11

Lecture 3:

Laboratory Diagnostics in ARV Therapy

delivered byDr. Madisa Mine, Botswana Ministry of

Health

KITSO AIDS Training Program

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Main Lab Tests in ARV Therapy

• HIV diagnosis:– Adults: ELISA test– Infants under 18 months: DNA PCR test

• Monitor viral suppression: Viral load

• Monitor recovery of immune system: CD4 cell count

• Monitor drug toxicity: chemistry and haematology

• ARV Resistance Assay

33

Detection of HIV Infection

• Antibody methods for diagnosing HIV- (ELISA) Enzyme Linked Immunosorbent

Assay- Western Blot- Rapid tests

• DNA PCR tests

• p24 antigen (used to screen blood donations)• HIV culture (used for research)

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Window Period

• Period between infection and first reliable detection of HIV by lab test.

• Window period varies by test and by individual.

• The majority of infected individuals are positive by ELISA, antigen, and/or DNA/RNA tests by 6-8 weeks after infection.

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Minimum Time from Infection to First Detection of HIV-1 Markers

5 10 15 20 25 30 35

ELISA

p24

DNA PCR

RNA (VL)

Detection of HIV: time (in days) after infection

KEY:

TIME WHEN HIV DETECTION BY TEST IS POSSIBLE

TIME PERIOD BEFORE DETECTION

Infection

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Diagnosis in Adults

• In adults, diagnosis of HIV infection is best determined by the detection of antibodies (markers) to the virus in blood.

• The antibodies are specific for particular virus proteins and are unique to HIV.

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ELISA Algorithm for Diagnosing HIV

2 Parallel ELISA Tests Negative

Patient uninfected or inwindow period of

infection

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2 Parallel ELISA Test Positive

Discordant Parallel ELISA Tests (positive/negative)

Repeat ELISAs on same sample

Positive

Patient infected

Western Blot

Positive Indetermina

te

Negative

Patient infected

Patient uninfected or in window period

of infection

Re-draw blood and repeat ELISA

Discordant

Negative

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Western Blot

• Most commonly used confirmatory test.

• Detects antibodies directed at specific HIV envelope and core proteins.

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Alternative Methods of Sero-diagnosis of HIV Infection

• Rapid tests

• Non-invasive testing methods– Saliva or urine

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Rapid Tests

• Quick, cheap, easy to use.

• Easy to store.

• A laboratory is not required.

• Combinations of rapid tests are highly sensitive and specific.

• However, interpretation does require trained personnel.

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Sample Required for ELISASample Required for ELISA

Five (5) milliliters of whole blood in plain or purple-top EDTA tubes.

Samples should be stored in the fridge at 4 0C and NOT frozen.

Samples should be sent to the lab within 24 hours.

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Labeling of Lab Test Tubes Labeling of Lab Test Tubes (all assays)(all assays)

• Tubes should be labeled with:

– PATIENT IDENTIFICATION NUMBER

(ID/Omang)

– Date of collection

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Filling out Lab Test Forms (all assays)

• Fill in patient information:• ID / Omang• Patient initials (as a cross-check for errors)• Patient gender (M or F)• Patient date of birth (day / month / year)

• Fill in sample information:• Date specimen drawn (day / month / year)• Time specimen drawn (24 hour clock)

• Site information:• Initials and signature or stamp of clinician

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Diagnosis in Infants

• Diagnosis of HIV by serology in infants under 18 months is complicated by the presence of maternal antibodies passed along both in utero and via breast milk.

• Hence detection of HIV nucleic acid in infant blood is done by DNA PCR.

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DNA PCR Assay

• PCR based on cellular proviral HIV DNA provides a qualitative result:

Positive or Negative

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Sample Required for DNA PCR

• .5-1.0 ml of whole blood in purple-top EDTA tubes w/ anticoagulant.

• Tube should be mixed well by inverting slowly 5-10 times IMMEDIATELY after collection to prevent clotting.

• Sample should be stored in the fridge and NOT frozen.

• The sample should be sent to the lab immediately.

• Turn-around-time is 2 weeks.

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Monitoring HIV Disease Progression

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HIV Disease Progression

• Progression can be monitored by:

Clinical markers:▪ HIV/AIDS-related conditions and

mortality.

Laboratory markers: Increase in blood virus levels (viral load) Decrease in CD4 cell count

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CD 4 and Viral Load Progression

Viral load

CD4

TIMEInfection

High

Low

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• Measures the number of virus particles per ml of blood by quantifying HIV RNA.

• With the standard test used in Botswana, 400 – 750,000 HIV copies per ml of blood can be detected.

• Measure viral load at start of therapy, after 3 months and every 3 months thereafter.

Viral Load

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Sample Collection for Viral Load

• Collect blood in 5-7 ml EDTA anticoagulant tube for adults, 3ml tube for infants.

• Tube should be mixed well by inverting slowly 5-10 times IMMEDIATELY after collection to prevent clotting.

• Store in the 4 0C fridge while awaiting transportation to lab.

• Sample should be transported to lab within 4-6 hours of collection in cool box with ice pack at 4 0C.

2323

Sample Collection for Viral Load

• If sample cannot reach testing labs within 6 hours, collection viral load sample in PPT tube.

• Local lab should centrifuge PPT tube samples to separate plasma and then ship to testing lab within 24 hours.

2424

Viral Load Results

• Turn-around time is 2 weeks.

• Detection limit of assay = 400 copies/ml.

• Undetectable result indicates viral load below 400 copies/ml.

• Recent study in Botswana:– median viral load in asymptomatic patients: 36,000

copies/ml.– median viral load in AIDS patients: 296,000 copies/ml.

• Potential 0.2-0.3 log inherent variability in viral load assay (60-80% due to biologic variation).

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CD4 Cell Counts

2626

CD4 Cell Counts

• CD4 cell count measures the number of CD4 cells per cubic milliliter of blood.

• The CD4 count is a measure of the degree of immuno-compromise and stage of HIV disease progression.

• The CD4 count is an important test for deciding whether ARV therapy is required and for monitoring the recovery of the immune system under treatment.

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CD4 Counts in Botswana

• Uninfected : 750 cells/ul

(IQR: 560-900)

• Asymptomatic HIV-1 positive: 350 cells/ul

(IQR: 268-574)

• Patients with AIDS : 121 cells/ul

(IQR: 50-250)

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Sample Collection for CD4 Count

• Three (3) ml of whole blood in EDTA anticoagulant (purple top tube).

• Tube should be mixed well by inverting slowly 5-10 times IMMEDIATELY after collection to prevent clotting.

• Sample should be transported to the lab within 24 hours.

• Store and transport sample at room temperature: 20-30 0C (cool box without ice pack).

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CD4 cell counts

• Turn-around time is 72 hours.

• Measure CD4 cell count:

– As an eligibility screen for therapy.

– At baseline and every 3 months on therapy.

– Off-therapy:

If CD4 count > 350: every 6 months.

If CD4 between 201 and 349: every 3 months.

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Interpretation of CD4 Counts

• Use absolute CD4 cell count in adults.

• Use CD4% in infants and children.

• Ignore CD8 count and CD4:CD8 ratio.

• 10-25 % variability in CD4 cell count due to:– biologic variation.– sampling/measurement error.

• Variation usually not clinically relevant.

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Monitoring for ARV Toxicity

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Chemistry and Haematology

• Before start of therapy: full blood count, liver function, kidney function, blood sugar.

• At start of therapy, 1 month, 3 months, then 3-monthly:– FBC– liver and kidney function– Glucose

• NVP-containing regimens: liver function tests are drawn two weeks after HAART initiation.

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Samples required for chemistry and haematology tests

Tests Type of tube Volume required Comments

FBC EDTA ( purple top) 3 ml Mix gently by inverting 5-10 times

LFT      

Creatinine      

Amylase Plain/no additive   Do not mix/ allow time to

Proteins, CO2 ( red top) 3-5 ml Clot before testing

CPK, TBil      

Lipase      

Cholesterol      

Triglycerides      

HDL/LDL  Plain (red)  3 ml Fasting sample/clotted

Lactate Heparin tube on ice  3 ml Mix gently by inverting 5-10 times

Glucose Sodium Fluoride (Grey Top)

2 ml Mix gently by inverting 5-10 times

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Other Tests

• Syphilis serology (5 ml plain red-top tube).

• Hepatitis B antigen (5 ml plain red-top tube).

– No longer part of baseline laboratory tests under Revised 2005 Guidelines.

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Resistance Assays

• A genotypic resistance assay is performed for patients failing second-line therapy to determine which drugs to which a patient may no longer be susceptible.

• The resistance assay, combined with expert opinion, will guide treatment decisions for third line therapy.

• The blood sample for resistance assays should be drawn while a patient is still on the failing regimen. Or no more than four weeks after discontinuation.

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Sample Collection for Resistance Assay

• Collect blood in 5-7 ml EDTA anticoagulant tube for adults, 3ml tube for infants.

• Tube should be mixed well by inverting slowly 5-10 times IMMEDIATELY after collection to prevent clotting.

• Store in the 4 0C fridge while awaiting transportation to lab.

• Sample should be transported to lab within 4-6 hours of collection in cool box with ice pack at 4 0C.

3737

Sample Collection for Resistance Assay

• If sample cannot reach testing labs within 6 hours, collection viral load sample in PPT tube.

• Local lab should centrifuge PPT tube samples to separate plasma and then ship to testing lab within 24 hours.

3838

Summary

• HIV diagnosis:– Adults: ELISA test– Infants under 18 months: DNA PCR test

• Monitor viral suppression: Viral load

• Monitor recovery of immune system: CD4 cell count

• Monitor drug toxicity: chemistry and haematology

• ARV Resistance Assay