Before going for HIV screening test it is very important for everyone to know the concept of window period. HIV Screening test may give false negative result during the window period. The window period is an interval of three weeks to six months between the time of HIV infection to the production of measurable antibodies to HIV.
Only test can determine whether a person has HIV infection or not, we can’t tell somebody’s HIV status by his or her physical appearances. There are varieties of laboratory test for HIV infection. Some test diagnose if a person is infected (HIV positive) or not (HIV negative), or other determine the severity of the HIV infection or measure how well the treatment is controlling the progress of the disease. The test through blood is the most common and urine test has been developed but it is not wide spread. Saliva test are also under development.
The most common test is blood test. The two main tests to diagnose HIV infection are the Enzyme Linked Immunosorbent Assay (ELISA) and the western Blot (W.B) analysis.
When HIV invades the body, the immune system produces large numbers of antibodies against it. The antibodies are very ineffective in neutralizing HIV as it cannot entry into cell which has been infected by HIV. But the presence of HIV antibodies help us to determine if a person is infected with HIV or not.
HIV antigen, obtain from HIV that has been grown in laboratory on tissue culture will be coated on laboratory dish. The individual serum is then added to the dish. The individual’s serum is then added to the dish, and allowed sufficient time to react with the HIV antigen. The serum is then washed away. If the antibodies to HIV were present, they would have bound to the HIV antigen in the dish despite the washing.
Now an enzyme that is capable of giving a colour reaction is added to the mixture. This enzyme is in a form that can tag on to the anti bodies if they are present. The sample is washed. Chemical that react with the enzyme to produce a colour change are now add to the dish. An HIV positive sample (Where enzymes, linked to the anti body are present) shows the colour change. In a negative sample, the colour change is absent for the enzymes have been washed away.
In most cases, the ELISA is accurate. However, there is an occasional false result. A false positive result (positive result in a non-affected person) can occur in people with such condition as blood cancer, auto immune disease like rheumatoid arthritis or lupus, or inflammation of the liver due to alcoholism or viral infection. A negative false result (Negative false result in an HIV infected person) can occur if the blood sample is taken in the six month window period following the infection when HIV’s anti bodies have not yet develop. In addition, there is always the possible of laboratory errors such as inadequate washing. For these reasons, an ELISA test needs to be confirmed if it is positive with a more sensitive test, such as a western blot analysis.
Western Blot analysis
Western blotting uses a technique called electrophoresis- the separation molecules based on their rates of migration in an applied electrical flied. A negative electrical charge is applied to all the molecules. The rate of migration of a molecule through the electrical field depends on its molecular weight.
HIV is obtained from tissue culture in the laboratory, and its component proteins are purified by various techniques. (In an infected individual, these HIV proteins act as an antigen, evoking an antibody response.) The mixture of proteins is then separated into individual proteins through electrophoresis in a semisolid medium such as Gel. The gel now contains bands of individual proteins; the band appearing in various locations depends on the rate of migration. Proteins with the higher molecular weights do not travel far and form bands near the starting point.
The proteins are then transferred (“blotted”) onto nitrocellulose membranes or filters paper, reproducing the band pattern found in the gel. The paper is then cut into two narrow strips. This is what is supplied to laboratories in the manufacturer’s test kit.
The individual’s serum is placed on the paper strip and allowed to react with the viral proteins. An HIV positive person’s serum contains antibodies against several of these virus proteins, and the specific of these viral proteins, and the specific antigens and the antibodies now bind. After washing and removal of unbound antibody, antibodies against the anti HIV antibody are now added. As in the ELISA test, these anti bodies are tagged with enzymes capable of giving a colour reaction when a specific chemical is added.
The colour reactions appear as bands on the paper where antigen and antibody are bound. There is no colour banding in areas where the paper only contains unbound antigen. The manufacturers of the test kit supply a referral standard against which the test can be scored.
The combination and intensity of the bands that are presents determine the result. A negative western blot result is one where no bands are present. What constitutes a positive Western blot result has been debated. According to the CDC, the result is positive if it contains at least two of the three bands considered to be of diagnostic significance: anti p-24, antigp-41 and anti-gp-120/160
Result that cannot be classified as negative or positive are classed as “indeterminate”. Some indeterminate results show reactively to one or two antigens, but this is not significant to be called positive. Sometimes there are odd bands in locations where there are no HIV proteins. These are presumed to be contaminant proteins with some similarities to antigens specific for antigens in the serum, and so there is a week blinding. (This is called “cross reaction”). Such contaminate proteins usually come from the cell of the tissue culture in which HIV was grown. Most indeterminate Western blot results are seen in people with no other evidence of HIV infection.
The western blot analysis, however, can give a false negative result if it is performed during the window period or in the terminal stage of AIDS when the immune system is in such disarray that antibodies are no longer produced in measurable levels. A false positive Western blot test is usually due to faulty laboratory technique.
Rapid screening Test
The ELISA test requires special equipment, so the blood specimen (sufficient for two ELISA test) is usually sent to laboratory. The result may not be known until a few days later, depending on how the particular laboratory handles the procedure. If it is positive, then a Western Blot analysis is needed for confirmation. Testing, therefore, requires at least two visits. During the second time, results are communicated, additional counselling is provided and a plan of treatment and support charted out.
Now rapid screening tests to detect antibody to HIV have been developed. The sensitivity of such test is comparable to that of ELISA. Several rapid HIV Test are use in many countries, although only one has been approved in the United States of America. These rapid tests produce result within 30 munities, and are useful in field work, or in rural areas where there may be no electricity available, let alone a laboratory. Confirmatory tests are usually done in laboratory elsewhere.
However, people who test positive by rapid test can be advised on the same day about the screening tests result, and of the need to prevent transmitting HIV to others if they tested positive. This is important because many people do not return for the second visit to find out their results. The CDC reported that, at publicly funded test site in the United States in 1996, 26% if those who tested positive and 33% of those test negative did not return for their second visit. In developing countries also, the rate of return visit is not uniformly satisfactory.
However, the practice of same day reporting may vary from clinic to clinic. Many health-care providers do not believe in communicating result of a positive ELISA or a rapid HIV test unless they are confirmed by the western blot analysis. Many firms believe that counselling should always accompany the communication of tests result.