Module 3: Overview of HIV Testing Technologies

Purpose To provide the participants with a basic knowledge of HIV testing

and how HIV rapid test results are interpreted.

Pre-requisite Module 1: Overview of HIV Infection

Modules

Module Time 1 hour 5 minutes

Learning At the end of this module, participants will be able to:

Objectives
• Discuss settings where HIV testing will be part of service
delivery during an era of expanded services
• 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

Module Overview

Activity/ Resources
Step Time Content
Method Needed
Slides 2-3; Prepared
1 2 min Presentation Module introduction
flipchart – content outline
Presentation;
2 5 min Discussion Expansion of HIV Testing Slides 4-6

3 15 min Exercise Spectrum of HIV Tests Slides 7-16;

Presentation; HIV Rapid Testing

4 30 min Slides 17-32
Discussion Technologies
Slide 33;
Interpreting Individual HIV Exercise Sheets:
5 5 min Exercise
Rapid Test Results Interpreting Individual HIV
Rapid Test Results
Key messages
6 8 min Q&A Summary Slides 34-35

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Material/Equipment Checklists:

PowerPoint slides or transparencies

Overhead projector or computer w/LCD projector

Flipchart

Multiple sets of samples showing reactive, non-reactive, and invalid test
results from each test approved for use in-country

Exercise sheets: Interpreting Individual HIV Rapid Tests

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 Teaching Guide

Slide Number Teaching Points

1 Module 3: Overview of HIV Testing Technologies

DISPLAY this slide before you begin the module. Make sure
participants are aware of the transition into a new module.

2 Learning Objectives
STATE the objectives on the slide.

3 Content Overview
EXPLAIN the topics that will be covered in this module.

Flipchart WRITE the content outline on a flipchart prior to training.

REFER to it frequently to orient participants to where they are in
the module.

4 HIV Testing Occurs in a Variety of Settings

DESCRIBE the graphic on the slide by making the following
points:
• HIV testing will occur in a variety of settings outside of
the laboratory
• The settings where testing will likely to occur during an
era of expansion of services include: Testing &
Counselling Centers (T & C), Antenatal Clinics (ANC),
Blood Banks, Surveillance programs, TB clinics,
hospitals, and Sexually Transmitted Infections (STI)
Clinics
HIGHLIGHT the following points:
• While all settings where testing occurs can triage
persons to treatment and care, tuberculosis (TB) clinics
and hospitals will be the primary venues for providing
anti-retroviral treatment to HIV infected persons, and for
providing care to HIV affected persons.
• T&C, ANC, Blood Banks, and surveillance are the
primary venues for providing prevention programs.

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Slide Number Teaching Points

5 Expansion of Testing Services

EXPLAIN the following points:

Testing will need to be integrated at all levels of testing
services, and that testing must be linked to referral
services, e.g., ANC and VCT.

To facilitate the expected high volume of testing, non-
traditional test sites will need to be incorporated into the
national testing strategy. These non-traditional sites
must however be linked back to the lab referral network
and a quality management system.

6 Use of HIV Testing Technologies in the Continuum of Care

A variety of tests are performed at different stages. HIV rapid
tests play an important role in initially identifying those who are
infected with the HIV virus.
Other tests, e.g., CD4 and viral load, play an important role in
determining whether therapy can be initiated, and once initiated,
if the drugs are working or not..

7 Spectrum of HIV Tests

POINT OUT this list reflects commonly performed test
associated with HIV. Some tests are for diagnostic purposes,
e.g., EIAs, rapid tests, Western Blot, and p24
Other tests are supplemental in monitoring disease progression,
such as CD4 and viral load.

8 Challenges of HIV Testing

EMPHASIZE the following points:

The ability of some test to detect early infections is sub-
optimal

Specialized testing is required to diagnose HIV infection
in infants younger than 18 months.

The variability of HIV viruses impacts upon the sensitivity
the specificity of HIV tests. Early generation of HIV test
kits could not detect antibodies produced against strains
of group O.

Cross reactivity with other health conditions or infections
decreases performance of the assay, e.g.,
cytomegalovirus and Epstein-Barr virus.

Some technologies require specific equipment that must
be properly maintained.

The skill required to accurately perform and interpret
tests varies; from minimal to high level of skill

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Slide Number Teaching Points

9 Enzyme Immunoassays (EIAs)

STATE the points on the slide

Some participants may not understand certain technical terms

such as microwell. Consider passing around a microtiter plate
for participants to see what is meant by “microwells.”

TIPS

10 Enzyme Immunoassays (EIAs) - Cont’d

DESCRIBE how Enzyme Immunoassay works.
• Sample is added to micro-well plate that has been
coated with HIV antigen(s).
• After a series of reagent additions, incubations and
washings, the plate is placed in reading device.
• The reading device measures the optical density of color
that develops if HIV antibody is present in the client’s
sample.
EMPHASIZE multiple factors can affect testing. As previously
described a certain level of technical skill AND functioning
equipment is a must.

11 HIV Rapid Tests

STATE the points on the slide.
HIGHLIGHT an added advantage for using rapid tests is the
ability to use whole blood.
EMPHASIZE that while HIV rapid tests, in general, is
considered to be low in complexity, all test must be
appropriately evaluated prior to use. It is equally important that
the test be validated for use in the environment where testing
will occur.
EMPHASIZE the need for appropriate training on use of test.

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Slide Number Teaching Points

12 Western Blot / Line Immunoassays

STATE The Western Blot is a supplemental test for confirming
HIV infection.
EXPLAIN the cellulose strip on the right.
DESCRIBE key issues involved:
• It lacks standardization in performance and
interpretation.
• Although considered a confirmation test, this assay has
a high range of indeterminate results.
• It is a complex test.
• It is very expensive.

13 HIV p24 Antigen

STATE the points on the slide.

14 CD4 T-Lymphocyte
STATE the points on the slide.
EMPHASIZE the need for instruments to be properly
maintained, such as the BD FASCount, and BD FASCaliber
seen on this slide.

15 Viral Load
STATE the points on the slide. The higher the viral load
(number of copies of HIV in the blood), the greater the
progression of the disease.
EMPHASIZE a number of Issues exist with this test:

Kits and reagents are expensive

Demanding molecular techniques

Concerns over contamination

Experienced technicians required

Difficult/complex assays

Need separate dedicated supplies, equipment (including
biosafety cabinets), and air conditioned rooms

Need constant source of electrical power

PCR-based technologies susceptible to genetic variation
and low copy number

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Slide Number Teaching Points

Define terms such PCR, and explain low copy number when
teaching participants without lab background.

TIPS

16 Complexity of HIV Tests Varies*

4 levels of complexity for HIV tests have been described in a
number of WHO reports. The complexity of tests varies, from
minima – level 1, to complex - level 4, in terms of equipment,
and technical skill.

17 HIV Rapid Tests provides excellent tool for expansion of

services
TRANSITION into HIV rapid tests.
STATE HIV rapid testing provides excellent tool for expansion of
services. The remaining module will focus on HIV rapid tests.

18 HIV Rapid Tests: Advantages

DISCUSS the advantages on the slide.
EMPHASIZE If clients do not obtain their HIV results, this is a
missed opportunity for therapy or preventative measures.
ASK participants to share any stories that illustrate the adverse
effects of having to wait a long time for test results.

For participants without any lab or medical background:

• EXPLAIN the term reagent. A reagent is a substance
TIPS used in a chemical reaction to detect or produce other
substances.
• EXPLAIN why some lab materials need refrigeration,
and others don’t, emphasizing the consequences of not
refrigerating materials that require refrigeration (kits e.g.
Capillus, and serum quality controls).

19 HIV Rapid Tests: Disadvantages

STATE the points on the slide
DISCUSS past and present problems in slow turn-around of
results from the laboratory, and the poor come-back rate of
clients to obtain their results (due to fear, cost issues, transport
issues, etc.).

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Slide Number Teaching Points

For participants without any lab or medical background:

• EXPLAIN that although the tests are not difficult, they
TIPS will require a lot of practice and supervision to become
proficient.
• EMPHASIZE they must not feel afraid or embarrassed to
stop at any time and ask questions.

20 Body Fluids Used for HIV Rapid Testing

EXPLAIN that HIV tests could be performed on a wide range of
body fluids. Serum, plasma, whole blood and oral fluids are
used the most.
ASK participants what each fluid is and the typical method for
obtaining each.

For participants without any lab or medical background:

• Briefly DESCRIBE these different fluids.
TIPS • INFORM them that the samples they will use for HIV
rapid testing will most likely be whole blood drawn from
their clients’ fingertips.

Slides 21, 22, 23, 25, 26, 29, and 30 are highly technical. We
recommend that you do not present these slides to lay
counsellors.
TIPS
If you do present these slides,
• EXPLAIN every technical term on the slides
• USE analogies and examples to help participants
understand.

21 Three Formats of HIV Rapid Tests

STATE that there are three main formats or types for rapid HIV
tests, and you will explain each format in the following slides.

22 How Immunoconcentration Works

EXPLAIN the graphic on the slide.
DESCRIBE how Immunoconcentration works.
• Flow-through (or immunoconcentration) devices are
usually cartridges, with HIV antigen attached to a
membrane.
• The specimen and individual reagents are each added to
the cartridge in a series of steps.
• Presence of HIV antibody is indicated by the
development of a colored spot or line.

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Slide Number Teaching Points

23 Tests Based on Immunoconcentration

STATE the devices mentioned in the slide (DESCRIBE more
fully ones used in-country).
DESCRIBE the graphics on slide.

Modify these slides if necessary:

• Delete tests not used in your country’s algorithm
Customization
Notes • Replace photos with in-country examples
24, 27, 28, 31

24 Reading Results: Genie II

EXPLAIN how to read results from Genie II.

If non-reactive, you will only see one visible dot in the
control region
• If reactive, you will see either one or two visible dots.
One dot for HIV 1, and the other for HIV 2
• At the control dot, Human IgG links to membrane-bound
anti-human IgG
Note: It will be helpful to physically point to the control dot.

For participants without lab or medical background, you may

need to explain the terms “reactive” and “non-reactive.”

TIPS • Reactive – Antibodies to HIV present in the client’s blood

• Non-reactive – No HIV antibodies detected

25 How Immunochromatography Works

EXPLAIN the graphic on the slide by describing how
Immunochromatography works.
• Specimen is applied to a pad (filter) where it mixes with
gold or selenium colloid-antigen conjugate. This mix
migrates through the nitrocellulose strip to immobilized
recombinant antigens and synthetic peptides at the
patient window.
• If HIV antibodies are present then a red line will form in
the test area of the strip.

SIMPLIFY the explanation when teaching participants without

lab background:

TIPS

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Slide Number Teaching Points

26 Tests Based on Immunochromatography

EXPLAIN how lateral flow devices work.
• Capillary flow (lateral flow) devices resemble dipsticks.
All of the necessary reagents are usually incorporated
into the test strip embedded in the device.
• Specimen (and sometimes buffer or a reagent) added to
the strip flows across the reagents, and a colored line
develops in the presence of antibody.
• Most lateral flow devices also have an internal control
that detects human IgG. This internal control indicates
that specimen was added to the test strip. If no human
IgG is detected, an internal control line does not develop
indicating an invalid test.
STATE the devices mentioned in the slide (DESCRIBE more
fully ones used in-country).

SIMPLIFY the explanation when teaching participants without

lab background:

TIPS
27 Reading Results: Determine
EXPLAIN how to read test results from Determine test kit.
• The reactive reaction shows two lines
o One for the control band.
o The other for the test. A band in the test area
means a reactive result.
• A non-reactive reaction will show a control band only.
• The control band (line) must always be present for the
test results to be valid.

28 Reading Results: OraQuick

ASK participants to explain OraQuick test results on the slide.

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Slide Number Teaching Points

29 How Particle Agglutination Works

DESCRIBE how particle agglutination works by describing the
graphic on the slide.
• The round circles represent antigen-coated latex
particles that bind to antibodies to HIV (represented by
the “Y”. Note: POINT TO each figure as you describe
the graphic.
• Agglutination or clumping occurs when the antibodies
bind to the antigen-coated particles
• Agglutination assays were among the first of the rapid
tests developed.
• Inexperienced persons or those who do not conduct the
tests frequently may have problem with differentiating
the coarseness or clumping of individual particles from
true agglutination. They sometimes “over-interpret”
agglutination, which result in a larger number of false-
positives.

SIMPLIFY the explanation when teaching participants without

lab background:

TIPS
30 Tests Based on Agglutination
STATE the devices mentioned in the slide (DESCRIBE more
fully ones used in-country).
DESCRIBE the 3 images on the slide, using Capillus as the
main example (as it is the most commonly used agglutination
test):
• Left – The blood is placed in the oval area, also called
the mixing well.
• Center – The specimen travels along the thin tubes in
the slide.
• Right – If the blood contains antibodies to the HIV virus,
visible clumping or agglutination can be seen.

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Slide Number Teaching Points

31 Reading Results: Capillus

DISCUSS the three different results on the slide.
• ASK participants to describe what they see in the slide.
• POINT OUT the clumping or agglutination in the reactive
sample, and the smooth liquid without clumps in the non-
reactive test.
• MENTION that unlike other test previously discussed,
there is no control line to let the tester know that the test
has worked correctly. However, the test kit includes a
positive and negative control, which can be used to
verify on a regular basis that the test is working properly

Transition STATE that there is a third possible result – the control line is
not present.
REMIND that when the control line fails to show, it indicates that
the test has failed. The result is therefore called “invalid.”
TRANSITION into next slide.

32 There Are Only Three Possible Outcomes for Single HIV

Antibody Tests
REVIEW the terms on the slide.
EMPHASIZE these are the terms when interpreting the outcome
of a single test.
EMPHASIZE if a test yields an invalid result; meaning no control
band or line is present, the test has failed. The test MUST be
repeated using a new test device.

If the tests used in the exercise are not in your country’s

algorithm, consider replacing them with tests that are.
Customization
Notes
33

33 EXERCISE – Interpreting Individual HIV Rapid Test Results

Exercise REFER participants to the exercise in their manual.
5 minutes ASK participants to spend 3 minutes, working alone, to identify
the results.
DISCUSS the answers as a group.

Consider having multiple sets of real samples available to pass

around, with each set containing three different test results
(reactive, non-reactive, and invalid) for each test approved in-
TIPS country. The more participants, the more sets you will need.

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Slide Number Teaching Points
34 Key Messages
STATE the points on the slide.

35 Summary
ASK participants to answer the questions on the slide.
ANSWER any questions the participants may have.

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