children <6 years of ageCD4 percentage varies less than CD4 numberBecause of this, CD4 percentage (rather than count) is often used in pediatric ART initiation and follow-up
34Pediatric Age-Specific HIV Classification: CD4+ Immunologic Categories Modified from: CDC Revised classification system for human immunodeficiency virus infection in children less than 13 years of age. MMWR, 1994; 43 (No. RR-12): p. 1–10.
35Case 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 undetectableEighteen months later, her viral load again rises to 330, though the next viral load is undetectableBACKGROUND INFORMATIONThese transient, low-level rises in her viral load have been termed “blips.”Blips have not been shown to be associated with ART failure, and ART should not be changed when blips occur.Consecutive, persistent elevations is viral load are clinically significant, and may indicate a need to change ART. At minimum, they should prompt a meticulous review of adherence.
36Drug ResistanceDefinition: growth of HIV despite presence of therapeutic levels of drug concentrationArises from genetic mutations of HIVAs the number of mutations increases, this leads to an increasing degree of resistance
37Treatment FailureDetected by laboratory evaluation before it becomes obvious clinicallyDetected by a few different methodsTreatment failure due to resistance is detected early by rise in virological meansViral load testing is the most sensitive, earliest means to detect ART failure.CD4 cell decline is intermittent in its ability to detect ART failure (between viral load, and clinical signs of failure, e.g., new OI).Where available, resistance testing is useful to help guide changes in ART when the current regimen is failing.
38Detection of Treatment Failure CD4 cell countViral load testingViral resistance testingGenotypePhenotypeChanges in CD4 count that would be evidence of treatment failure include:Decline after previous increase; return to pre-ART baseline; failure to rise when ART is startedChanges in viral load that would be evidence of treatment failure include:Possibilities: failure to achieve 1 log drop in viral load within first 4 weeks treatment; failure to achieve undetectable viral load within first 4-6 months of ART; persistent detectable virus after becoming undetectable; rise after achieving a stable, low level of persistent virusResistance testing should be ordered when:available, a change in ART is being considered, and while patient is still taking the failing regimen
39Indications for Resistance Testing Recently acquired infection (primary HIV infection)Chronically infected treatment-naïve patientsPatients receiving ART who show evidence of virologic failure (rising viral load)The rationale to have resistance testing in primary HIV infection is that recently infected persons show high level of resistance to ARVs probably due to a wider dissemination of resistant viruses in the community.In patients receiving ARVs resistance testing guides therapy changes.
40Types of Resistance Testing GenotypePhenotypeVirtual phenotypeGenotype – analyzes genetic sequence of virus to detect presence of mutationsPhenotype – measures ability of the virus to grow in vitro in presence of varying drug concentrations.Virtual phenotype – a proprietary process that compares genotype results to a database of hundreds of comparable patients, in an attempt to predict phenotypic response
41Toxicity Monitoring Goals Assure safety of ARVsDetect specific organ dysfunctions:Known to be associated with specific ARVsThat can be detected before they become clinically apparentTo prevent or limit adverse health effects
42Ethiopian Guidelines for Laboratory Monitoring of Patients Taking ART These are the laboratory guidelines from the “Guidelines for use of antiretroviral drugs in Ethiopia, Jan 2005”
43Key Points Laboratory tests determine: Laboratory tests measure: The diagnosis of HIV infectionTiming for initiating ART and OI prophylaxisLaboratory tests measure:HIV’s effect on the immune and other body systemsResponse to and toxicity of treatmentsLaboratory testing is key to determine HIV diagnosis, optimize ART effect, and minimize ART toxicityStep 3: Key Points, Questions (Slide 43) – 5 minutes