Module 1

Ethiopian Public
Health Institute

Overview of HIV Testing

Technologies
 Module 1

Ethiopian Public

Overview of HIV Testing

Health Institute

Technologies

Overview of HIV Testing Technologies

 Learning Objectives
At the end of this module, you will be able to:
• Discuss the spectrum of testing technologies for
HIV
• Explain the advantages and disadvantages of HIV
rapid tests
• Accurately recognize individual test result as
reactive, non-reactive, or invalid

The Ethiopian Public

Lab workers Health workers Health Institute
Counselors
 Content Overview
• Expansion of HIV rapid testing
• Spectrum of HIV diagnostic tests
• Challenges with HIV testing
• Spectrum of HIV testing technologies
• Advantages and disadvantages of HIV rapid
testing
• Three formats of rapid tests
• Reading individual test results
The Ethiopian Public
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Counselors
HIV Testing Occurs in a Variety of Settings

T&C

ANC
Blood Provide
Prevent HIV Banks Provide ARV
ARV
treatment
treatment to
to
Infections Surveillance
HIV-infected
HIV-infected
persons
persons
TB Clinics

Hospitals Provide care to

HIV-affected
STI Clinics persons

Lab workers Health workers Counselors

 Expansion of Testing Services
• Integrate HIV
laboratory services
and diagnostics fully
into national health
laboratory structures
• Facilitate testing in
non-traditional
settings
• Consider all our
testing options

Lab workers Health workers Counselors

Use of HIV Testing Technologies in the Continuum of
Care
Use HIV rapid diagnostic test to identify
an HIV infection

Initiate treatment with ARVs

minimal diagnostics required

Monitor effectiveness of ARVs

with diagnostics (viral load,
CD4) and safety with basic
laboratory tests The Ethiopian Public
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 Spectrum of HIV Tests
• HIV diagnosis (Antibody/Antigen testing)
– Enzyme Immunoassays (EIAs)
– Rapid tests
– Western blot (WB)
• Early diagnosis in infants
– P24
– DNA PCR
• Initiation and monitoring of ART
– CD4
– Viral Load
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Detecting HIV-infection with various formats and
generations

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 Challenges of HIV Testing
• Early detection of seroconversion
• Early detection in infants born to HIV positive
mothers
• Effect of HIV subtypes on test performance
• Impact of other health conditions on test
performance
• Product specific equipment
• Technical skill

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 Enzyme Immunoassays (EIAs)
• Quantitative assay to measure HIV antibodies
– Most detect antibodies to HIV-1 and HIV-2
– Antigens coated in microwells
– HIV Antigen / Antibody reaction is detected by color change
– Intensity of color reflects amount of antibody present in
serum
• Some assays can detect both HIV antibody and HIV
antigen (shorter window period using 4th generation)
• Issues:
– Skilled lab technician
– Large volume testing
– Properly maintained equipment required
– Initial cost
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 Enzyme ImmunoAssays (EIAs) – Cont’d

After several incubation and wash steps, a An automated reader gives a

color reaction occurs if HIV antibody is measurement of optical density
present (presence of color) for each well

Lab workers Health workers

 HIV Rapid Tests
• Qualitative assay to detect HIV antibodies
• Most detect HIV 1 and HIV 2
• As reliable as EIAs
• Issues:
– Small volumes
– Validation of use
– Appropriate training

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Western Blot / Line Immunoassays
• Used as supplemental test for
confirmation (only difficult
cases)
• Detects antibodies to specific
HIV antigens on cellulose strip
• Issues:
– Multiple standards for
performance and
interpretation
– Expensive
– Limited commercial
availability
Lab workers Health workers
 HIV p24 Antigen
• Core protein of the virus
• EIA detects p24 antigen before antibody can be
detected
 Detected 2 to 3 weeks after HIV infection
 Detected about 6 days before antibody tests become reactive
• Used for:
– Diagnosis of pediatric HIV-1 infections
– Blood bank safety (high incidence countries)
• Issues:
– Level 4 complexity
– Properly maintained equipment required
– Not suitable for diagnosis of established infection

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 CD4 T-Lymphocyte
• CD4 T-lymphocyte
counts used for:
– Determining clinical
prognosis
– Assessing criteria for
antiretroviral therapy
• Manual and automated
methods
• Issues:
– Requires high level of
technical skill for test
performance and
interpretation
– Properly maintained
equipment

Lab workers Health workers

 Viral Load
• Quantitative molecular assay measures amount of
HIV in blood products
• Used to:
– Predict disease progression
– Monitors response to anti-retrovirals
• Issues:
– Relatively expensive
– Labor-intensive
– Special facilities

The Ethiopian Public

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 Complexity of HIV Tests Varies*
• Level 1: No additional equipment and little or no
laboratory experience needed
• Level 2: Reagent preparation or a multi-step process
is required; Centrifugation or optimal equipment
• Level 3: Specific skills such as diluting are
required
• Level 4: Equipment and trained laboratory
technician are required.

*WHO Reports
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HIV Rapid Tests
provides
excellent tool for
expansion of
services

Lab workers Health workers Counselors

 HIV Rapid Tests: Advantages
• Increases access to prevention (VCT) and
interventions (PMTCT)
• Supports increased number of testing sites
• Same-day diagnosis and counseling
• Robust and easy to use
• Test time under 30 minutes
• Most require no refrigeration
• None or one reagent
• Minimal or no equipment required
• Minimum technical skill
The Ethiopian Public
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 HIV Rapid Tests: Disadvantages

• Small numbers for each test run

• Quality Assurance/Quality Control at multiple
sites
• Test performance varies by product
• Refrigeration required by some products, e.g.,
Capillus
• Reader variability in interpretation of results
• Limited end-point stability of test results

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Body Fluids Used for HIV Rapid Testing

• Serum
• Plasma
• Whole blood
• Oral fluids

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 Three Formats of HIV Rapid Tests

• Immunoconcentration (flow-through device)

• Immunochromatography (lateral flow)
• Particle agglutination

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 How Immunoconcentration Works
HIV antibody links to bound HIV peptide
antigens forming the color spot

Internal Control
HIV-1 peptide

HIV-2 peptide

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 Tests Based on Immunoconcentration
Flow-Through Devices:
– Stat-Pak

Top view

Side view

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Reading Results: Stat-Pak

Non-reactive Reactive

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How Immunochromatography Works

Add Control
Test
Sample Conjugate Line line

IgG Antibodies Colloidal gold HIV antigen Anti-IgG/gold

HIV antibodies conjugated to antibodies
HIV antigen

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 Tests Based on Immunochromatography

Lateral Flow Devices

– First Response
– Unigold Control
– Vikia HIV Antigen
– Determine
– KHB rapid
– Abon
Sample pad
– SD Bioline
Specimen Flow
– Stat Pack

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 Reading Results

Non-
Reactive

Reactive

Sample Pad Test line Control line

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 How Particle Agglutination Works
Anti-HIV antibodies bind to the antigen-coated latex
particles.

Antigen

Antibody

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 Tests Based On Agglutination
Agglutination devices:
• Capillus

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Reading Results: Capillus

Non-
reactive

Weak
Reactive

Strong
Reactive

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 There Are Only Three Possible
Outcomes for Single HIV Antibody Tests
Reactive or “Positive”
– Test band
– Control band
Non-reactive or “Negative”
– Control band only
Invalid
– No control band present
– Test has failed. Repeat with new device.
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Exercise: Interpreting Individual HIV
Rapid Test Results

– Refer to Participant Manual

– Work alone to determine individual test
results
– 3 Minutes

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 Key Messages
• HIV rapid tests can be as reliable as EIA
• All tests require attention to training,
supervision, and monitoring at points of
service.
• As testing is expanding and decentralized,
training, supervision, and monitoring must
follow accordingly and become all the more
important.

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Counselors
 Summary
• Where is HIV rapid testing likely to occur
during an era of expansion of services?
• What is the intended use for: EIAs, Western
Blot, p24 Antigen, CD4, Viral Load
• What are rapid tests?
• Why we use rapid tests?
• How do you read a result – reactive, non-
reactive or invalid?

The Ethiopian Public

Lab workers Health workers Health Institute
Counselors
The Ethiopian Public
Health Institute