Lab support in hiv treatment and management

LABORATORY SUPPORT IN LABORATORY SUPPORT IN MANAGEMENT AND MANAGEMENT AND TREATMENT OF HIV TREATMENT OF HIV

INFECTIONINFECTIONDr Abhijit Chaudhury MD,DNB, Dr Abhijit Chaudhury MD,DNB, Diplomate of American Board.Diplomate of American Board.

Professor, Dept of Microbiology.Professor, Dept of Microbiology.

Importance Of Laboratory in Importance Of Laboratory in Management of HIV InfectionManagement of HIV Infection

DIAGNOSISDIAGNOSIS ASSESS THE NEED FOR THERAPYASSESS THE NEED FOR THERAPY ASSESS THE EFFECTIVENESS OF ARTASSESS THE EFFECTIVENESS OF ART HELP IN DECISION MAKING FOR HELP IN DECISION MAKING FOR

CHANGE OF THERAPY (Treatment CHANGE OF THERAPY (Treatment Failure)Failure)

DRUG TOXICITY MONITORING DRUG TOXICITY MONITORING (BIOCHEMISTRY)(BIOCHEMISTRY)

Basic ToolsBasic Tools

Nucleic Acid Testing (NAT).Nucleic Acid Testing (NAT). CD4+ Lymphocyte Count.CD4+ Lymphocyte Count. Phenotype Testing for Drug Phenotype Testing for Drug

Resistance.Resistance.

NATNAT

NAT is the detection of DNA/RNA in NAT is the detection of DNA/RNA in patient’s sample.patient’s sample.

PCR : Useful for detection and PCR : Useful for detection and identification of a wide variety of identification of a wide variety of organisms in patient’s samples. organisms in patient’s samples. Highly specific and sensitive. Even a Highly specific and sensitive. Even a few organisms present can be few organisms present can be detected. detected.

Polymerase Chain ReactionPolymerase Chain Reaction

Amplification technique.Amplification technique. A huge number of copies of DNA/ A huge number of copies of DNA/

RNA can be made from a single copy.RNA can be made from a single copy. 30-40 cycles will generate ~ 1 30-40 cycles will generate ~ 1

million copies.million copies. Results are available in 24-48 hrs.Results are available in 24-48 hrs.

Basic Steps of PCRBasic Steps of PCR

Denaturation: The double stranded DNA is Denaturation: The double stranded DNA is melted to open the single strands at 94 C.melted to open the single strands at 94 C.

Annealing( 54 C) Primer is added( Small Annealing( 54 C) Primer is added( Small piece of DNA of a particular organism) which piece of DNA of a particular organism) which binds with portions of the sample DNA binds with portions of the sample DNA ( Template) and starts copying the template.( Template) and starts copying the template.

Extension ( 72 C): Under the influence of the Extension ( 72 C): Under the influence of the added Taq polymease enzyme both strands added Taq polymease enzyme both strands of the DNA get copied. One DNA becomes 2!of the DNA get copied. One DNA becomes 2!

NATNAT

Commercially 4 types of test formats are Commercially 4 types of test formats are available for HIV:available for HIV:

1. PCR to Detect Proviral DNA1. PCR to Detect Proviral DNA 2. RT-PCR: PCR amplification of cDNA 2. RT-PCR: PCR amplification of cDNA

generated from viral RNA ( Target generated from viral RNA ( Target amplification). Can detect up to 40 amplification). Can detect up to 40 copies/ml of RNA.copies/ml of RNA.

3. bDNA Assay: Nucleic acid capture assay 3. bDNA Assay: Nucleic acid capture assay using signal amplification. Isothermic using signal amplification. Isothermic process. Reliable to 75 copies/mlprocess. Reliable to 75 copies/ml

NATNAT

4. 4. NASBA ( Nucleic acid sequence based NASBA ( Nucleic acid sequence based assay) : Isothermic nucleic acid assay) : Isothermic nucleic acid amplification with internal controls. Can amplification with internal controls. Can detect minimum of 176 copies/ml of RNA.detect minimum of 176 copies/ml of RNA.

5.5.PProduct roduct EEnhanced nhanced RTRT (PERT) Assay (Not (PERT) Assay (Not available commercially). 10available commercially). 1066 -10 -107 7

times more sensitive than RT-PCR, can times more sensitive than RT-PCR, can detect 10 viral particles.detect 10 viral particles.

NAT-ImportanceNAT-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 quantificationUsed for virus quantification Used for drug resistance testingUsed for drug resistance testing No commercial NAT kit is available for No commercial NAT kit is available for

detection or quantification of HIV 2.detection or quantification of HIV 2.

CD4+ T- Lymphocyte Count.CD4+ T- Lymphocyte Count.

The Gold Standard of CD4 cell count The Gold Standard of CD4 cell count is the Flow Cytometry Method using is the Flow Cytometry Method using Fluorescent Activated Cell Sorter.Fluorescent Activated Cell Sorter.

The absolute count in Indian The absolute count in Indian population varies from ~ 700-900/cu population varies from ~ 700-900/cu mm. mm.

CD4+ Lymphocyte CountCD4+ Lymphocyte Count

The major indicator of immune function The major indicator of immune function Most recent CD4 count is best predictor ofMost recent CD4 count is best predictor of

disease progressiondisease progression CD4 count usually is the important considerationCD4 count usually is the important consideration

in decision to start ART in decision to start ART Important in determining response to ARTImportant in determining response to ART

Adequate response: CD4 increase 100-150 cells/µL per Adequate response: CD4 increase 100-150 cells/µL per yearyear

CD4 monitoringCD4 monitoring Check at baseline (x2) and at least everyCheck at baseline (x2) and at least every

3-6 months3-6 months

CD4+ Lymphocyte CountCD4+ Lymphocyte Count

Exact CD4 count at which to initiate Exact CD4 count at which to initiate therapy not known, but evidence points therapy not known, but evidence points to starting at higher countsto starting at higher counts

Current recommendation: ART for all Current recommendation: ART for all patients with CD4 counts of <350 patients with CD4 counts of <350 cells/µLcells/µL, certain others regardless of , certain others regardless of count.count.

Clinical Category and/orClinical Category and/orCD4 CountCD4 Count

RecommendationRecommendation

History of AIDS-defining History of AIDS-defining illnessillness

CD4 count of <350 cells/µLCD4 count of <350 cells/µL Pregnant womenPregnant women HIV-associated nephropathyHIV-associated nephropathy Hepatitis B coinfection, Hepatitis B coinfection,

when HBV treatment is when HBV treatment is indicatedindicated

Initiate ARTInitiate ART

Indications for Initiating Indications for Initiating ART: ART:

Chronic InfectionChronic Infection

Clinical Category and/or Clinical Category and/or CD4 CountCD4 Count

RecommendationRecommendation

CD4 count of >350 CD4 count of >350 cells/µLcells/µL, asymptomatic, , asymptomatic, without conditions listed without conditions listed aboveabove

Optimal time to initiate ART Optimal time to initiate ART is not well defined; consider is not well defined; consider individual patient individual patient characteristics and characteristics and comorbiditiescomorbidities

Indications for Initiating ART: Indications for Initiating ART: Chronic InfectionChronic Infection

VIRAL LOADVIRAL LOAD

HIV RNAHIV RNA Less important than CD4 count, but may influence Less important than CD4 count, but may influence

decision to start ART and help determine frequency of CD4 decision to start ART and help determine frequency of CD4 monitoringmonitoring

> 50,000 copies of RNA/ml is an indication for initiation of > 50,000 copies of RNA/ml is an indication for initiation of ART.ART.

Critical in determining response to ARTCritical in determining response to ART Goal of ART: HIV RNA below limit of detection (ie, <40 to <80 Goal of ART: HIV RNA below limit of detection (ie, <40 to <80

copies/mL, depending on assay)copies/mL, depending on assay) RNA monitoringRNA monitoring

Check at baseline (x2) Check at baseline (x2) Immediately before initiating ARTImmediately before initiating ART 2-8 weeks after start or change of ART (Normal response 2-8 weeks after start or change of ART (Normal response

is tenfold decrease in HIV RNA level within 4-8 weeks)is tenfold decrease in HIV RNA level within 4-8 weeks) Every 3-4 months with stable patientsEvery 3-4 months with stable patients

Treatment FailureTreatment FailureA. VIROLOGIC FAILUREA. VIROLOGIC FAILURE

Incomplete virologic response: Incomplete virologic response: In patient on initial ART, HIV RNA >400 In patient on initial ART, HIV RNA >400

copies/mL after 24 weeks on therapy or copies/mL after 24 weeks on therapy or >50 copies/mL by>50 copies/mL by48 weeks (confirm with second test)48 weeks (confirm with second test)

Virologic rebound:Virologic rebound: Repeated detection of HIV RNARepeated detection of HIV RNA

after virologic suppressionafter virologic suppression(eg, >50 copies/mL)(eg, >50 copies/mL)

Treatment FailureTreatment FailureB. IMMUNOLOGIC FAILUREB. IMMUNOLOGIC FAILURE

Failure to achieve and maintain adequate Failure to achieve and maintain adequate CD4 response despite virologic suppressionCD4 response despite virologic suppression There is no specific definition for immunologic failure, There is no specific definition for immunologic failure,

although some studies have focused on patients who fail although some studies have focused on patients who fail to increase CD4 T-cell counts above a specific threshold to increase CD4 T-cell counts above a specific threshold (e.g. >350 or 500 cells/mm3) over a specific period of (e.g. >350 or 500 cells/mm3) over a specific period of time (e.g. 4–7 years).time (e.g. 4–7 years).

Persistently low CD4 count while on Persistently low CD4 count while on suppressive ART is associated with suppressive ART is associated with increased risk for increased risk for AIDS-related complicationsAIDS-related complications

Resistance TestingResistance Testing

Before initiation of ART:Before initiation of ART: Resistance testing (genotype) recommended for all at entry to Resistance testing (genotype) recommended for all at entry to

carecare Recommended for all pregnant womenRecommended for all pregnant women Transmitted resistance in 6-16% of HIV-infected patientsTransmitted resistance in 6-16% of HIV-infected patients Identification of resistance mutations may optimize treatment Identification of resistance mutations may optimize treatment

outcomesoutcomes In absence of therapy, resistance mutations may declineIn absence of therapy, resistance mutations may decline

over time and become undetectable by current assays, butover time and become undetectable by current assays, butmay persist and cause treatment failure when ART is startedmay persist and cause treatment failure when ART is started

Patients with virologic failure:Patients with virologic failure: Perform while patient is taking ART, or ≤4 weeks afterPerform while patient is taking ART, or ≤4 weeks after

discontinuing therapydiscontinuing therapy Interpret in combination with history of ARV exposureInterpret in combination with history of ARV exposure

and ARV adherenceand ARV adherence

Resistance Testing- Resistance Testing- GENOTYPINGGENOTYPING

Detects drug resistance mutations in Detects drug resistance mutations in specific genesspecific genes(eg, reverse transcriptase and protease)(eg, reverse transcriptase and protease)

Sequencing or probingSequencing or probing Results within 1-2 weeks Results within 1-2 weeks Interpretation of mutations and cross-Interpretation of mutations and cross-

resistance is complexresistance is complex Consultation with specialists is Consultation with specialists is

recommendedrecommended

Resistance Testing-Resistance Testing-PHENOTYPINGPHENOTYPING

Measures the ability of viruses to grow in Measures the ability of viruses to grow in various concentrations of ARV drugsvarious concentrations of ARV drugs

Results within 2-3 weeksResults within 2-3 weeks More expensive than genotypingMore expensive than genotyping The ratio of the IC50s of the test and The ratio of the IC50s of the test and

reference viruses is reported as the fold reference viruses is reported as the fold increase in IC50, or fold resistanceincrease in IC50, or fold resistance

Interpretation may be complexInterpretation may be complex Consultation with specialists is Consultation with specialists is

recommendedrecommended

Coreceptor Tropism AssayCoreceptor Tropism Assay

Should be performed when CCR5 Should be performed when CCR5 antagonist is being considered antagonist is being considered Maraviroc should be given only to patients Maraviroc should be given only to patients

with exclusive CCR5 tropismwith exclusive CCR5 tropism Current commercially available tropism Current commercially available tropism

assay is 100% sensitive for CXCR5 clones assay is 100% sensitive for CXCR5 clones that make up ≥0.3% of the populationthat make up ≥0.3% of the population

Consider in patients with virologic failure Consider in patients with virologic failure on a CCR5 antagonist.on a CCR5 antagonist.

List Of Lab Tests at Initiation of List Of Lab Tests at Initiation of TherapyTherapy

BASELINE EVALUATION:BASELINE EVALUATION: HIV antibody HIV antibody CD4 cell count CD4 cell count Plasma HIV RNAPlasma HIV RNA Resistance test (genotype)Resistance test (genotype) CBC, chemistry profile, BUN, Cr, transaminaseCBC, chemistry profile, BUN, Cr, transaminase Fasting glucose and lipidsFasting glucose and lipids RPR or VDRLRPR or VDRL Hepatitis A, B, C serologyHepatitis A, B, C serology Toxoplasma IgGToxoplasma IgG

List Of Lab Tests at Initiation of List Of Lab Tests at Initiation of Therapy: Additional TestsTherapy: Additional Tests

Tuberculin skin test (TST) or IFN-Tuberculin skin test (TST) or IFN-γγ release assayrelease assay

Chest X ray (if symptoms, or positive Chest X ray (if symptoms, or positive Tuberculin test or IFN-Tuberculin test or IFN-γγ release assay release assay))

Gynecologic exam with Pap smearGynecologic exam with Pap smear Testing for Chlamydia and gonorrhoeaTesting for Chlamydia and gonorrhoea

WHO Recommendations for WHO Recommendations for Resource Limited SettingsResource 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.

ConclusionConclusion

Laboratory support is absolutely essential Laboratory support is absolutely essential in the management of HIV infected in the management of HIV infected patient.patient.

There is an urgent need to establish an There is an urgent need to establish an HIV laboratory at SVIMS with the minimum HIV laboratory at SVIMS with the minimum capabilities as recommended by WHO capabilities as recommended by WHO ( Diagnostic, CD4 count, Viral load ( Diagnostic, CD4 count, Viral load estimation).estimation).

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