1 Lecture 3: Laboratory Diagnostics in ARV Therapy delivered by Dr. Madisa Mine, Botswana Ministry...
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Transcript of 1 Lecture 3: Laboratory Diagnostics in ARV Therapy delivered by Dr. Madisa Mine, Botswana Ministry...
11
Lecture 3:
Laboratory Diagnostics in ARV Therapy
delivered byDr. Madisa Mine, Botswana Ministry of
Health
KITSO AIDS Training Program
22
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)
44
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.
55
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
66
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.
77
ELISA Algorithm for Diagnosing HIV
2 Parallel ELISA Tests Negative
Patient uninfected or inwindow period of
infection
88
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
99
Western Blot
• Most commonly used confirmatory test.
• Detects antibodies directed at specific HIV envelope and core proteins.
1010
Alternative Methods of Sero-diagnosis of HIV Infection
• Rapid tests
• Non-invasive testing methods– Saliva or urine
1111
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.
1212
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.
1313
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
1414
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
1515
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.
1616
DNA PCR Assay
• PCR based on cellular proviral HIV DNA provides a qualitative result:
Positive or Negative
1717
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.
1818
Monitoring HIV Disease Progression
1919
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
2020
CD 4 and Viral Load Progression
Viral load
CD4
TIMEInfection
High
Low
2121
• 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
2222
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).
2525
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.
2727
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)
2828
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).
2929
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.
3030
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.
3131
Monitoring for ARV Toxicity
3232
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.
3333
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
3434
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.
3535
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.
3636
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