AIDS Tests Health Article

Diagnostic tests

Diagnostic blood tests for AIDS are usually given to persons in high-risk populations, pregnant females, health care and public service workers who have been exposed to HIV, or those who have symptoms associated with AIDS. The condition of testing positive for HIV antibody in the blood is called seroconversion, and persons who are HIV-positive are called seroconverters.

AIDS tests used to diagnose infection fall into two categories, screening tests and confirmatory tests. It is possible to diagnose HIV infection by isolating the virus from the blood. However, viral culture is expensive, not widely available, and time consuming. Screening tests detect the presence of antibodies to several HIV antigens. These tests are inexpensive, widely available, and accurate in detecting 99.9% of HIV infections. However, approximately 0.2% of persons without HIV infection will test positive. In order to eliminate these false positives, persons should be tested in duplicate and positive results followed by a confirmatory test.

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA). This test is the most commonly used method to screen blood for transfusions as well as to diagnose patients. An ELISA test for HIV works by attaching two or more HIV antigens to a plastic well or beads. A sample of the patient's blood serum is added and incubated. If antibodies to HIV antigens are present, they will bind to the tube, or bead. After washing to remove excess proteins, an anti-human immunoglobulin conjugated to an enzyme is added. After incubation, excess antibody is washed away and a chemical called a substrate is added. The enzyme reacts with the substrate to form a colored product that indicates a positive test.

The latest generation of ELISA tests are 99.5% sensitive to HIV. Occasionally, the ELISA test will be positive for a patient without symptoms of AIDS from a low-risk group. Because this result is likely to be a false positive, the ELISA must be repeated on the same sample of the patient's blood. If the second ELISA is positive, the result should be confirmed by the Western blot or other confirmatory test.

WESTERN BLOT (IMMUNOBLOT). The Western blot or immunoblot test is used as a reference procedure to confirm the diagnosis of HIV infection. In Western blot testing, HIV antigens of different size are purified from HIV cultures and separated from each other by the process of electrophoresis. (In electrophoresis, protein molecules are suspended in a gel and separated by applying an electric current through the gel.) The HIV antigens are transferred to a nylon membrane or nitrocellulose filter. The patient's serum is added to this, and if antibodies are present they will bind to the corresponding viral antigens. The membrane is washed and anti-human immunoglobulin conjugated to an enzyme is added. After washing again, the color-developing substrate is added, which causes colored bands to appear where the serum antibodies are attached to the membrane. Western blots can detect antibodies to several HIV antigens, but results must be interpreted with caution because antibodies produced against other viruses may cross-react. A test is considered positive when antibodies are seen against antigens from at least two of the three major HIV antigens (p24, gp41, and gp120/160).

When used in combination with ELISA testing, Western blot testing is 99.9% specific. It can, however, yield false negatives in patients with very early HIV infection and in those infected by HIV-2. In some patients the Western blot yields indeterminate results.

IMMUNOFLUORESCENCE ASSAY (IFA). This method is sometimes used to confirm ELISA results instead of the Western blot test. An IFA test detects the presence of HIV antibody in a sample of the patient's serum by incubating the serum with H9 cells infected with HIV virus. The cells are grown in tissue culture and transferred to glass slides, which are frozen. After incubating with the patient's serum, the slide is washed to remove the serum and fluorescein-conjugated anti-human immunoglobulin is added. After incubation, the unbound conjugate is removed by washing the slide. The slide is examined using a fluorescent microscope. The conjugate causes the HIV-infected cells coated with antibody to have a green fluorescence. This test can detect antibody of the IgM class that is produced within seven to 10 days after infection.

RADIOIMMUNOPRECIPITATION ASSAY (RIPA). A third confirmatory HIV testing method is the radioimmunoprecipitation assay (RIPA). This test also uses H9 lymphocytes infected with HIV. The cells are grown in tissue culture media containing radioactive methionine. This causes the viral antigens to be radioactive. A lysate is prepared from the cultured cells and incubated with serum. If antibodies are present in the serum they will bind to the radioactive viral antigens. These radioactive immune complexes are isolated onto sepharose beads coated with Staphylococcal protein-A. The beads are precipitated and tested for radioactivity, the presence of which indicates a positive test.

VIRAL LOAD TESTS. Tests for viral load measure the amount of virus in the blood either by quantifying nucleic acid or p24 antigen (p24 antigen capture assay). They may be used as confirmatory tests for HIV infection, but are more often used to determine the progression of HIV disease to AIDS and to determine the onset of drug resistance, both of which are signaled by an increase in the concentration of circulating viruses. The nucleic acid based tests for viral load include the reverse transcriptase-polymerase chain reaction (RT-PCR) test, branched DNA signal amplification method (bDNA), and the nucleic acid sequence-based amplification method (NASBA). DNA amplification methods can detect as little as 50 copies of viral RNA per mL of plasma and can detect infection during the "window phase," when anti-body levels are too low to produce a positive test result. In the RT-PCR assay, guanidinum isothocyanate is added to the patient's serum or plasma. The RNA is precipitated with isopropanol, and the RNA is resuspended and incubated in a medium containing reverse transcriptase, heat stable DNA polymerase (Taq polymerase), oligonuclotide primers tagged with biotin, and nucleotide triphosphates. The reverse transcriptase produces a double stranded DNA copy of the viral RNA. This DNA copy serves as the template for the polymerase chain reaction. Heat is used to separate the target DNA strand, a process known as denaturation. The temperature is lowered and the primers bind to the target sequence, a process called annealing. Heat stable DNA polymerase fills in the sequence by adding nucleotide triphosphates to the 3' end of the primer, a process called extention. This makes a new copy of the double stranded DNA. The cycle is repeated, making use of the newly synthesized DNA molecule as a template. If the process is repeated 30 times there will be over one billion copies of the target DNA. The amplified DNA, called amplicons, are denatured into single strands and are detected by means of an enzyme-conjugated DNA probe which hybridizes to the amplicons.

P24 ANTIGEN CAPTURE ASSAY. The p24 antigen capture assay is also used to measure viral load. Found in the viral core of HIV, p24 is a protein that can be measured by enzyme immunoassay. Generally, p24 is detected early in infection (before antibody production) but then falls to undetectable levels shortly after antibody production. The p24 assay is useful in detecting HIV infection before seroconversion, and for this reason it is used along with ELISA when testing donor blood for HIV. A return to detectable levels occurs when the virus becomes activated. Therefore, the test is used to identify patients who have become unresponsive to antiviral therapy and to indicate progression to AIDS. The test is not a useful screening test for HIV, since only about 20-30% of patients are positive in the early stages of HIV infection. Beads coated with monoclonal antibodies against p24 antigen are mixed with serum and incubated. After washing to remove unbound serum proteins, the beads are mixed with a second antibody to p24 derived from a rabbit. The beads are washed again, and an enzyme-conjugated anti-rabbit immunoglobulin is added. A final wash step is performed and substrate is added. The amount of color formed is proportional to the p24 antigen level of the serum.

BLOOD DONOR TESTING. Blood donated for transfusion is tested for HIV-1 and HIV-2 by ELISA, p24 antigen capture, and RT-PCR. For the latter, donor samples are pooled and tested for the presence of virus. This process detects the rare donors units that are negative for anti-HIV but potentially infective. This process has reduced the window phase from 22 days (ELISA alone) down to 11 days. It is estimated that the risk of receiving a transfusion of HIV positive blood in the United States is less than 1 in 562,500 when ELISA and p24 antigen testing are both used.

In 1999, the U.S. Food and Drug Administration (FDA) approved an HIV home testing kit. The kit contains multiple components, including material for specimen collection, a mailing envelope to send the specimen to a laboratory for analysis, and provides pre- and post-test counseling. It uses a finger prick process for blood collection. The results are obtained by the purchaser through a toll-free telephone number using a personal identification number (PIN). Post-test counseling is provided over the telephone by a licensed counselor. The only kit approved by the FDA as of 2001 was the Home Access test system.