I'm studying Clinical Medicines

1. Module 10.1
Role of the Laboratory in HIV/AIDS
Treatment, Control & Prevention
KMTC Machakos

2. Envelope
Core
RT

3. Laboratory Diagnosis of HIV
Infection
CDC
Group I
CDC
Group II/III
CDC
Group IV
(Weeks) (Years)
Window period
Infection
Time
Level
Serological markers of HIV infection
p24 antigen
p24 antigen
IgM antibody
gp41 antibody (IgG)
p24 antibody (IgG)

4. Laboratory Diagnosis
 Diagnosis is important for
 prevention of spread
 Care efforts
 Short course of ART decreases spread from infected
mothers to infants
 In tuberculosis patients knowledge of and therapy for
HIV can decrease morbidity

5. EIA/Western Blot
Viral Culture
p24 antigen Detection
DNA PCR
RT PCR
Rapid antibody tests
Assay Development Chronology
1980
1985
1990
1995
2000

6. Detection of anti-HIV antibodies
Objectives:
 Transfusion
 Surveillance
 Diagnosis

7. Antibody Detection by ELISA
 Important screening assay
 easy
 useful for large sample numbers
 highly sensitive and specific
 useful for blood product screening
 useful for diagnosing and monitoring patients
 useful for determining disease prevalence
 useful for research

8. Antibody Detection
i) Simple/Rapid Tests
 Common in small laboratories and small HIV
testing centres esp VCT, PITC, PMTC,etc
 Uses following method
 Agglutination
 Immunofiltration
 Immunochromatographic

9. Benefits of HIV Rapid Testing
 Sensitivity and specificity equal to ELISA
 No electricity or machinery
 No highly skilled technical staff
 Whole blood, plasma, or serum
 Very small amounts of blood suitable for
finger prick
 Provision of same day/same hour results
 Inbuilt controls

10. Assessment and Comparison of Suitability of Rapid
HIV Test Kits in Small Laboratories
Sensitivity 98 – 99%
<98%
3
1
Specificity >98%
95 - 98%
3
1
Incubation Room temp.
Other temp.
3
1
Shelf life >1 year
6 months – 1 year
3
1
Storage temperature Ambient – RT
Refrigeration 2-8oC
3
1
Price/test (USD) <1.0
1 –2
3
2
Ease of performance Easy
Less easy
3
2
Time to complete test <10 min
10 – 45 min
3
2
Washer required No
Yes
3
1
Reader required No
Yes
3
1
Score

11. Definitions
 Sensitivity – ability of the test method to detect correctly sample
that contains HIV antibody, a/a+c, expressed as percentage
 Specificity- ability of the test method to detect correctly sample that
do not contain antibody to HIV, d/b+d, expressed as percentage
 Positive predictive value (PPV) – the probability that when the test
is reactive, the specimen does contain antibody to HIV, a/a+b
 Negative predictive value (NPV) – the probability that when the
test is negative, the specimen does not contain antibody to HIV,
d/c+d
 Note: Predictive values vary with prevalence of HIV in the
population
Reference method
+ - Total
+ a (true pos) b (false pos) b + b
- C (false neg) D (true neg) C + d
Total A + c B + d N (a + b + c + d)
Test
method

12. Antibody Detection
Screening tests
ii) EIA (long ELISA)
 Most commonly used in big laboratories
 Now at 4th generation tests- sensitivity and specificity
very good
 Solid phase coated with recombinant antigens and/or
peptides and similar antigens conjugated to a
detecting enzyme
 IgG and IgM detected (detection of IgM may reduce
the 2-4 week window period)
 Antigens used are mixtures of HIV-1and HIV-2

13. Antibody Detection
Confirmatory tests
 Western Blot
 Gold Standard
 Electrophoretic separation of antigens
 Specific antibodies to each viral antigen
 Indirect Immunofluorescent Antibody Assay (IFA)
 Fluorescent microscopy to visualize HIV infected cells.
 Polymerase Chain reaction (PCR)
 DNa sequencing of genes

14. Western Blot Testing
 Confirmatory test
 Antibody profile given to a number of Ag
 Viral lysate separated into components by
PAGE electrophoresis

15. PCR
 Polymerase chain reaction
 can amplify small amounts of viral nucleic acid so they can
be detected by hybridization with nucleic acid probes
 mononuclear cells from patient separated by ficoll hypaque
gradient centrifugation
 cells are then lysed and double stranded DNA is separated
into single strands
 primers are added which are specific for viral DNA
 addition of DNA polymerase and nucleotides causes
amplification of viral DNA
 alternately heated and cooled to allow for
amplification, dissociation reannealing with primers
and amplification etc again 30 cycles

16. PCR
 Polymerase chain reaction
 can amplify small amounts of viral nucleic acid so they can
be detected by hybridization with nucleic acid probes
 mononuclear cells from patient separated by ficoll hypaque
gradient centrifugation
 cells are then lysed and double stranded DNA is separated
into single strands
 primers are added which are specific for viral DNA
 addition of DNA polymerase and nucleotides causes
amplification of viral DNA
 alternately heated and cooled to allow for
amplification, dissociation reannealing with primers
and amplification etc again 30 cycles

17. Evaluation of PCR Testing
 Very sensitive
 can detect non replicating viral genomes
 1-2 ml only needed so ideal for newborns
 useful in window period
 useful for typing HIV-1 and HIV-2
 false positives if carry over from previous samples
 if Ab negative but PCR positive repeat PCR
 not used for routine screening
 used in therapy efficacy measurements

18. Testing of Neonates
 IgGs in baby are from mother for first few months
and don’t indicate infection
 Most infants from HIV positive mothers will initially
test positive – using antibody detection
 Not sensitive in first 3 months of life
 p24 Ag detection after disrupting Ag-Ab complexes
with dilute HCl
 PCR testing - some positive by 38 days, most not
positive until 6 months – Most recommended

19. ELISA
Schematic Presentation
HIV antigens
Solid phase
(Well)
Antibodies to HIV
(From the sample)
Enzyme
Conjugate
Stop solution
Formation of specific antigen/antibody complex
Base for enzyme (peroxidase) activity
Hydrogen peroxide for enzyme to digest and produce colour
Stops enzyme activity and changes colour
Colour intensity read by spectrophotometer
Substrate
(Colour reagent)

20. Specimen Handling for ELISA
 Test procedure will indicate whether serum,
plasma, or both can be used for testing
 Most kits recommend
 Separating the serum/plasma from the red
cells shortly after collection
 Specific time that the specimen can be
stored at 2-8o
 Mixing thawed specimens well before use

21. Quality Control for ELISA
Technique
 Use: Negative Controls
 1 HIV-1 Pos. Control
 1 HIV-2 Pos. Control
 1 HIV-1 Group O Pos. Control
 Mix specimens and controls well before use
 Adhere to incubation times and temperatures
 Avoid splashing liquids from 1 well to another
 Neg. Control readings must be below the Negative
Cutoff Value
 Pos. Control readings must be above the specified
value for the kit

22. sensitive
Report Consider +ve
A
+ve
-ve
specific
B
+ve
-ve
sensitive
A
+ve
-ve
Report
Indeterminate Report
sensitive
C
+ve
-ve
+ - -
Low risk
A
+ve
-ve
sensitive
specific
B
+ve
-ve
Report
Report
TRANSFUSION
TRANSFUSION
SURVEILLANCE
DIAGNOSIS
DIAGNOSIS
I II III
WHO HIV Antibody Testing Strategies
+ - +
High risk
Sensitivity >97%
Specificity >95%
SERIAL TESTING
PARALLEL TESTING

23. Determine Assay
Test Principle
 HIV-1 & HIV-2 antigens
 Lateral flow
Test Components
 Test Pads
 Desiccant
 Instruction manual
 Chase Buffer (Whole Blood)

24. Testing kit

25. Pull off protective foil

26. Blood Sample 50ul

27. Chase Buffer

28. Results
 Reactive 2 lines of any intensity appear in
both the control and patient areas.
 Non-reactive 1 line appears in the control area
and no line in the patient area.
 Invalid No line appears in the control area.
Do not report invalid results. Repeat test with a new test device even if a
lineappears in the patient area.

29. PATIENT CONTROL
HIV-1/2
472U100
7A
Ab (POS)
or
No Ab (NEG)
Solution
P
Ag/Ab complex
Lateral Flow – Schematic Presentation
Sample pad
Selenium
Conjugate
Colour reagent HIVAg
Anti-human
immunoglobulins
C
C
No reaction
Client
#
Client
#
7A
PATIENT CONTROL
HIV-1/2
472U100
P

30. Unigold
Lab workers Health workers Counselors
4
Uni-Gold: Collecting Specimen
3. Collect specimen using the disposable pipette

31. Unigold Testing
Lab workers Health workers Counselors
5
Uni-Gold: Adding Specimen and
Reagent to Test Device
4. Add 2 drops (approx. 60µl) of
specimen to the sample port in
the device
5. Add 2 drops (approx. 60µl) of the
appropriate wash reagent to sample
port

32. Wait for reaction
Lab workers Health workers Counselors
6
Uni-Gold: Getting Results
6. Wait for 10 minutes (no longer than
20 min.) before reading the results
7. Read and record the results and
other pertinent info on the worksheet

33. Unigold Results
Lab workers Health workers Counselors
7
Uni-Gold: Test Interpretation
Reactive Invalid
Non-reactive

34. Any questions?

35. THANK YOU