Over the years, tests to detect HIV/AIDS, and how far the disease has progressed, have been refined and become more accurate. Dr Sangeeta Joshi explains the tests and what they reveal

Human immunodeficiency viruses (HIV) cause a serious disorder of the immune system in which the normal immunity against infections breaks down, leaving the person more prone to a variety of infections and other conditions. Acquired immunodeficiency syndrome (AIDS) is the final stage of HIV infection. There are two types of HIV: 1 and 2. In India, HIV-1 is the predominant infection; however, HIV-2 infection and mixed infections with both HIV-1 and 2 are occasionally seen.

HIV-1 and 2 are retroviruses, that is, they contain RNA which is transcribed into DNA. Once the HIV enters the human body via infected blood, semen, vaginal secretions etc, the virus immediately targets cells which display the viral receptors - CD4.These receptors are seen on lymphocytes and other cells. The virus enters these cells, forms DNA, integrates into the host genome, multiplies and persists for many years.

After a period of 3-12 weeks, the host mounts an immune response against the virus which is detected as antibody in the blood. This stage is called seroconversion. Current routine laboratory diagnosis of HIV is mainly based on the detection of these specific anti-HIV antibodies. The period following the entry of HIV into the body and the appearance of detectable levels of antibodies with the available tests is called the ‘window period’. During the window period, the patient is highly infectious but the antibody test is negative. This is also a worrying time for patients who have had an exposure and then want to be tested for HIV.

Many persons with HIV do not get tested until late in their infection. Approximately 40-50% of patients with HIV infection are diagnosed to have AIDS within one year of first testing HIV positive. Testing for HIV is complex because of its implications. Since it is transmitted through the sexual route, and the infection remains for life, a number of social, moral, ethical and legal issues are related to a positive test result.

Before any person is tested he should be counselled and his consent needs to be taken. Once the HIV results are available, the person would have to be counselled again (post-test counselling). The confidentiality of the test results should be maintained.

Before moving on to the testing modalities, a word about sensitivity and specificity:

Sensitivity is the accuracy with which a test can confirm the presence of an infection. Tests with high sensitivity show few false negatives and are used as screening tests for blood donors.

Specificity is the accuracy with which a test can confirm the absence of an infection. Tests with high specificity show few false positives and are used for the diagnosis of HIV infection in an individual.

Tests for HIV

Techniques used in the detection of HIV include: screening tests, supplemental tests, detection of viral antigen, viral RNA/DNA and direct isolation of the virus.

Standard screening tests for HIV include detection of anti-HIV antibodies in blood by enzyme immunoassays (EIA/ELISA). These tests use virus antigens (proteins) to detect the circulating antibodies. The bound antibodies are detected by a colorimetric reaction. These tests become positive 3-12 weeks after infection and have more than 99% sensitivity and greater than 95% specificity. The tests can detect infection with HIV-1 and HIV-2. However, false positives may occur in people with multiple blood transfusions, malignancies, alcoholic hepatitis etc. False negatives can occur very early in the infection or in patients who do not produce enough detectable antibodies or very late in the infection.

Rapid tests: Apart from the ELISA tests which take two to three hours to perform and require specialised equipment, several rapid tests are available which give the result within half an hour. Rapid tests give a visual reaction and include dot-blot tests and particle agglutination. The rapid tests detect HIV-1 and 2 and do not require specialised equipment and can be easily done in smaller laboratories. However, specificity of these tests is lower than ELISA. These tests may not detect infection when the antibody level is very low.

Supplemental tests are those tests which confirm the presence of HIV infection. Here, since the screening tests may give false positive results, a combination of three screening tests with different antigens or different principles are used before a positive result is declared. As per the national HIV testing policy of India, a healthy individual reactive in three different systems of testing (rapid, ELISA) is confirmed to be having HIV infection. The other supplemental tests like Western Blot / Immunofluorescence are used to resolve discordant results of ELISA/ rapid tests.

In the Western Blot (WB) test, viral antigens are separated and transferred on a nitrocellulose paper. The specific antibodies bind to the different antigens and give a band. Criteria for a positive result differ between manufacturers, but usually include the presence of at least two specific bands. A negative WB should not have any bands. Sometimes we get bands in the WB strip which does not fit in the positive category; these are termed indeterminate reactions and may indicate early infection.

Western Blot (WB) tests were initially used as the gold standard and confirmatory test for HIV but now it is used for resolving discordant screening results. WB tests are highly specific as they detect HIV antibodies to specific HIV proteins. But they are more laborious to perform and expensive.

Detection of viral antigen, RNA/DNA: During the window period, the individual is highly infectious but anti-HIV antibody negative. The p24 antigen is a core protein of the HIV and is one of the earliest proteins to be produced. The p24 antigen or HIV RNA are present prior to or in the early stages of seroconversion. HIV infection can be detected on an average nine days earlier by p24 antigen detection than by antibody EIAs. Nowadays, there are fourth-generation ELISA kits available which detect p24 antigen along with antibodies and thus reduce the window period. The p24 antigen appears in the blood within two weeks of exposure and remains for 8-12 weeks until its corresponding antibody appears.

Detection of the viral RNA or DNA can be done by the polymerase chain reaction (PCR). This is done in laboratories with specialised equipment. Here the HIV RNA/DNA is amplified from the blood cells. This technique can detect few copies of the viral genome. This method is highly sensitive and useful in confirming HIV in indeterminate samples or in the neonate.

Isolation of the virus may be performed on blood and other body fluids. This is done in specialised laboratories, is costly, time consuming and remains mostly as a research tool.

HIV testing in the neonate: Antibodies from the mother can cross over to the newborn and persists for months. Hence there is no purpose in doing the anti-HIV antibody testing in the newborn to find out if the child is infected. In such cases p24 antigen testing or DNA/RNA testing would be more appropriate.

Once an individual has been diagnosed with HIV, s/he needs to be on follow up to monitor the state of her or his immune system that is, the CD4 cells and also to monitor the amount of virus in the blood (HIV viral load or HIV RNA Quantitative test). The decision to give anti HIV treatment and prophylactic antibiotics would depend upon the above two test results.

HIV test results – apprehensions and fears

A negative HIV antibody result: Most infected persons will develop detectable HIV antibody within three months of exposure. A negative HIV test usually indicates the absence of HIV infection. If the initial negative test was done within the first three months after exposure, repeat testing should be considered after three months time post exposure. The appropriate timing for follow-up testing would depend upon the time of exposure, the risk behaviour of the person and the person’s anxiety. The timing of the follow-up testing should be such that the person must be assured that the exposure did not lead to infection. If the follow-up testing is negative, then the person is not likely to be HIV infected.

Persons with ongoing exposure: Persons with continued HIV risk behaviour pose a special challenge for follow-up testing. Periodic follow-up testing may have to be done in such persons. In such cases, a repeated interaction with the person gives a chance to monitor the HIV status, start early treatment and give opportunities for HIV prevention counselling.

Persons with no identifiable risk: These individuals come in for testing with a lot of fears and doubts about the transmission. Such persons should be repeatedly counselled regarding the disease and its prevention.

Knowledge of HIV infection status can benefit the health of individual persons and the community. Thus, HIV testing should be made as convenient as possible; it should be confidential, voluntary, and done after appropriate counselling.

(Dr Sangeeta Joshi is Consultant Microbiologist, Manipal Hospital, Bangalore)

InfoChange News & Features, March 2008