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Lab support in hiv treatment and management
 

Lab support in hiv treatment and management

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  • Genotyping assays detect drug resistance mutations that are present in the relevant viral genes. Certain genotyping assays involve sequencing of the entire reverse transcriptase and protease genes, whereas others use probes to detect selected mutations that are known to confer drug resistance. Genotypic assays can be performed rapidly, and results can be reported within 1-2 weeks of sample collection. <br />
  • Phenotypic assays measure the ability of a virus to grow in different concentrations of antiretroviral drugs. Reverse transcriptase and protease gene sequences derived from patient plasma HIV RNA are inserted into the backbone of a laboratory clone of HIV, either by cloning or by in vitro recombination. Replication of the recombinant virus at different drug concentrations is monitored by expression of a reporter gene and is compared with replication of a reference HIV strain. The drug concentration that inhibits 50% of viral replication (i.e., the median inhibitory concentration [IC]50) is calculated, and the ratio of the IC50 of test and reference viruses is reported as the fold increase in IC50 (i.e., fold resistance). <br /> Automated, recombinant phenotypic assays are commercially available with results available in 2–3 weeks. However, phenotypic assays cost more to perform than genotypic assays. In addition, interpretation of phenotypic assay results is complicated by incomplete information regarding the specific resistance level (i.e., fold increase in IC50) that is associated with drug failure, although clinically significant fold increase cutoffs are now available for some drugs [711]. Again, consultation with a specialist can be helpful for interpreting test results. <br /> Further limitations of both genotypic and phenotypic assays include lack of uniform quality assurance for all available assays, relatively high cost, and insensitivity for minor viral species. If drug-resistant viruses are present but constitute &lt;10%–20% of the circulating virus population, they probably will not be detected by available assays. Thislimitation is important because, after drugs exerting selective pressure on drug-resistant populations are discontinued, a re-emergence of wild-type virus as the predominant plasma population often occurs, resulting in a decrease of the proportion of virus with resistance mutations to below these thresholds [12-14]. This reversion to predominantly wild-type virus often occurs in the first 4–6 weeks after drugs are stopped. Prospective clinical studies have shown that, despite this plasma reversion, reinstitution of the same antiretroviral agents (or those sharing similar resistance pathways) is usually associated with early drug failure, and the virus present at failure is derived from previously archived resistant virus [15]. Therefore, resistance testing is of greatest value when performed before or within 4 weeks after drugs are discontinued (AII). Because detectable resistant virus may persist in the plasma of some patients for longer periods of time, resistance testing beyond 4 to 6 weeks after discontinuation may still reveal mutations. Yet, the absence of detectable resistance in such patients must be interpreted with caution in designing subsequent antiretroviral regimens. <br />

Lab support in hiv treatment and management Lab support in hiv treatment and management Presentation Transcript

  • LABORATORY SUPPORT IN MANAGEMENT AND TREATMENT OF HIV INFECTION Dr Abhijit Chaudhury MD,DNB, Diplomate of American Board. Professor, Dept of Microbiology.
  • Importance Of Laboratory in Management of HIV Infection      DIAGNOSIS ASSESS THE NEED FOR THERAPY ASSESS THE EFFECTIVENESS OF ART HELP IN DECISION MAKING FOR CHANGE OF THERAPY (Treatment Failure) DRUG TOXICITY MONITORING (BIOCHEMISTRY)
  • Basic Tools    Nucleic Acid Testing (NAT). CD4+ Lymphocyte Count. Phenotype Testing for Drug Resistance.
  • NAT NAT is the detection of DNA/RNA in patient’s sample. PCR : Useful for detection and identification of a wide variety of organisms in patient’s samples. Highly specific and sensitive. Even a few organisms present can be detected.
  • Polymerase Chain Reaction     Amplification technique. A huge number of copies of DNA/ RNA can be made from a single copy. 30-40 cycles will generate ~ 1 million copies. Results are available in 24-48 hrs.
  • Basic Steps of PCR    Denaturation: The double stranded DNA is melted to open the single strands at 94 C. Annealing( 54 C) Primer is added( Small piece of DNA of a particular organism) which binds with portions of the sample DNA ( Template) and starts copying the template. Extension ( 72 C): Under the influence of the added Taq polymease enzyme both strands of the DNA get copied. One DNA becomes 2!
  • NAT Commercially 4 types of test formats are available for HIV: 1. PCR to Detect Proviral DNA 2. RT-PCR: PCR amplification of cDNA generated from viral RNA ( Target amplification). Can detect up to 40 copies/ml of RNA. 3. bDNA Assay: Nucleic acid capture assay using signal amplification. Isothermic process. Reliable to 75 copies/ml 
  • NAT 4. NASBA ( Nucleic acid sequence based assay) : Isothermic nucleic acid amplification with internal controls. Can detect minimum of 176 copies/ml of RNA. 5.Product Enhanced RT (PERT) Assay (Not available commercially). 106 -107 times more sensitive than RT-PCR, can detect 10 viral particles.
  • NAT-Importance     Used for definitive diagnosis of HIV infection in infants and children aged under 18 months. No role in diagnosis in adults Used for virus quantification Used for drug resistance testing No commercial NAT kit is available for detection or quantification of HIV 2.
  • CD4+ T- Lymphocyte Count.   The Gold Standard of CD4 cell count is the Flow Cytometry Method using Fluorescent Activated Cell Sorter. The absolute count in Indian population varies from ~ 700-900/cu mm.
  • CD4+ Lymphocyte Count     The major indicator of immune function Most recent CD4 count is best predictor of disease progression CD4 count usually is the important consideration in decision to start ART Important in determining response to ART   Adequate response: CD4 increase 100-150 cells/µL per year CD4 monitoring  Check at baseline (x2) and at least every 3-6 months
  • CD4+ Lymphocyte Count   Exact CD4 count at which to initiate therapy not known, but evidence points to starting at higher counts Current recommendation: ART for all patients with CD4 counts of <350 cells/µL, certain others regardless of count.
  • Indications for Initiating ART: Chronic Infection Clinical Category and/or CD4 Count  History of AIDS-defining illness  CD4 count of <350 cells/µL  Pregnant women  HIV-associated nephropathy Recommendation  Hepatitis B coinfection, when HBV treatment is indicated Initiate ART
  • Indications for Initiating ART: Chronic Infection Clinical Category and/or CD4 Count CD4 count of >350 cells/µL, asymptomatic, without conditions listed above Recommendation Optimal time to initiate ART is not well defined; consider individual patient characteristics and comorbidities
  • VIRAL LOAD  HIV RNA    Less important than CD4 count, but may influence decision to start ART and help determine frequency of CD4 monitoring > 50,000 copies of RNA/ml is an indication for initiation of ART. Critical in determining response to ART   Goal of ART: HIV RNA below limit of detection (ie, <40 to <80 copies/mL, depending on assay) RNA monitoring     Check at baseline (x2) Immediately before initiating ART 2-8 weeks after start or change of ART (Normal response is tenfold decrease in HIV RNA level within 4-8 weeks) Every 3-4 months with stable patients
  • Treatment Failure A. VIROLOGIC FAILURE  Incomplete virologic response:   In patient on initial ART, HIV RNA >400 copies/mL after 24 weeks on therapy or >50 copies/mL by 48 weeks (confirm with second test) Virologic rebound:  Repeated detection of HIV RNA after virologic suppression (eg, >50 copies/mL)
  • Treatment Failure B. IMMUNOLOGIC FAILURE  Failure to achieve and maintain adequate CD4 response despite virologic suppression   There is no specific definition for immunologic failure, although some studies have focused on patients who fail to increase CD4 T-cell counts above a specific threshold (e.g. >350 or 500 cells/mm3) over a specific period of time (e.g. 4–7 years). Persistently low CD4 count while on suppressive ART is associated with increased risk for  AIDS-related complications
  • Resistance Testing  Before initiation of ART:       Resistance testing (genotype) recommended for all at entry to care Recommended for all pregnant women Transmitted resistance in 6-16% of HIV-infected patients Identification of resistance mutations may optimize treatment outcomes In absence of therapy, resistance mutations may decline over time and become undetectable by current assays, but may persist and cause treatment failure when ART is started Patients with virologic failure:   Perform while patient is taking ART, or ≤4 weeks after discontinuing therapy Interpret in combination with history of ARV exposure and ARV adherence
  • Resistance Testing- GENOTYPING      Detects drug resistance mutations in specific genes (eg, reverse transcriptase and protease) Sequencing or probing Results within 1-2 weeks Interpretation of mutations and crossresistance is complex Consultation with specialists is recommended
  • Resistance TestingPHENOTYPING       Measures the ability of viruses to grow in various concentrations of ARV drugs Results within 2-3 weeks More expensive than genotyping The ratio of the IC50s of the test and reference viruses is reported as the fold increase in IC50, or fold resistance Interpretation may be complex Consultation with specialists is recommended
  • Coreceptor Tropism Assay  Should be performed when CCR5 antagonist is being considered    Maraviroc should be given only to patients with exclusive CCR5 tropism Current commercially available tropism assay is 100% sensitive for CXCR5 clones that make up ≥0.3% of the population Consider in patients with virologic failure on a CCR5 antagonist.
  • List Of Lab Tests at Initiation of Therapy  BASELINE EVALUATION:          HIV antibody CD4 cell count Plasma HIV RNA Resistance test (genotype) CBC, chemistry profile, BUN, Cr, transaminase Fasting glucose and lipids RPR or VDRL Hepatitis A, B, C serology Toxoplasma IgG
  • List Of Lab Tests at Initiation of Therapy: Additional Tests     Tuberculin skin test (TST) or IFN-γ release assay Chest X ray (if symptoms, or positive Tuberculin test or IFN-γ release assay) Gynecologic exam with Pap smear Testing for Chlamydia and gonorrhoea
  • WHO Recommendations for Resource Limited Settings    In the absence of a CD4 cell count, a total lymphocyte count (TLC) below 1200 cells/mm3 in patients with symptomatic HIV disease has been recommended as a guide to the initiation of ART. Data suggest that a TLC below 1200 cells/mm3 as a surrogate for a CD4 count below 200 cells/mm3 has high positive predictive value but poor negative predictive value and that it cannot be used alone in asymptomatic patients to determine treatment eligibility. WHO considers that TLC should be gradually eliminated from the adult ARV guidelines.
  • Conclusion   Laboratory support is absolutely essential in the management of HIV infected patient. There is an urgent need to establish an HIV laboratory at SVIMS with the minimum capabilities as recommended by WHO ( Diagnostic, CD4 count, Viral load estimation). THE END