Module 3: Overview of HIV Testing Technologies 1 Participant Manual

2005

Module 3

Overview of HIV Testing
Technologies


Purpose
To provide you with a basic knowledge of HIV testing and how
HIV rapid test results are interpreted.
Pre-requisite
Modules
Module 1: Overview of HIV Infection
Learning
Objectives
At the end of this module, you will be able to:
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
Content Outline 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

Handouts Exercise #1: Interpreting Individual HIV Rapid Tests
Exercise #2: Interpreting Individual HIV Rapid Tests

Notes on
Customization
Delete examples of HIV rapid tests not used in your countrys
algorithm and replace with in-country examples.
Module 3: Overview of HIV Testing Technologies 2 Participant Manual
2005

HIV Testing
Occurs in a
Variety of Settings


HIV testing occurs 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
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.

Expansion of
Testing Services

Testing will need to be integrated at all levels of testing services,
and 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 laboratory referral network and a quality
management system.

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.

STI Clinics
TB Clinics
Hospitals
T&C
ANC

Blood
Banks
Surveillance
Provide care
to HIV-
affected
persons
Provide ARV
treatment to
HIV-infected
persons



Prevent HIV
Infections

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2005
Spectrum of HIV
Tests

The list below 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.
HIV diagnosis (Antibody/Antigen testing)
Enzyme Immunoassays (EIAs)
Rapid tests
Western blot (WB)
Early diagnosis in infants
p24
DNA/RNA PCR
Initiation and monitoring of ART
CD4
Viral Load

Challenges of HIV
Testing

There are several challenges associated with HIV testing:
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. However, people
have limited access to this testing.
Some tests may not be able to detect antibodies
produced against specific HIV subtypes. For example,
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.
Personnel need a certain level of skill to accurately
perform and interpret tests varies (from minimal to high
level)

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Enzyme
Immunoassays
(EIAs)

EIA is a quantitative assay that measure HIV antibodies. Most
EIAs can detect antibodies to HIV-1 and HIV-2. Here is 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 clients
sample.
Multiple factors can affect testing such as skilled lab technician,
large volume testing, and properly maintained equipment. A
certain level of technical skill AND functioning equipment is a
must.

HIV Rapid Tests

HIV rapid tests are qualitative assays that detect HIV antibodies.
Most of them can detect HIV 1 and HIV 2. These tests are as
reliable as EIAs.
One advantage of HIV rapid tests is its ability to use whole
blood. While HIV rapid tests in general are considered to be low
in complexity, all tests must be appropriately evaluated prior to
use and personnel be properly trained. It is equally important
that the test be validated for use in the environment where
testing will occur.

Western Blot /
Line
Immunoassays

The Western Blot is a supplemental test for
confirming HIV infection. It detects antibodies
to specific HIV antigens on cellulose strip.
key issues involved include:
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.

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2005
HIV p24 Antigen

HIV p24 antigen is the core protein of the virus. EIA detects p24
antigen before antibody can be detected. p24 is usually
detected 2 to 3 weeks after HIV infection, and detected about 6
days before antibody tests become reactive.
A modified p24 antigen assay is used for diagnosis of pediatric
HIV-1 infections and blood bank safety (high incidence
countries).
The issues for this type of testing include high level of
complexity (i.e., level 4) and equipment used for this testing
must be properly maintained.

CD4 T-
Lymphocyte

CD4 T-lymphocyte counts are
used for determining clinical
prognosis, assessing criteria
for antiretroviral therapy, and
monitoring therapy. CD4
counts may be performed
using either manual or
automated methods.
Performing CD4 T-lymphocyte
counts requires high level of
technical skill for test
performance and
interpretation. It is also very
important to have properly
maintained instruments such
as the BD FACSCount, and
BD FACSCalibur.


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Viral Load

This is a quantitative molecular assay that measures amount of
HIV in blood products. The higher the viral load (number of
copies of HIV in the blood), the greater the progression of the
disease.
This assay is used to predict disease progression, assist with
deciding when to initiate anti-retroviral therapy, and monitor
response to anti-retrovirals.
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

Complexity of HIV
Tests Varies

Four 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.
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


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

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HIV Rapid Tests:
Advantages
HIV rapid tests have the following 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 (a substance used in a chemical
reaction to detect or produce other substances)
Minimal or no equipment required
Minimum technical skill


Information Box

Certain kits and reagents require refrigeration as specified by
the manufacturers. If they are not stored according to
manufacturers instructions, the quality of the tests will be
compromised.


HIV Rapid Tests:
Disadvantages

HIV rapid tests also have a few 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 the results, i.e., reading
should be done in a short time window
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.).
If clients do not obtain their HIV results, this is a missed
opportunity for therapy or preventative measures.

Body Fluids Used
for HIV Rapid
Testing

HIV tests could be performed on a wide range of body fluids.
Serum, plasma, whole blood and oral fluids are used the most.
The samples used for HIV rapid testing will most likely be whole
blood drawn from clients fingertips.

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Information Box

Serum, plasma, whole blood, oral fluids: what is the
difference?
Serum is the liquid part of the blood without the red blood cells.
Plasma is liquid part of the blood containing an anticoagulant
without the red blood cells.


Three Formats of
HIV Rapid Tests

There are three main formats or types for rapid HIV tests:
Immunoconcentration (flow-through device)
Immunochromatography (lateral flow)
Particle agglutination
Read on to find out more about each format.

How
Immunoconcentra
tion Works

HIV antibody links to bound HIV peptide antigens forming the
color spot

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.

Tests Based on
Immunoconcentra
tion

Some examples of flow-
through devices are
Multi-Spot and Genie II.



HIV-1 peptide
HIV-2 peptide
Internal Control
Side view
Top view
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Reading Results:
Genie II

If the result is non-reactive,
you will only see one visible
dot in the control region
If the result is 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



Information Box

Definition of reactive and non-reactive
Reactive means antibodies to HIV are present in the clients
blood.
Non-reactive means there are no HIV antibodies detected.


How
Immunochromato
graphy 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.

Add
Sample
Conjugat
C Contro
l
line
T Test
Line
IgG Antibodies
HIV antibodies
Colloidal gold
conjugated to
HIV antigen
Anti-
IgG/gold
antibodies
HIV antigen
Non-reactive Reactive
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Tests Based on
Immunochromato
graphy

Some examples of
lateral flow devices
include:
Determine
Hema-Strip
OraQuick
Unigold


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.

Reading Results:
Determine


The reactive reaction shows two lines: one for the control band,
and 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.

Sample pad
HIV Antigen
Control
Specimen Flow
Non-
Reactive
Sample Pad Control line Test line
Reactive
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Reading Results:
OraQuick


The test device on the far left indicates a non-reactive result,
while the device in the center and the one on the far right show
reactive results.

How Particle
Agglutination
Works

Anti-HIV antibodies bind to the antigen-coated latex particles.

Agglutination assays were among the first of the rapid tests
developed. The round circles represent antigen-coated latex
particles that bind to antibodies to HIV (represented by the Y).
Agglutination or clumping occurs when the antibodies bind to
the antigen-coated particles.
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.

Non-
Reactive
Reactive
Antigen
Antibody
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Tests Based on
Agglutination

Examples of agglutination
devices include:
Capillus
Serodia









Look at the three images above:
On the left The blood is placed in the oval area, also
called the mixing well.
In the center The specimen travels along the thin tubes
in the slide.
On the right If the blood contains antibodies to the HIV
virus, visible clumping or agglutination can be seen.

Reading Results:
Capillus


Look at these slides. Did you notice the clumping or
agglutination in the reactive sample, and the smooth liquid
without clumps in the non-reactive test?
Unlike other test previously discussed, there is no control line in
Capillus 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.

Non-
reactive
Weak
Reactive
Strong
Reactive
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Besides reactive and non-reactive, there is a third possible
result the control line is not present. When the control line fails
to show, it indicates that the test has failed. The result is
therefore called invalid.

There Are Only
Three Possible
Outcomes for
Single HIV
Antibody Tests

In summary, the three possible outcomes for a single HIV
antibody test are:
Reactive or Positive when both test band and control
band are present.
Non-reactive or Negative when only the control band
is present.
Invalid when no control band is present.
If a test yields an invalid result, the test has failed. The test
MUST be repeated using a new test device.

Exercises

Interpreting Individual HIV Rapid Test Results
At the end of this module, you will find two exercise sheets.
Study the examples and write your interpretation of the test
results in the space provided.




Key message
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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Module Module Module Module Review Review Review Review
Find out how much you have learned by answering these questions.
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 _____________________________________________________________
- CD4 _____________________________________________________________
- Viral Load _______________________________________________________

What are rapid tests?
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________

Why use rapid tests?
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
Module 3: Overview of HIV Testing Technologies 15 Participant Manual
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Exercise #1: Interpreting Individual HIV Rapid Tests


Instructions: Interpret the test results in the following examples.
Write your interpretation of the test result on the line
provided below each example.








________________ ________________ ________________








________________ __________________ __________________
Module 3: Overview of HIV Testing Technologies 16 Participant Manual
2005

Exercise #2: Interpreting Individual HIV Rapid Tests


Instructions: Interpret the test results in the following examples.
Write your interpretation of the test result on the line
provided below each example.



____________________________________________________________

____________________________________________________________

____________________________________________________________