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Back to Infections Lab Tests

HIV test

Several tests exist to detect the presence of HIV, the virus that causes AIDS. They are used for different purposes:

  • to test whether an individual is infected with HIV
  • to screen donated blood or organs
  • to measure the amount of free virus in a patient's blood in order to monitor the progress of the disease under Wilkinson antiretroviral drug therapy

Some terminology:

the window of a test is the time period after initial infection during which the test cannot yet detect the presence of HIV. The shorter the better.

the sensitivity of a test is the percentage of HIV positive cases that are correctly identified as positive by the test.

the specificity of a test is the percentage of HIV negative cases that are correctly identified as negative by the test. The higher the better.

Antibody tests

The body produces antibodies when infected with HIV, and the antibody tests detect the presence of these rather than the presence of HIV itself.

In 1996, the CDC described the case of an HIV-infected man who persistently tested negative on the antibody tests [1]. This is considered to be extremely rare.

The ELISA test

The ELISA test is the first screening test commonly employed; it has a high sensitivity but not a very high specificity. The test proceeds by the general ELISA method: the patients 400-fold diluted serum is poured over a plate to which known HIV particles were attached. The HIV-antibodies in the serum (if present) will bind to the HIV particles. The rest of the serum is washed away. Then a specially prepared new antibody, which attaches to antibody and to which an enzyme was bound, is poured over the plate. The new antibody which did not attach to any antibody is washed away. The enzyme action causes a color change. This allows to detect whether any of the new antibody remained, which is only possible if the patient's serum contained HIV-antibodies.

The low specificity of the test comes from the fact that sometimes there are cross-reacting antibodies: these are antibodies which attach to HIV particles "by accident", even though the body has never encountered HIV before. If an ELISA test is positive, the result is commonly confirmed with a Western-Blot test.

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The Western-Blot test

The Western-Blot test uses the general Western Blot procedure. HIV-infected cells are opened and the contained proteins are entered into a slab of gel to which a voltage is applied. Different proteins will move with different velocities in this field, depending on their size and electrical charge. Once the proteins are well separated, they are transferred to another plate and the procedure continues similar to ELISA: the patient's diluted serum is poured over the plate, HIV-antibodies bind to certain proteins on the plate, others are washed away, and enzyme-linked antibodies detect the presence of bound antibody. Since the proteins were separated, it is possible to see exactly to which HIV proteins the patients has antibodies (which the regular ELISA test cannot accomplish). The test is considered positive if antibodies to several major HIV proteins are present.

The combination of ELISA and Western Blot is the standard test employed for diagnosing HIV infection. All blood and organ donations are also screened with these tests. The window for the newer versions of these tests is stated in the literature to be on average 22 days. The patients gets the results usually 2 days to 2 weeks after the blood withdrawal.

OraQuick

OraQuick is another antibody-based test; it needs only one drop of blood from the finger and gives results in 20 minutes. Here the blood is mixed with a solution and then a measuring strip is inserted; the solution slowly travels upwards on the strip until it reaches a region where HIV proteins are attached. If the blood contains HIV antibodies, they will bind here, later causing a color reaction.

Orasure

Orasure is an HIV test which uses mucosal trasudate from the tissues of cheeks and gums. It is an antibody test which first employs ELISA, then Western Blot.

There is also a urine test; it employs both the ELISA and the Western Blot method.

Home Access Express HIV-1 Test

Home Access Express HIV-1 Test is an approved home test: the patient collects a drop of blood and mails the sample to a laboratory; the results are obtained over the phone.

There are also several unapproved immediate home-test products on the U.S. market.

Antigen tests

Antigen tests directly detect the presence of a part of the virus (an antigen), by applying specific antibody.

p24 Antigen test

The p24 antigen test detects the presence of the p24 protein of HIV (also known as CA), a major core protein of the virus. It uses a variant of the ELISA strategy: a well is coated with monoclonal antibodies specific to the p24 protein. The patient's blood is poured over the plate, and p24 protein will stick to the antibody; the rest is washed away. Then enzyme-linked antibody to p24 is applied to the plate; the enzyme action then causes a color change if p24 was present in the sample.

This test is now used routinely to screen blood donations, thus reducing the window to about 16 days. It is not useful for general diagnostics, as it has very low sensitivity and only works during a certain time period after infection; once the body produces antibodies against HIV, the test becomes useless.

A variant of the p24 test first applies heat to denature the p24 protein and thus separate it from its antibody. This test is used to monitor the viral load and disease progression, as a cheaper alternative to the nucleic acid based methods described next. Nucleic acid based tests
In the Amplicor RT-PCR test, the viral RNA is extracted from the patient's plasma and is treated with reverse transcriptase so that the RNA of the virus is transcribed into DNA. The polymerase chain reaction (PCR) is applied, using two primers thought to be unique to the virus's genome. After the PCR amplification process is completed, which takes some time, the resulting amplified segments bind to specific oligonucleotides bound to the vessel wall and are then made visible with a probe that's bound to an enzyme. The amount of virus in the sample can be quantified.

Branched DNA plasma test

In the Quantiplex bDNA or branched DNA test plasma is centrifugated to concentrate the viruses, which are then opened to release the RNA. Special oligonucleotides are added which bind to viral RNA and to certain oligonucleotides bound to the wall of the vessel. In this way, viral RNA is fastened to the wall. Then new oligonucleotides are added which bind at several locations to this RNA; and other oligonucelotides which bind at several locations to those oligonucleotides. This is done to amplify the signal. Finally, oligonucleotides that bind to the last set of oligonucleotides and that are bound to an enzyme are added; the enzyme action causes a color reaction which allows to quantify the viral RNA in the original sample. Currently, version 3.0 of the Quantiplex is in use; it is claimed to detect viral loads as low as 50 per millilitre.

Nucleic acid based tests are now routinely used to determine the viral load, the amount of free virus in the blood. This is an important variable when monitoring a drug therapy.

In the U.S. donated blood is also screened with nucleic acid based tests, shortening the window to about 12 days. Since these tests are relatively expensive, the blood is screened by first pooling some 10-20 samples, testing these together, and if the pool tests positive, each sample is retested individually.

Other

The CD4 T-cell count is a procedure where the number of CD4 T-cells in one microlitre of blood are counted in a standard medical lab test after a blood draw.

This test does not check for the presence of HIV. Instead, it is used to estimate viral activity and immune system function in HIV+ people. This test is also used occasionally to estimate immune system function for people whose CD4 T cells are impaired for reasons other than HIV infection, which include several blood diseases, several genetic disorders, and the side effects of many chemotherapy drugs.

Generally speaking, the lower the number of T cells, the lower the immune system's function will be. Normal T4 counts are about 1000 CD4+ T cells per microliter, although the counts may fluctuate by 10% or more even in healthy people, depending on recent infection status, nutrition, exercise and other factors -- even the time of day. Women tend to have somewhat lower counts than men.

Symptoms of T4 cell immune collapse are almost never seen until the number drops below 500. In HIV+ people, AIDS is officially diagnosed when the count drops below 200 cells.

Similar symptoms of immune collapse are generally seen in people with very low T4 cell counts, no matter whether this immunosuppression is caused by HIV or by cancer or by any other disease. However, the long-term treatment differs substantially, because it needs to address the cause of the immunosuppression.

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