maj

1. LABORATORY ASSAYS FOR THE DIAGNOSIS
OF
HUMAN IMMUNODEFICIENCY VIRUS
INFECTIOUS BLOCK
APRIL 2018
Dr. B. NYOMBI

2. • All the pillars of HIV treatment, disease control and
prevention depend on reliable laboratory derived data
• Early diagnosis of HIV infection is a key strategy for limiting
the further spread of the virus
• Provides HIV infected pregnant women with the option of
seeking medication that can prevent transmission of the
virus to their infants
• For their children, dry blood spot diagnosis of infection in
infancy can trigger early life prolonging treatment
LABORATORY ASSAYS FOR THE DIAGNOSIS
OF HIV INFECTION

3. Key diagnostic Challenges
• Lack of capacity for HIV sequencing and resistance testing
• Limited access to RNA and DNA polymerase chain
reaction for viral load testing
• Limited access to infant diagnosis of HIV infection
• The inadequate implementation of QA and proficiency
testing for viral load testing, serology and lymphocyte
subset enumeration

4. LABORATORY ASSAYS
Competence in the following HIV methods should be
present:
• Rapid tests for the purposes of training and QA
• ELISA
• Confirmatory testing including Western Blot
• Capacity to confirm equivocal diagnoses
• Ultrasensitive p24 antigen testing(depending on testing
algorithms)
• Viral load determination using reverse transcriptase
polymerase chain reaction (RT-PCR)
• Dry blood spot analysis for infant diagnosis

5. CHARACTERISTICS OF THE HIV
• HIV has high affinity to CD4+ lymphocytes and monocytes
• HIV binds the CD4+ cells and becomes internalized where
it replicates itself by generating a copy of viral DNA by the
aid of reverse transcriptase
• Reverse transcription is error prone resulting to genetic
mutations which result into different HIV genotype and
drug resistance
• HIV binding and fusion utilize the two co-receptors CCR5
and CXCR4 along with CD4
• Viral DNA becomes incorporated into the host DNA by aid
of integrase, causing latent infection and enabling further
replication
• Viral DNA is transcribed into multiple copies of viral RNA

6. HIV LIFE CYCLE
AND
TARGETS FOR ANTIRETROVIRAL THERAPY
Reverse
Transcriptase
Inhibitors
Protease
Inhibitors
Integrase
Inhibitors
Entry/Fusion
Inhibitors
Various PI
NRTIs,
NNRTIs
T-20

7. HIV
A
B
C
D
F
G
H
J
K
?
Type 2
Type 1
Group O
Group M
Group N CRF
HIV GENOTYPIC CLASSIFICATION
Group P

8. Sequence of appearance of laboratory marker for
Laboratory diagnosis of HIV-1 infection

9. Laboratory Diagnostic methods for HIV infection
• Serological methods for detection of antibody
– Rapid Tests
– ELISA
– Western blot
• Antigen detection methods
– P24 antigen capture test
– Polymerase Chain Reaction (PCR) used in diagnosis:
acute HIV infection, HIV infection in infants
• Recent Infection
– HIV-1 Limiting Antigen Avidity EIA

10. ELISA TEST
• Different ELISA testing techniques used are
– Indirect
– Competitive
– Sandwich
• ELISA test is useful for
– Screening blood and blood products
– Diagnosis and monitoring patients
– Determining prevalence of infection
– Research investigations

12. Evolution of ELISA
• HIV immunoassays based on different design principles are
generally grouped into “generations”:
• 1st generation – All antigens used to bind HIV antibodies are
from a lysate of HIV-1 viruses grown in cell culture
• 2nd generation - Recombinant protein antigens alone used
to bind HIV antibodies designed for specific HIV-1 and HIV-
2 antigenic epitopes that improves sensitivity
• 3rd generation – Synthetic peptide or recombinant protein
antigens are used to bind HIV IgM and IgG antibodies and
increase sensitivity during early seroconversion
• 4th generation—Synthetic peptide or recombinant protein
antigens as 3rd generation and monoclonal antibodies to
detect p24 antigen

13. 1st and 2nd Generation ELISA

14. 3rd Generation ELISA

15. 4th Generation ELISA

16. WESTERN BLOT ASSAY
• Utilises a lysate prepared from HIV virus
• The lysate is electrophorized to separate out the HIV
proteins (Antigens)
• The separated proteins are blotted on nitrocellulose
paper
• The paper is cut into strips and reacted with test serum
samples
• Specific bands form where the antibodies reacted and
shown by indicators such binding of anti-antibodies
tagged with enzyme that catalyse a substrate to produce
a coloured product

17. CREATING WESTERN BLOT STRIPS
HIV lysate proteins
are separated by
size using gel
electrophoresis
Proteins are
transferred
(blotted) onto the
surface of a
membrane
Strips are
incubated with
patient serum and
antihuman IgG
conjugated with
an enzyme (and
chromagen)
The membrane is
cut into strips

18. HIV WESTERN BLOT BANDING PATTERN
env gp160
gp120
gp 41
gag p55
p18
p24
pol p65
p51
p31

19. WESTERN BLOT BANDING
*
*
*
* - 2 of the bands must be present to be interpreted as positive

20. Western blot assay
• Gold Standard
• WB should be viewed as a supplemental test
which can be used to confirm positive/discordant
results obtained from EIA.
• Advantages - Specific interaction of antibody and
antigen can be directly visualized.
• Disadvantages:Technically demanding, Expensive
Subject to interpretation
* Presence or absence of bands
* Intensity of those bands

21. Limiting Antigen (LAg ) Assay
• Estimates number of recent infections in a defined cohort
• Only HIV Positive samples
• Antibodies produced early in HIV Infection show a low
avidity for the antigen
• Antibody Avidity increases progressively with time after
exposure to an immunogen
• The avidity of the antibody can be measured in the
presence or absence of a denaturing agent that will elute
low-avidity antibody from the antigen-antibody complex
• Low avidity – recent infection
• Higher avidity – long term infection

22. Limiting Antigen (LAg ) Assay

23. HIV Testing Strategies
• Parallel testing means samples are tested
simultaneously by two different tests.
• Serial testing means samples are tested by a first
test (screening test). The results of the first test
determine whether additional testing is required.
• HIV EIA testing: the samples are screened on the
first assay and the samples that are positive and
confirmed on another assay or two other assays.
• If two or three assays are used, all assay principles
and test methods must be different.

24. • Strategy 1: All samples are tested with one ELISA. All
reactive results are considered infected and all non-reactive
results are considered uninfected. Settings:
transfusion/transplant services and prevalence/surveillance
• Strategy 2: All samples are tested first with one ELISA. Any
sample found to be reactive with the first test must be
tested by the second ELISA. Samples that are reactive on
both ELISA tests are considered HIV Infected. Samples that
are non-reactive on the first ELISA are not tested further.
• Strategy 3: Similar to 2, except a third test is performed. All
samples that are reactive on the first ELISA (Screen) is tested
on the second (confirmatory 1) ELISA, samples reactive on
the second ELISA is tested on the third ELISA (confirmatory
2). Samples that are positive on all 3 ELISA’s are considered
Infected. Samples that are non-reactive on the first ELISA
are considered uninfected and not tested further.

25. What is an Algorithm?
Combination and sequence of specific tests used in a
given algorithm

27. How do you know which tests to use in an
algorithm?
• Test must be readily available in the country
• Test performance in country
• The tests must be validated prior to use
• Must be able to detect HIV 1 and HIV 2
• The sensitivity and specificity of the assay
determines which test will be used first

28. New HIV Diagnostic Algorithm

29. Verification of test methods
Before selecting test methods for use, these must be
verified by the laboratory prior to placing in use
What is meant by verification?
Verification is process of establishing evidence that provides
high degree of assurance that a product or system
accomplishes its intended requirements
Verification is the process assessing:
• Precision
• Accuracy
• Sensitivity
• Specificity
• Linearity
• Analytical Measurement

30. Verification of test methods
• Verification of the test method: test the kit using known
reactive and non reactive samples.
• Compare the results of the new method with test method
already in use. Use Proficiency testing panels to validate
the test method.
• Test between 50 – 500 samples if resources available.
• Determine sensitivity and specificity of the test using
special formula. Sensitivity and Specificity should be
greater than 99%
• Perform precision and accuracy testing
• Write out report
• Approve report

31. • Precision testing
– Closeness of agreement between independent
test/measurement results obtained under stipulated conditions.
– Test approx. 20 samples over a period of a few days (5 days) using
the same operator and the same equipment OR test the approx.
20 samples in the same day but in different test runs using same
operator and same equipment.
– Analyze the results and calculate mean, Standard Deviation and
%CV. The acceptable %CV can be obtained from package insert.
• Accuracy
– Is the true value of a substance being measured. Verification of
accuracy is the process of determining that the test system is
producing true valid results
– Test approx. 20 samples over a period of a few days (5 days) using
the same operator and the same equipment OR test the approx.
20 samples in the same day but in different test runs using same
operator and same equipment. Analyze the results. The final
result should be the same for each sample tested on each day or
in each test run.

32. ACCURACY AND PRECISION
• Accuracy is also used as a statistical measure of how
well a binary classification test correctly identifies or
excludes a condition
• The accuracy is the proportion of true results (both
true positives and true negatives) in the population
• Accuracy may be determined from Sensitivity and
Specificity, provided prevalence is known, using the
equation:
• Accuracy = (sensitivity)(prevalence) + (specificity)(1 − prevalence)

33. Reference Test
Gold standard
Positive Negative
New Test
Positive
True
Positive
False
Positive
(Type I
error)
→ Positive
predictive
value
Negative
False
Negative
(Type II
error)
True
Negative
→
Negative
predictive
value
↓
Sensitivity
↓
Specificity
Sensitivity and Specificity

34. Sensitivity and Specificity
Sensitivity - The ability of a test to accurately detect a true positive.
Specificity - The ability of a test to accurately detect a true negative
• When choosing the test methods to be used in an algorithm, sensitivity and
specificity of the test is key to selecting which assay will be used first, second
and/or third
• The first test should be the most sensitive - all samples that are truly reactive
should be accurately identified by the first test
• The second test should be more specific and also sensitive. Samples that are
falsely reactive in the first test should be accurately identified as a true non
reactive and samples that are truly reactive should be accurately identified by
the second test
• The same principle applies to the third test as for the second.
• Sensitivity and specificity should be greater than 99%, 100% is optimal.