Laboratory
Tests for HIV
Professor Alan
Smith - Head of Virology answers questions relating
to HIV-testing:
How do you test?
What are you testing for? Why are there different tests?
What is the difference between antigen and antibody?
HIV, the Human Immunodeficiency Virus like any other
organism be it bacteria such as those causing cholera,
typhoid or tuberculosis; or a parasite such as those
causing malaria are all foreign to the human body and
are recognised as such by our immune system. Any foreign
substance, such as those given as examples above, are
termed ANTIGENS
because they act as a stimulus to the body to produce
ANTIBODIES.
Both of these entities are highly specific, that is
to say, one type of antigen will cause the production
of a specific antibody; for instance the antibody produced
in response to the 'flu virus will be different from
the antibody produced in response to hepatitis virus
or the HI-virus.
 From
the above it is easy to see that we do not have HIV
antibodies if we have not been exposed to (had in our
body) the HIV (antigen).
The laboratory has various tests for all manner of
antigens and antibodies. For measles, influenza, mumps,
etc, etc.
The ELISA is a
technique for identifying the presence of these various
antigens or antibodies in a patient's blood or other
body fluid. To detect the presence of a specific antibody
in blood we use the appropriate antigen in the ELISA
method.
ELISA is an acronym
for Enzyme Linked Immune Serum Assay.
Before the antigen (HIV) enters the body there are
no HIV antibodies present. The period from the time
HIV enters and there are detectable levels of HIV antibody
present is variable but on average in excess of three
weeks.
This period is known as "the window period"
during this time there may be vary large numbers of
the virus in the blood (the viral load will be high)
even though the person will test negative to the ELISA
antibody test. As the level of antibody production rises
so the numbers of virus will diminish.
The importance of the laboratory ELISA
is that it uses automated robotic machines to remove
the risk of human error; it also has programmed periods
when the patient's serum is exposed to the test antigen
at predetermined temperatures. This has become the "gold
standard" against which all other tests (such as
rapid tests) are measured.
The ELISA uses
an enzyme/substrate colour reaction, which will detect
very weak levels of antibody present in the blood.
The ELISA, by using
antibody as its reference substance, can also be used
to detect the presence of antigen in the blood.
One problem with all laboratory tests for antibody
or antigen is the risk of cross-reactions; these can
result in a false positive result but cannot cause a
false negative result.
Cross-reactions:
As mentioned earlier antibodies are highly specific
for antigens, however, mistaken identity is always a
possibility. Just as an eyewitness can be mistaken at
the scene of a crime. To protect against this possibility
in the laboratory each batch of 90 tests includes "controls"
these are samples known to be negative or known positives.
This can be likened to the police identification parade
where a number of volunteers of similar identity are
paraded, with the suspect, for identification by the
witness.
A further protective method used in all laboratories
is the retesting of all positive samples by a second
and different ELISA.
In the virology dept at KEVIII hospital all positive
tests are retested on a different ELISA
machine with a test kit from a different supplier. Furthermore
another laboratory technologist does the repeat tests.
Any discordant samples, that is to say positive in one
assay and negative in the second, are subjected to an
array of further tests including molecular techniques.
Rapid testing:
Rapid testing has the advantages of bringing the laboratory
to the patient's bedside or into the clinic situation.
As in most compromise situations there are trade-offs
to be kept in mind.
Most rapid tests are based upon the physics of capillary
action and microscopic particles migrating along a membrane
surface. The antigen, to be used to detect the presence
of antibody, is fixed to microscopic particles that
are on the surface of a membrane "mat". Whole
blood from a finger prick is usually collected in a
narrow bore glass tube, coated with a substance to prevent
the blood from clotting. This is then transferred to
the test device and as it migrates along the "mat",
in much the same way that spilt coffee spreads over
a table cloth, the blood and microscopic particles are
exposed to reagents to develop a colour reaction if
the sample is positive. Often there is a test line that
develops to indicate the test is working correctly.
It is most important, in all of these rapid tests,
to observe the manufacturers instructions precisely
especially with regard to volumes to be used and duration
of the period before reading the result.
As in laboratory ELISAs
it is most important (even more important) to confirm
positive results. A common problem in the field is that
workers do not appreciate that repeat tests must be
done by the use of a different test, repeating a positive
with the same test kit only serves as a duplicate test
NOT a confirmatory test.
Rapid tests serve a purpose for specific uses they
do not have the high degree of sensitivity and specificity
as laboratory ELISA
tests nor can they include the extensive controls utilized
in laboratory based tests.
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