AIDS Tests (Encyclopedia of Medicine)
AIDS tests, short for acquired immunodeficiency syndrome tests, cover a number of different procedures used in the diagnosis and treatment of HIV patients. These tests are sometimes called AIDS serology tests. Serology is the branch of immunology that deals with the contents and characteristics of blood serum. Serum is the clear light yellow part of blood that remains liquid when blood cells form a clot. AIDS serology evaluates the presence of human immunodeficiency virus (HIV) infection in blood serum and its effects on each patient's immune system.
AIDS serology serves several different purposes. Some AIDS tests are used to diagnose patients or confirm a diagnosis; others are used to measure the progression of the disease or the effectiveness of specific treatment regimens. Some AIDS tests can also be used to screen blood donations for safe use in transfusions.
In order to understand the different purposes of the blood tests used with AIDS patients, it is helpful to understand how HIV infection affects human blood and the immune system. HIV is a retrovirus that enters the blood stream of a new host in the following ways:
- by sexual contact
- by contact with infected body fluids (such as blood and urine)
(The entire section is 4131 words.)
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AIDS Tests (Encyclopedia of Nursing & Allied Health)
AIDS tests, short for acquired immunodeficiency syndrome tests, cover a number of different procedures used in the diagnosis and treatment of HIV-infected patients. Tests that measure antibodies to the human immunodeficiency virus (HIV) are called AIDS serology tests. Serology is the branch of immunology that deals with the identification and measurement of antibodies in serum which indicate the presence of disease or immunity to disease. Serum is the normally clear light yellow noncellular portion of blood that forms after the sample is allowed to clot. Some AIDS tests measure HIV antigens or nucleic acid rather than antibodies produced in response to HIV infection. AIDS tests evaluate the presence of HIV in blood serum, and the effects of HIV infection on the patient's immune system.
AIDS serology tests have several uses. Some AIDS tests are used to diagnose patients or confirm a diagnosis; others are used to measure the progression of the disease or the effectiveness of specific treatment regimens. Some AIDS tests can also be used to screen blood donations for safe use in transfusions.
In order to understand the different purposes of the blood tests used for AIDS patients, it is helpful to understand how HIV infection affects the immune system. HIV is a retrovirus that enters the blood stream of a new host in the following ways:
- sexual contact, including oral and anal intercourse
- entry of HIV infected body fluids (such as blood or urine) through a cut or break in the skin
- transmission during pregnancy
- using or being pricked by a needle that had previously been used by or on an infected person
- transfusion of infected blood products
A retrovirus is a virus that contains two identical strands of RNA and a unique enzyme called reverse transcriptase that converts the viral RNA to DNA within the host cell. Another viral enzyme called integrase inserts this proviral DNA into the host cell genome. Other viral proteins control the process of transcription which forms RNA copies of the inserted DNA, production of structural viral proteins, assembly of immature virus particles, maturation, and release (budding) from the host cell. The entire process takes 12-24 hours.
The primary host cell for HIV is the T helper cell. The HIV envelope contains a glycoprotein called gp120, which binds to a surface molecule on the T helper cell called CD4. Other types of lymphocytes which lack the CD4 molecule are not infected. In addition to T helper cells, HIV can infect phagocytic cells (macrophages, monocytes, and dendritic cells) which serve as reservoirs and spread the virus throughout the body. The virus can also infect certain tissue cells such as neurons, which in part accounts for some of the underlying pathology of HIV disease.
HIV disease begins as a flu-like illness two to six weeks after infection which subsides without treatment. Antibodies to the viral envelope appear eight to 12 weeks after infection. Most patients enter a phase of clinical latency, which on average lasts eight to 10 years. This is followed by gradual loss of CD4 positive lymphocytes (T helper cells). T helper cells produce a substance called interleukin-2 (IL-2), which stimulates other cells (T cells and B cells) in the human immune system to respond to infections. Without the IL-2, the immune response collapses and patients become susceptible to a wide range of infections. Depletion of T helper cells signals the onset of opportunistic infections, malignancy, dementia, and a constellation of other diseases associated with AIDS.
There are no medical restrictions on administering AIDS tests. Most tests are performed on blood, but a screening test using urine is available. Health care professionals should always follow standard precautions recommended by the Centers for Disease Control (CDC) to reduce the possibility of accidental needlestick injury or exposure to the patient's blood and body fluids. This includes wearing latex gloves, washing hands before and following venipuncture, and using disposable needles and safety devices.
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.
In addition to tests for viral antigens and antibodies, other blood tests are needed to evaluate and manage patients with HIV disease. The most important of these is the CD4 positive lymphocyte count. This test measures the number of T helper cells in the blood. A CD4 count of less than 200/microL or 14% of the total lymphocyte count in a person who is HIV positive constitutes a diagnosis of AIDS. A falling CD4 positive lymphocyte count parallels a rise in viral replication and correlates with both a risk of opportunistic infection and drug resistance in patients receiving highly active antiviral therapy (HAART).
It is important for doctors treating AIDS patients to measure the CD4 positive lymphocyte count on a regular basis. Experts consulted by the U.S. Public Health Service recommend the following frequency of serum testing based on the patient's CD4+ level:
- CD4+ count more than 600 cells/microL: Every six months.
- CD4+ count between 200-600 cells/microL: Every three months.
- CD4+ count less than 200 cells/microL: Every three months.
When the CD4+ count falls below 200 cells/microL, the doctor will put the patient on a medication regimen to protect him or her against opportunistic infections.
BETA2-MICROGLOBULIN (b2M). Beta2-microglobulin is a protein found on the surface of all human cells with a nucleus. It is released into the blood when a cell dies.
Although rising blood levels of [.beta]2[.Mu] are found in patients with cancer and other serious diseases, a rising [.beta]2[.Mu] blood level can be used to measure the progression of AIDS.
GENOTYPIC DRUG RESISTANCE TEST. Genotypic testing can help determine whether specific gene mutations, common in people with HIV, are causing drug resistance and drug failure. The test looks for specific genetic mutations within the virus that are known to cause resistance to anti-viral drugs. For example the drug 3TC, also known as lamivudine (Epivir), is not effective against strains of HIV that have a mutation at a particular position, known as M184V, in their reverse transcriptase enzyme. If the genotypic resistance test shows a mutation at position M184V, it is likely that person is resistant to 3TC and not likely to respond to treatment. Genotypic tests are only useful when the patient is already taking antiviral medication, and the viral load is greater than 1,000 copies per mL of plasma. The cost of the viral drug resistance testing is usually between $300 and $500, and is usually not covered by insurance plans, including Medicare.
PHENOTYPIC DRUG RESISTANCE TESTING. Phenotypic testing directly measures the in vitro sensitivity of a patient's HIV strains to particular drugs and drug combinations. The test measures the concentration of a drug required to inhibit viral replication by 50% and 90%. This is the same method used by researchers to determine whether a drug might be effective against HIV before using it in human clinical trials. Phenotypic testing is a more direct measurement of resistance than genotypic testing. Unlike genotypic testing, phenotypic testing does not require a high viral load, but it is recommended that persons already be taking antiretroviral drugs. The cost is between $700 and $900 and is usually not covered by insurance plans, including Medicare.
AIDS serology in children
Children born to HIV-infected mothers may acquire the infection through the mother's placenta or during the birth process. Public health experts recommend the testing and monitoring of all children born to mothers with HIV. Diagnostic testing in children older than 18 months is similar to adult testing, with ELISA screening confirmed by Western blot. Younger infants can be diagnosed by direct culture of the HIV virus, PCR testing, and p24 antigen testing. These techniques allow a pediatrician to identify 50% of infected children at or near birth, and 95% of cases in infants three to six months of age.
In addition to diagnostic testing for HIV infection, many laboratory tests are important to the proper management and treatment of patients with HIV disease and AIDS. These include the complete blood count (CBC), cultures and serological tests for opportunistic infections, Pap smear for cervical cancer, imaging studies, nerve conduction studies, and tests for nutritional status.
Preparation and aftercare are important parts of AIDS diagnostic testing. Doctors are now advised to take the patient's emotional, social, economic, and other circumstances into account and to provide counseling before and after testing. Patients are generally better able to cope with the results if the doctor has spent some time with them before the blood test explaining the basic facts about HIV infection and testing. Many doctors now offer this type of informational counseling before performing the tests.
The risks of AIDS testing are primarily related to disclosure of the patient's HIV status rather than to any physical risks connected with blood testing. Some patients are better prepared to cope with a positive diagnosis than others, depending on their age, sex, health, resources, belief system, and similar factors.
Normal results for ELISA, Western blot, IFA, and PCR testing are negative for HIV antibody.
Normal results for CD4+ lymphocytes: 500-1200/microL, 34-67% of all lymphocytes.
The following abnormal results of AIDS tests indicate progression of the disease:
- Percentage of CD4 positive lymphocytes: less than 14% of all lymphocytes.
- CD4+ lymphocyte count: less than 200 cells/microL.
- Viral load test: Levels more than 5000 copies/mL.
Health care team roles
Nurses and phlebotomists (workers who draw blood) are usually the health care professional that draw the blood for AIDS tests. However, it is the physician that recommends specific treatment and prescribes needed medication. HIV tests are performed by clinical laboratory scientists, CLS (NCA) or medical technologists, MT (ASCP). It is often the role of a trained counselor to tell the patient the test results. Regardless of the results, nurses, health care educators, and counselors often are responsible for educating the patient about safe sex practices and risk factors for contracting HIV, along with the possible need for periodic repeat testing.
Antibody protein in the blood that identifies and helps remove disease organisms or their toxins. Antibodies are secreted by B cells. AIDS diagnostic tests work by demonstrating the presence of HIV antibody in the patient's blood.
Antigenny substance that stimulates the body to produce antibodies.
B cell type of white blood cell derived from bone marrow. B cells are sometimes called B lymphocytes. They secrete antibody and have a number of other complex functions within the human immune system.
CD4 type of protein molecule in human blood that is present on the surface of 65% of human T cells. CD4 is a receptor for the HIV virus. When the HIV virus infects cells with CD4 surface proteins, it depletes the number of T cells, B cells, natural killer cells, and monocytes in the patient's blood. Most of the damage to an AIDS patient's immune system is done by the virus' destruction of CD4+ lymphocytes. CD4 is sometimes called the T4 antigen.
Complete blood count (CBC) routine analysis performed on a sample of blood taken from the patient's vein with a needle and vacuum tube. The measurements taken in a CBC include a white blood cell count, a red blood cell count, the red cell distribution width, the hematocrit (ratio of the volume of the red blood cells to the blood volume), and the amount of hemoglobin (the blood protein that carries oxygen).
Electrophoresis method of separating complex protein molecules suspended in a gel by running an electric current through the gel.
Enzyme-linked immunosorbent assay (ELISA) diagnostic blood test used to screen patients for AIDS or other viruses. The patient's blood is mixed with antigen attached to a plastic tube or bead surface. A sample of the patient's blood serum is added, and excess proteins are removed. A second antibody coupled to an enzyme is added, followed by a chemical that will cause a color reaction that can be measured by a special instrument.
Human immunodeficiency virus (HIV) transmissible retrovirus that causes AIDS in humans. Two forms of HIV are now recognized: HIV-1, which causes most cases of AIDS in Europe, North and South America, and most parts of Africa; and HIV-2, which is chiefly found in West African patients. HIV-2, discovered in 1986, appears to be less virulent than HIV-1, but may also have a longer latency period.
Immunofluorescent assay (IFA) blood test sometimes used to confirm ELISA results instead of using the Western blotting. In an IFA test, HIV antigen is mixed with a fluorescent compound and then with a sample of the patient's blood. If HIV antibody is present, the mixture will fluoresce when examined under ultraviolet light.
Lymphocyte type of white blood cell that is important in the formation of antibodies. Doctors can monitor the health of AIDS patients by measuring the number or proportion of certain types of lymphocytes in the patient's blood.
Macrophage large white blood cell, found primarily in the bloodstream and connective tissue, that helps the body fight off infections by ingesting the disease organism. HIV can infect and kill macrophages.
Monocyte large white blood cell that is formed in the bone marrow and spleen. About 4% of the white blood cells in normal adults are monocytes.
Opportunistic infectionn infection that develops only when a person's immune system is weakened, as happens to AIDS patients.
Polymerase chain reaction (PCR) test performed to evaluate false-negative results to the ELISA and Western blot tests. In PCR testing, numerous copies of a gene are made by separating the two strands of DNA containing the gene segment, marking its location, using DNA polymerase to make a copy, and then continuously replicating the copies. The amplification of gene sequences that are associated with HIV allows for detection of the virus by this method.
Retrovirus virus that contains a unique enzyme called reverse transcriptase that allows it to replicate within new host cells.
Seroconversionhe change from HIV-negative to HIV-positive status during blood testing. Persons who are HIV-positive are called seroconverters.
Serologyhe analysis of the contents and properties of blood serum.
Serumhe part of human blood that remains liquid when blood cells form a clot. Human blood serum is clear light yellow in color.
T cellsymphocytes that originate in the thymus gland. T cells regulate the immune system's response to infections, including HIV. CD4 lymphocytes are a subset of T lymphocytes.
Viral load test new blood test for monitoring the speed of HIV replication in AIDS patients. The viral load test is based on PCR techniques and supplements the CD4+ cell count tests.
Western blot technique that is used to confirm ELISA results. HIV antigen is purified by electrophoresis and attached by blotting to a nylon or nitrocellulose filter. The patient's serum is reacted against the filter, followed by treatment with developing chemicals that allow HIV antibody to show up as a colored patch or blot. If the patient is HIV-positive, there will be stripes at specific locations for two or more viral proteins. A negative result is blank.
WBC differential white blood cell count in which the technician classifies the different white blood cells by type as well as calculating the number of each type. A WBC differential is necessary to calculate the absolute CD4+ lymphocyte count.
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Ken R. Wells