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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