HIV test
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HIV test kits used both to screen donor blood, blood components and cellular products, and to diagnose, treat and monitor persons with HIV and AIDS are regulated in the United States by the FDA.
HIV tests to detect antibodies, antigens or RNA in serum, plasma, oral fluid, dried blood spot or urine have been approved by FDA (http://www.fda.gov/cber/products/testkits.htm) for donor screening, diagnosis, prognosis and patient monitoring.
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Terminology
The window period is the time from infection until a test can detect any change. The average window period with antibody tests is 22 days. Antigen testing cuts the window period to approximately 16 days and NAT further reduces this period to 12 days. FDA 2001 (http://www.fda.gov/bbs/topics/ANSWERS/2001/ANS01103.html)
Antibody tests are reported as positive or negative. Performance of these tests is described in terms of:
- sensitivity the percentage of the results that will be positive when HIV is present
- specificity the percentage of the results that will be negative when HIV not present.
All diagnostic tests have limitations, and sometimes their use may produce erroneous or questionable results.
- false positive results indicate that HIV is present when, in fact, it is not.
- false negative results do not identify HIV that is present.
Nonspecific reactions, hypergammaglobulinemia, or the presence of antibodies directed to other infectious agents that may be antigenically similar to HIV can produce false positive results. Autoimmune diseases, such as systemic lupus erythematosus, can also cause false positive results. Indeed, the scientific literature has documented more than 60-70 different factors other than past or present infection with HIV that can cause a positive reaction on HIV tests - including flu, flu vaccination, herpes simplex, rheumatoid arthritis, malaria, tuberculosis, antibodies to leprosy - and even pregnancy. A list of the factors known to cause false-positive results, together with references to the medical literature for each factor, is available at http://www.aliveandwell.org/html/questioning/66reas.html.
Principles
Screening donor blood and cellular products
Tests selected to screen donor blood, blood components and cellular products provide a high degree of confidence that HIV is not present. A combination of antibody, antigen and nucleic acid tests are used by blood banks in Western countries. The WHO estimated that in 2000 inadequate blood screening in some countries resulted in 1 million new HIV infections.
Diagnosis of HIV infection
Different tests are selected for the diagnosis of HIV infection in a particular person, which requires a high degree of confidence that HIV is present. In the United States, this is achieved by an algorithm using two tests to detect antibodies, which are the body’s response to infection. If antibodies are detected by an initial test based on the ELISA method, then a second test based on the Western blot procedure determines the size of the antigens in the test kit binding to the antibodies. This testing algorithm is claimed to have an accuracy of 99.5%, which means that testing 100,000 people not infected with HIV would be expected to produce 500 false positive results. The predictive value of this testing algorithm depends on the prevalence of HIV infection in the group being tested. If 10% of the group being tested is infected with HIV, less than 2% of the positive results will be false positive. But if less that 1 in 7,500 people in the group being test are infected with HIV, then over 90% of the positive results will be false positive.
Human Rights
The UNAIDS/WHO policy statement on HIV Testing (http://www.unaids.org/html/pub/una-docs/hivtestingpolicy_en_pdf.htm) states that conditions under which people undergo HIV testing must be anchored in a human rights approach, which pays due respect for ethical principles. According to these principles, the conduct of HIV testing of individuals must be:
- confidential
- accompanied by counselling
- conducted with informed consent, meaning both informed and voluntary
The reality is that stigma and discrimination continue to stop people, who may have been exposed to HIV infection, from consenting to HIV testing.
HIV testing and counselling has a pivotal role in preventing HIV infection around the world.
Antibody tests
Antibody tests are specifically designed for the routine testing of HIV in adults, are inexpensive, and are very accurate. If a person does not have not a realistic risk of infection, then these tests are not necessary.
Antibody tests give false negative results during the window period of between three weeks and six months from the time of HIV infection until the immune system produces detectable amounts of antibodies. During this window period an infected person can transmit HIV to others, without their HIV infection being detectable using an antibody test. Antiretroviral therapy during the window period can delay the formation of antibodies and extend the window period beyond 12 months. Template:Conference reference
A person exposed to HIV would be expected to produce antibodies that specifically bind to HIV. The development of antibody tests for HIV has been complicated because all human blood has large quantities of antibodies that bind to components of the antibody test kits. For this reason, the blood to be tested is diluted in an attempt to detect antibodies that have been produced after exposure to HIV. Manufacturers of all these test kits state that, "There is no single recognised standard for establishing the presence or absence of antibodies to HIV in human blood."
ELISA
The ELISA test was the first screening test commonly employed. It has a high sensitivity. The low specificity of the test is because antibodies attach to antigens in the test kits "by accident", even though the person has never been exposed to HIV. About 80% of positive ELISA tests are followed by a negative Western-Blot test, and therefore regarded as false positive.
The test proceeds by the general ELISA method: the person's serum is diluted 400 fold and applied to a plate to which HIV antigens have been attached. Some of the antibodies in the serum may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. Then a specially prepared "secondary antibody"—an antibody that binds to human antibodies—is applied to the plate, followed by washes. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. As the ELISA results are reported as a number, the most controversial aspect of this test is deciding the "cut off" point between positive and negative.
Western blot
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, while their electrical charge is levelled by a substance, called sodium lauryl sulfate. Once the proteins are well separated, they are transferred to a membrane and the procedure continues similar to ELISA: the person's diluted serum is applied to the membrane and antibodies in the serum may attach to some of the HIV proteins. Antibodies which do not attach are washed away, and enzyme-linked antibodies with the capability to attach to the person's antibodies first detect to which HIV proteins the person has antibodies.
There no universal criteria for interpreting the Western blot test: the number of viral bands which must be present vary from definition to definition. If no viral bands are detected, the result is negative. If at least one viral band for each of the GAG, POL, and ENV gene-product groups are present, the result is positive. Such a criteria, however, is not sensitive enough for clinical use, as the absence of antibodies to p24 or to p31 is relatively common even in patients who are clearly infected with HIV. Thus the three-gene-product approach to Western blot interpretation has not been adopted for public health or clinical practice. The Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and CDC standard ignores p31 results, and interprets the presence of gp41 and gp120/160 bands as a positive result. Tests in which less than the required number of viral bands are detected are reported as indeterminate: a person who has an indeterminate result should be retested, as later tests may be more conclusive. Almost all HIV-infected persons with indeterminate Western-Blot results will develop a positive result when tested in one month; persistently indeterminate results over a period of six months suggests the results are not due to HIV infection.
The Western blot test is usually performed after a positive ELISA test, because many samples that are ELISA negative have antibodies to one or more of the proteins in the Western blot test, and would give inaccurate results, especially when interpreted according to criteria which require a complete absence of all viral and non-viral bands to report a Western-Blot result as negative rather than indeterminate.
Rapid or point-of-care tests
Rapid Antibody Tests are qualitative immunoassays intended for use as a point-of-care test to aid in the diagnosis of HIV infection. These tests should be used in conjunction with the clinical status, history, and risk factors of the person being tested. The specificity of Rapid Antibody Tests for in low-risk populations has not been evaluated. These tests should be used in appropriate multi-test algorithms designed for statistical validation of rapid HIV test results.
If no antibodies to HIV are detected, this does not mean the person has not been infected with HIV. It may take several months after HIV infection for the antibody response to reach detectable levels, during which time rapid testing for antibodies to HIV will not be indicative of true infection status. A comprehensive risk history and clinical judgement should be considered before concluding that an individual is not infected with HIV.
OraQuick is an antibody test that provides results in 20 minutes. The blood, plasma or oral fluid is mixed in a vial with developing solution, and the results are read from a sticklike testing device.
Orasure is an HIV test which uses mucosal transudate 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 is a FDA-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 have been a number of cases of fraudulent tests being sold via mail order or the Internet to the general public. In 1997, a California man was indicted on mail fraud and wire charges for selling supposed home test kits. In 2004, the US Federal Trade Commission asked Federal Express and US Customs to confiscate shipments of the Discreet home HIV test kits, produced by Gregory Stephen Wong of Vancouver, BC. In February 2005, the US FDA issued a warning against using the rapid HIV test kits and other home use kits marketed by Globus Media of Montreal Canada.
Interpreting antibody tests
ELISA testing alone cannot be used to diagnose AIDS, even if the recommended investigation of reactive specimens suggests a high probability that the antibody to HIV 1 is present. In the United States, such positive tests are not reported out unless confirmed by a Western Blot test.
The ELISA antibody tests were developed to provide a high level of confidence that donated blood was NOT infected with HIV. It is therefore not possible to conclude that blood rejected for transfusion because of a positive ELISA antibody test is in fact infected with HIV. Usually, retesting the donor in several months will produce a negative ELISA antibody test.
All laboratory tests can produce 'false alarms'. This is a positive result in the screening test, which, on confirmatory testing is shown to be negative. False reactions are a recognised complication of all biological tests and are perfectly normal. They are of no significance for the health of the donor.
It is known that false positive results due to factors unrelated to exposure to the HIV virus are common with the ELISA test. False positives can be caused by antibodies to viruses other than HIV, antibodies produced by pregnancy, and other medical conditions. A false positive results DOES NOT indicate that you are infected with the AIDS virus, nor does it indicate a condition of significant risk to your health.
The specificity of viral markers depends on the success of virus isolation and purification. Without fully demonstrated success in virus isolation and purification, identification of viral markers is extremely hazardous and can lead to severe misinterpretation of clinical data. A dramatic illustration of this is to be found in current HIV research. In this case, the virus (HIV) has never been properly isolated, since sedimentation in sucrose gradient at the density of 1.16 g/mL was erroneously considered to yield pure virus, systematically ignoring that material sedimenting at that density contains large amounts of cell debris and microvesicles. Therefore, proteins and nucleic acids found in such 1.16 bands are very likely to be of cellular origin and cannot be used as viral markers. Such a faulty methodology has had extremely serious consequences, i.e. the world-wide use of HIV-antibody tests, Elisa and Western Blot, which dangerously lack specificity, as demonstrated in 1993 by Papadopulos et al (http://www.theperthgroup.com/SCIPAPERS/biotek8.html). Template:Journal reference
Antigen tests
The p24 antigen test detects the presence of the p24 protein of HIV (also known as CA), a major core protein of the virus. Monoclonal antibodies specific to the p24 protein are mixed with the person's blood. Any p24 protein in the person's blood will stick to the monoclonal antibody and enzyme-linked antibody to the monoclonal antibodies to p24 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 before the body produces antibodies to the p24 protein.
Nucleic acid based tests
Nucleic acid based tests amplify and detect a 142 base target sequence located in a highly conserved region of the HIV gag gene. Since 2001, donated blood in the United States, has been 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. A different version of this test is intended for use in conjunction with clinical presentation and other laboratory markers of disease progress for the clinical management of HIV-1 infected patients.
In the 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 with sufficient accuracy to detect three fold changes.
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. Monitoring the effects of antiretroviral therapy by serial measurements of plasms HIV-1 RNA with this test has been validated for patients with viral loads greater than 25,000 copies per millilitre. reference (http://www.fda.gov/cber/PMAsumm/P0000280S.pdf)
Other tests used in HIV/AIDS treatment
The CD4 T-cell count is not an HIV test, but rather 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. It is used monitor the immune system function in HIV+ people. Declining CD4 T-cell counts are considered to be a marker of the progression of HIV infection. In HIV+ people, AIDS is officially diagnosed when the count drops below 200 cells or when certain opportunistic infections occur. This use of a CD4 count as an AIDS criterion occurred in 1992; the value of 200 was chosen because it corresponded with an increased likelihood of opportunistic infections. Lower levels of CD4 counts in people with AIDS are indicators that prophylaxis against certain types of opportunistic infections should be instituted.
Low CD4 T-cell counts are associated with a variety of conditions, including many viral infections, bacterial infections, parasitic infections, sepsis, tuberculosis, coccidioidomycosis, burns, trauma, intravenous injections of foreign proteins, malnutrition, over-exercising, pregnancy, normal daily variation, psychological stress, and social isolation
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 between 500 and 1500 CD4+ T cells per microliter and the counts may fluctuate 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 200. Similar symptoms of immune collapse are generally seen in people with very low T4 cell counts, whether this immunosuppression is caused by HIV, cancer, or some other disease. However, the long-term treatment differs substantially, because it needs to address the cause of the immunosuppression.
See also
External links
- UNAIDS issues HIV testing policy (http://www.unaids.org/html/pub/una-docs/hivtestingpolicy_en_pdf.htm) June 2004
- HIV Testing August 5, 2004 (http://www.thebody.com/nmai/testing.html)
- HIV Antibody Assays September 2001 (http://hivinsite.ucsf.edu/InSite?page=kb-02-02-01)
- http://www.hivtest.org/ - CDC HIV test resources
- US FDA list of approved HIV tests (http://www.fda.gov/cber/products/testkits.htm)