Introduction to HIV Testing

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Introduction to HIV Testing
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Define laboratory diagnosis of HIV
infection Understand different HIV antibody
assays Understand assay characteristics
sensitivity specificity predictive value false
positive and false negative results Understand
counselling issues relating to provision of
results cognisant of test characteristics (see
above) Understand different testing algorithms in
particular serial versus parallel
testing Understand and discuss the concepts of
confidential and anonymous testing and informed
consent
Introduction to HIV Testing
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HIV infection is diagnosed largely by the
detection of antibodies against HIV in the blood
of infected patients There are three main types
of HIV antibody tests ELISA Western Blot
Rapid HIV tests
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ELISA
Enzyme linked immunosorbent assay HIV antibodies
if present in the test serum are sandwiched
between HIV antigen, fixed to the test well, and
enzymes, added to the test well following
binding of test antibodies to HIV antigen The
test well is washed to remove any unbound
enzyme A colour reagent is added Any bound
antigen catalyzes a change in colour of the
reagent The presence of HIV antibodies is
inferred from a change in colour of the reagent
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ELISA
Enzyme linked immuno sorbent assay
ve
Colour reagent
Pts antibody
HIV Ag Coated plate
Enzyme
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Introduction to HIV Testing
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Positive
Controls
Negative
Module 1 Sub Module 3 PPT02
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Western Blot Assay
Detects different antibodies within the test
serum capable of reacting with a variety of viral
proteins. Viral proteins are separated into
bands according to their molecular weight on an
electrophoresis gel. They are then transferred
or blotted onto nitrocellulose paper which is
incubated with the patients serum.
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Western Blot Assay
HIV antibodies react with specific HIV proteins
and bind to the nitrocellulose paper at precisely
the same point to which the target protein
migrated. Bound antibodies are then detected by
colouriometric techniques.
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Western Blot
Introduction to HIV Testing
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Indeterminate
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Rapid Tests
Rapid tests can employ a variety of techniques
including Particle agglutination Lateral
flow membranes Through flow membranes
Comb-dipstick based systems Most have
sensitivities and specificities of 99 and 98
respectively
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Rapid Tests
Advantages quicker to perform do not require
batching do not require specialised equipment
or trained personnel results delivered on the
same day Only WHO recommended rapid HIV
antibody tests should be used to ensure quality
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An Assay for Every Occasion
There are different requirements for different
antibody assays. The choice of antibody assay
will be determined by three factors The
objective of the assay The sensitivities and
specificity of the assay The prevalence of HIV
in the population being tested
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Objectives of Antibody Testing
Transfusion and transplant safety (for the
safety of the recipient) Surveillance (for the
knowledge of the population) Diagnosis of
clinical HIV disease (for the knowledge of the
individual) HIV assays used to meet one of these
objectives may be inappropriate for another
objectives, e.g. blood service testing for the
clinical diagnosis of HIV infection
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Test Characteristics
Each biological test has the capacity to give
false positive and false negative results The
accuracy of a biological assay is described by
three parameters Sensitivity Specificity
Predictive value
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Sensitivity
The capacity of an assay to detect a true
case A highly sensitive test will give very few
false negative results A highly sensitive test
is used when there is absolute need to have very
few false negative results, e.g. blood
transfusion service
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Specificity
The capacity of an assay to accurately define a
true non-case A highly specific test will give
very few false positive results Highly specific
tests are used when there is absolute need to
have very few false positive results, e.g. in the
diagnosis of HIV infection in an individual
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Predictive Value
The probability that a particular assay will
accurately predict the HIV status of an
individual varies with the prevalence of HIV
within population False negative results will
be less common in low prevalence populations
False positive results will be less common in
high prevalence countries False negative
results will be more common in low prevalence
countries False positive results will be more
common in low prevalence countries
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Predictive Value
In high prevalence populations a patient who
tests positive has a greater likelihood of truly
being infected In low prevalence populations a
patient who tests negative has a greater
likelihood of truly being uninfected
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Calculating the Accuracy of HIV tests
Introduction to HIV Testing
A people with HIV who test positive (true
positive) B people without HIV who test positive
(false positive) C people with HIV who test
negative (false negative) D people without HIV
who test negative (true negative) AC all
people who truly are infected with HIV BD all
people who truly are not infected with HIV
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Testing Strategies
Repeat HIV testing is recommended for all
positive results as false positive results may
occur The gains in accuracy of repeat HIV
testing must be weighed up against the increased
cost WHO UNAIDS currently recommend three
testing strategies to maximise accuracy while
minimising cost
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Strategy One
All blood is tested with one ELISA or rapid test
which is highly sensitive All positive (or
indeterminate) results are considered
infected All negative results are considered
uninfected This strategy is used in
Transfusion/transplantation services
Surveillance (in high prevalence areas)
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Strategy Two
All blood is tested with an ELISA or rapid
test If this first result is positive then a
second confirmatory HIV assay is
performed Confirmatory tests differ in
methodology and target proteins as much as
possible Discordant results are checked using
the same assays and if the results remain
discordant then the sample is considered
indeterminate
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Strategy Two
Generally used for the clinical diagnosis of HIV
infection Can be used for HIV surveillance in
low prevalence countries, repeat testing is
recommended to reduce rates of false positive
results All indeterminate results are reported
and analysed separately in annual surveillance
reports
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Strategy Three
Similar to Strategy Two except that a third
test is performed on all positive samples,
including samples with discordant results
(initially test positive and on repeat test
negative) The 3 assays should employ different
antigen preparation and methodologies If a
samples third test is interdeterminate then the
result will be considered indeterminate
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Strategies Two and Three
Sequence of Tests The more sensitive test is
used as the initial tests Whereas the more
specific tests are used as the secondary tests
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Serial Versus Parallel Testing
Serial tests (most common) The first test
uses a highly sensitive assay to reduce false
negative results Second test is not performed
unless the first test is positive Parallel
tests Two antibody tests are always
performed The tests differ in antigenic
targets and methodologies One test is highly
sensitive and the other test is highly specific
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Serial Versus Parallel Testing
For discordant results Strategy 2 both
initial tests repeated Strategy 3 a third
test (known as a tie-breaker) may be
used Parallel testing NOT recommended by WHO
primarily because of increased cost
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Serial Versus Parallel Testing
Advantages of parallel testing include
Reduction in the risk of false negative results
in high prevalence populations Only a single
finger prick is required Favourable perception
that two tests are better than one Reduction
in the stigma of the patient being called back
for the second test
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Algorithm for use of HIV Rapid Test in Testing
Counselling Services
NEGATIVE test resultCounsel for negative result
Positive test result
Positive test result
Second HIV rapid test
Second HIV rapid test
POSITIVE test resultCounsel for positive result
Negative test result
Introduction to HIV Testing
INCONCLUSIVE resultRepeat testing in 6 weeks
NEGATIVE test resultCounsel for negative result
Positive test result
Second HIV rapid test
POSITIVE test resultCounsel for positive result
Negative test result
Report result as INCONCLUSIVEREFER to Reference
Laboratory
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Diagnosing HIV Infection
HIV antibody assays cannot be used to
diagnose Acute HIV infection HIV infection
in the newborn
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Acute HIV Infection
The Window period Follows acute infection
with HIV, before HIV antibodies can be detected
in the patients blood stream Patient is
highly infectious, despite testing HIV antibody
negative, HIV is replicating rapidly in all body
compartments Typically up to 12 weeks duration
but may be shorter in more sensitive HIV antibody
assays (particularly those incorporating HIV p24
antigen see later)
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Diagnosis in Acute Infection
In acute infection assays are used that detect
part of the virus HIV p24 antigen - Detects
viral protein p24 - Highly specific (gt95) but
low sensitivity (80) HIV proviral DNA -
Detects HIV DNA which has been integrated into
the genome of peripheral blood mononuclear
cells - Based on PCR technology - Highly
specific (98) and highly sensitive (gt99) - It
is available in research settings only - Ability
to reliably detect HIV-2 and non-subtype B HIV-1
has not been fully determined
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Diagnosis in Acute Infection
HIV RNA A viral load test used in monitoring
patients with known HIV infection Truly
positive results generally are greater than
100,000 copies/m, whereas false positives are
generally less than 1,000 copies/mL Generally
not recommended for diagnosis of acute HIV
infection given significant rates of false
positive results (up to 10 of cases)
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Diagnosis in the Newborn
Due to transmission of maternal antibodies HIV
antibody tests cannot be used to diagnose HIV
infection in the newborn Maternal antibodies can
be detected for up to 18 months Non-antibody
assays for early detection of HIV infection in
the newborn include HIV p24 antigen Viral
culture Detection of viral genes (either HIV
DNA or HIV RNA)
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Confidential Anonymous Testing
Voluntary client must give informed consent
for the HIV test to be performed after pre-test
counselling and in the absence of
coercion Confidentiality is maintained by
three general methods Linked testing
Linked anonymous testing Unlinked anonymous
testing
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Confidential Anonymous Testing
Introduction to HIV Testing
WHO Guidelines for Using HIV Testing
Technologies in Surveillance Selection,
Evaluation, and Implementation 2001
WHO/CDS/EDC/2001.16
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Quality Assurance Programme
It is important that all VCT programmes develop
a quality assurance programme relating to HIV
testing This must involve an external reference
laboratory, each month 5 of the total sample
load is sent to the reference laboratory for
confirmation
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Phases in the Development of HIV Testing
Programmes
Pre-analytical Training staff Lab safety
Specimen collection Selection of kits
availability of kits Expiratory dates
Storage
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Phases in the Development of HIV Testing
Programmes
Analytical Written procedures manual
Testing performance Correct use of reagents
Inclusion of internal controls Quality control
monitoring procedure
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Phases in the Development of HIV Testing
Programmes
Post-analytical Interpreting results
Transcription of results Entering data and
tracking data Maintaining records Reviewing
quality control
Introduction to HIV Testing