AIDS TESTS

Definition

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 sometimes are 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.

Purpose

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 also can 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)
by transmission during pregnancy, or
through transfusion of infected blood products

A retrovirus is a virus that contains a unique enzyme called reverse transcriptase that allows it to replicate within new host cells. The virus binds to a protein called CD4, which is found on the surface of certain subtypes of white blood cells, including helper T cells, macrophages, and monocytes. Once HIV enters the cell, it can replicate and kill the cell in ways that are still not completely understood. In addition to killing some lymphocytes directly, the AIDS virus disrupts the functioning of the remaining CD4 cells. CD4 cells ordinarily 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, T cells do not reproduce as they normally would in response to the HIV virus, and B cells are not stimulated to respond to the infection.

Precautions

In some states such as New York, a signed consent form is needed in order to administer an AIDS test. As with all blood tests, healthcare professionals should always wear latex gloves and avoid being pricked by the needle used in drawing blood for the tests. It may be difficult to get blood from a habitual intravenous drug user due to collapsed veins.

Description

Diagnostic Tests

Diagnostic blood tests for AIDS usually are given to persons in high-risk populations who may have been exposed to HIV or who have the early symptoms of AIDS. Most persons infected with HIV will develop a detectable level of antibody within three months of infection. The condition of testing positive for HIV antibody in the blood is called seroconversion, and persons who have become HIV-positive are called seroconverters.

It is possible to diagnose HIV infection by isolating the virus itself from a blood sample or by demonstrating the presence of HIV antigen in the blood. Viral culture, however, is expensive, not widely available, and slow-it takes 28 days to complete the viral culture test. More common are blood tests that work by detecting the presence of antibodies to the HIV virus. These tests are inexpensive, widely available, and accurate in detecting 99.9% of AIDS infections when used in combination to screen patients and confirm diagnoses.

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

This type of blood test is used to screen blood for transfusions as well as diagnose patients. An ELISA test for HIV works by attaching HIV antigens to a plastic well or beads. 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 addition of a substance that will cause the enzyme to react by forming a color. An instrument called a spectrophotometer can measure the color. The name of the test is derived from the use of the enzyme that is coupled or linked to the second antibody.

The latest generations 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 test.

WESTERN BLOT (IMMUNOBLOT)

The Western blot or immunoblot test is used as a reference procedure to confirm the diagnosis of AIDS. In Western blot testing, HIV antigen is purified by electrophoresis (large protein molecules are suspended in a gel and separated from one another by running an electric current through the gel). The HIV antigens are 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. A commercially produced Western blot test for HIV-1 is now available. It consists of a prefabricated strip that is incubated with a sample of the patient's blood serum and the developing chemicals. About nine different HIV-1 proteins can be detected in the blots.

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 Western blotting. An IFA test detects the presence of HIV antibody in a sample of the patient's serum by mixing HIV antigen with a fluorescent chemical, adding the blood sample, and observing the reaction under a microscope with ultraviolet light.

POLYMERASE CHAIN REACTION (PCR)

This test is used to evaluate the very small number of AIDS patients with false-negative ELISA and Western blot tests. These patients are sometimes called antibody-negative asymptomatic (without symptoms) carriers, because they do not have any symptoms of AIDS and there is no detectable quantity of antibody in the blood serum. Antibody-negative asymptomatic carriers may be responsible for the very low ongoing risk of HIV infection transmitted by blood transfusions. It is estimated that the risk is between 1 in 10,000 and 1 in 100,000 units of transfused blood.

The polymerase chain reaction (PCR) test can measure the presence of viral nucleic acids in the patient's blood even when there is no detectable antibody to HIV. This test works by amplifying the presence of HIV nucleic acids in a blood sample. 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. It is questionable whether PCR will replace Western blotting as the method of confirming AIDS diagnoses. Although PCR can detect the low number of persons (1%) with HIV infections that have not yet generated an antibody response to the virus, the overwhelming majority of infected persons will be detected by ELISA screening within one to three months of infection. In addition, PCR testing is based on present knowledge of the genetic sequences in HIV. Since the virus is continually generating new variants, PCR testing could yield a false negative in patients with these new variants.

In 1999, the U.S. Food and Drug Administration (FDA) approved an HIV home testing kit. The kit contained 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. Other tests have been in development that would allow patients to monitor their own therapy in the home without sending out for results.

Prognostic Tests

Blood tests to evaluate patients already diagnosed with HIV infection are as important as the diagnostic tests. Because AIDS has a long latency period, some persons may be infected with the virus for 10 years or longer before they develop symptoms of AIDS. These patients are sometimes called antibody-positive asymptomatic carriers. Prognostic tests also help drug researchers evaluate the usefulness of new medications in treating AIDS.

BLOOD CELL COUNTS

Doctors can measure the number or proportion of certain types of cells in an AIDS patient's blood to see whether and how rapidly the disease is progressing, or whether certain treatments are helping the patient. These cell count tests include:

Complete blood count (CBC). A CBC is a 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 (WBC), a red blood cell count (RBC), 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). Although CBCs are used on more than just AIDS patients, they can help the doctor determine if an AIDS patient has an advanced form of the disease. Specific AIDS-related signs in a CBC include a low hematocrit, a sharp decrease in the number of blood platelets, and a low level of a certain type of white blood cell called neutrophils.
Absolute CD4+ lymphocytes. A lymphocyte is a type of white blood cell that is importantin the formation of an immune response. Because HIV targets CD4+ lymphocytes, their number inthe patient's blood can be used to track the course of the infection. This blood cell count is considered the most accurate indicator for the presence of an opportunistic infection in an AIDS patient. The absolute CD4+ lymphocyte count is obtained by multiplying the patient's white blood cell count (WBC) by the percentage of lymphocytes among the white blood cells, and multiplying the result by the percentage of lymphocytes bearing the CD4+ marker. An absolute count below200-300 CD+4 lymphocytes in 1 cubic millimeter (mm³) of blood indicate that the patient is vulnerable to some opportunistic infections.
CD4+ lymphocyte percentage. Some doctors think that this is a more accurate test than the absolute count because the percentage does not depend on a manual calculation of the number of types of different white blood cells. A white blood cell count that is broken down into categories in this way is called a WBC differential.

It is important for doctors treating AIDS patients to measure the lymphocyte count on a regular basis. Experts consulted by the United States Public Health Service recommend the following frequency of serum testing based on the patient's CD4+ level:

CD4+ count more than 600 cells/mm³: Every six months.
CD4+ count between 200-600 cells/mm³: Every three months.
CD4+ count less than 200 cells/mm³: Every three months.

When the CD4+ count falls below 200 cells/mm³, the doctor will put the patient on a medication regimen to protect him or her against opportunistic infections.

HIV VIRAL LOAD TESTS

Another type of blood test for monitoring AIDS patients is the viral load test. It supplements the CD4+ count, which can tell the doctor the extent of the patient's loss of immune function, but not the speed of HIV replication in the body. The viral load test is based on PCR techniques and can measure the number of copies of HIV nucleic acids. Successive test results for a given patient's viral load are calculated on a base 10 logarithmic scale.

ORAL HIV TESTS

Scientists have developed oral HIV tests that can be conducted with saliva samples. However, one of the unintended effects of these tests is the misperception that HIV can be transmitted through saliva.

RAPID HIV TESTS

Researchers constantly work on more rapid tests for HIV that can be done in physician offices or by less skilled people and more convenient locations in developing countries. A finger-stick test that can be read quickly from a whole blood sample had shown promising results in the fall of 2003. Another test, called the VScan test kit, requires no refrigeration or electricity and can safely be stored at room temperature. Even if the positive results must be confirmed by ELISA or Western blotting, an accurate initial rapid test can help screen populations for HIV antibodies.

BETA₂-MICROGLOBULIN (BETA₂M)

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 ₂M are found in patients with cancer and other serious diseases, a rising ₂M blood level can be used to measure the progression of AIDS.

P24 ANTIGEN CAPTURE ASSAY

Found in the viral core of HIV, p24 is a protein that can be measured by the ELISA technique. Doctors can use p24 assays to measure the antiviral activity of the patient's medications. In addition, the p24 assay is sometimes useful in detecting HIV infection before seroconversion. However, p24 is consistently present in only 25% of persons infected with HIV.

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 certain drugs used in HIV treatment. 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 on the reverse transcriptase protein-amino acid 184-known as M184V (MV, methionine to valine). So if the genotypic resistance test shows a mutation at position M184V, it is likely the person is resistant to 3TC and not likely to respond to 3TC treatment. Genotypic tests are only effective if the person is already taking antiviral medication and if the viral load is greater than 1,000 copies per milliliter (mL) of blood. The cost of the test, usually between $300 and $500, is usually now covered by many insurance plans.

PHENOTYPIC DRUG RESISTANCE TESTING

Phenotypic testing directly measures the sensitivity of a patient's HIV to particular drugs and drug combinations. To do this, it measures the concentration of a drug required to inhibit viral replication in the test tube. 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. Also, 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 now covered by many insurance plans.

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.

Preparation

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.

Aftercare

If the test results indicate that the patient is HIV-positive, he or she will need counseling, information, referral for treatment, and support. Doctors can either counsel the patient themselves or invite an experienced HIV counselor to discuss the results of the blood tests with the patient. They also will assess the patient's emotional and psychological status, including the possibility of violent behavior and the availability of a support network.

Risks

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

Normal results for ELISA, Western blot, IFA, and PCR testing are negative for HIV antibody.

Normal results for blood cell counts:

WBC differential: Total lymphocytes 24-44% of the white blood cells.
Hematocrit: 40-54% in men; 37-47% in women.
T cell lymphocytes: 644-2200/mm³, 60-88% of alllymphocytes.
B cell lymphocytes: 82-392/mm³, 3-20% of all lymphocytes.
CD4+ lymphocytes: 500-1200/mm³, 34-67% of alllymphocytes.

Abnormal Results

The following results in AIDS tests indicate progression of the disease:

Percentage of CD4+ lymphocytes: less than 20% of all lymphocytes.
CD4+ lymphocyte count: less than 200 cells/mm³.
Viral load test: Levels more than 5000 copies/mL.
-2-microglobulin: Levels more than 3.5 mg/dL.
P24 antigen: Measurable amounts in blood serum.

Key Terms

Antibody

A 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.

Antigen

Any substance that stimulates the body to produce antibodies.

B cell

A 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

A 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)

A 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). CBCs are a routine blood test used for many medical reasons, not only for AIDS patients. They can help the doctor determine if a patient is in advanced stages of the disease.

Electrophoresis

A method of separating complex protein molecules suspended in a gel by running an electric current through the gel.

Enzyme-linked Immunosorbent Assay (ELISA)

A 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)

A 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 also may have a longer latency period.

Immunofluorescent Assay (IFA)

A 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

A 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

A 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

A 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 Infection

An infection that develops only when a person's immune system is weakened, as happens to AIDS patients.

Polymerase Chain Reaction (PCR)

A 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

A virus that contains a unique enzyme called reverse transcriptase that allows it to replicate within new host cells.

Seroconversion

The change from HIV- negative to HIV-positive status during blood testing. Persons who are HIV-positive are called seroconverters.

Serology

The analysis of the contents and properties of blood serum.

Serum

The part of human blood that remains liquid when blood cells form a clot. Human blood serum is clear light yellow in color.

T cells

Lymphocytes 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

A 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

A technique developed in 1979 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

A 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.

For More Information: Please ask your attending physician on your next visit.

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