Transcript Detection of HIV

KITSO AIDS Training Program
Lecture 3:
Laboratory Diagnostics in ARV
Therapy
delivered by
Dr. Madisa Mine, Botswana Ministry of
Health
1
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
2
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)
3
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.
4
Minimum Time from Infection to First
Detection of HIV-1 Markers
TIME PERIOD BEFORE DETECTION
KEY:
TIME WHEN HIV DETECTION BY TEST
IS POSSIBLE
ELISA
p24
DNA PCR
RNA (VL)
Infection
5
10
15
20
25
30
35
5
Detection of HIV: time (in days) after infection
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.
6
ELISA Algorithm for Diagnosing HIV
2 Parallel ELISA
Tests Negative
Patient uninfected or in
window period of
infection
7
2 Parallel ELISA Test Positive
Patient
infected
Discordant Parallel ELISA Tests
(positive/negative)
Discordant
Repeat ELISAs on same sample
Positive
Positive
Western Blot
Indeterminate
Patient infected
Negative
Negative
Re-draw blood and
repeat ELISA
Patient uninfected
or in window period
of infection
8
Western Blot
• Most commonly used confirmatory test.
• Detects antibodies directed at specific HIV
envelope and core proteins.
9
Alternative Methods of
Sero-diagnosis of HIV Infection
• Rapid tests
• Non-invasive testing methods
– Saliva or urine
10
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.
11
Sample 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.
12
Labeling of Lab Test Tubes
(all assays)
• Tubes should be labeled with:
– PATIENT IDENTIFICATION NUMBER
(ID/Omang)
– Date of collection
13
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
14
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.
15
DNA PCR Assay
• PCR based on cellular proviral HIV
DNA provides a qualitative result:
Positive or Negative
16
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.
17
Monitoring HIV Disease
Progression
18
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

19
CD 4 and Viral Load Progression
High
Viral load
CD4
Low
Infection
TIME
20
Viral Load
• 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.
21
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.
22
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.
23
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).
24
CD4 Cell Counts
25
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.
26
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)
27
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).
28
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.
29
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.
30
Monitoring for ARV Toxicity
31
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 3monthly:
– FBC
– liver and kidney function
– Glucose
• NVP-containing regimens: liver function tests are
drawn two weeks after HAART initiation.
32
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
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 5-1033 times
CPK, TBil
Lipase
Cholesterol
Triglycerides
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.
34
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.
35
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.
36
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.
37
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
38