Understanding Fourth-Generation HIV Tests: Accuracy, Types, and Benefits
The most accurate and reliable routinely used diagnostic HIV test is a laboratory test that can detect both HIV antibodies and the p24 antigen. These are called combination or "combo" tests because they detect both substances. They are referred to as “fourth-generation” tests, incorporating more advanced technology than earlier generations.
HIV antibodies are produced by the human body in response to HIV infection. In the weeks following exposure to HIV, the immune system recognizes some components of the virus and begins to generate HIV antibodies to damage, neutralize, or kill it (this period is known as ‘seroconversion’). These antibodies persist for life.
The p24 antigen is a structural protein that makes up most of the HIV viral core. High levels of p24 are present in the blood during the short period between HIV infection and seroconversion, before fading away. Since the p24 antigen is usually detectable a few days before HIV antibodies, a test that can detect p24 has a slightly shorter window period than a test that only detects antibodies.
Fourth-generation combination tests are typically performed on plasma or serum—two components of blood that are separated from whole blood using laboratory equipment. A blood sample is taken from a vein in the arm using a needle. Samples from multiple individuals are analyzed simultaneously in a laboratory machine.
These tests are commonly referred to as “fourth-generation” tests or ELISA (enzyme-linked immunosorbent assay). First- and second-generation laboratory tests are no longer in use, while the third-generation test, which only detects antibodies, is not recommended for routine use in the UK.
Examples of fourth-generation tests include the Abbott Architect HIV Ag/Ab Combo, ADVIA Centaur HIV Ag/Ab Combo, Genscreen ULTRA HIV Ag-Ab, VIDAS HIV panel, and Vitros HIV combo. In product names, “Ag/Ab” indicates that the test detects both antigen and antibody.
Some tests are described as fifth-generation. They detect the same biomarkers but can also distinguish between samples that are positive for the p24 antigen and those positive for antibodies, as well as differentiate between HIV-1 and HIV-2. Examples include the Bio-Rad BioPlex 2200 HIV Ag/Ab and Elecsys HIV Duo.
Window Periods and Accuracy
The window period refers to the time immediately after infection during which tests may not detect antibodies or the p24 antigen, potentially leading to false negative results. It's challenging to determine the exact duration of the window period for any test, as it varies among individuals and is a complex area of research. Recently infected individuals would need to know precisely when they were exposed to HIV and provide multiple blood samples over the following days and weeks for accurate assessment.
Nevertheless, it is estimated that the median window period for fourth-generation tests is 18 days, with half of all infections detectable between 13 and 24 days after exposure. While this period may occasionally be longer, 99% of HIV-infected individuals would be detectable within 44 days of exposure.
Fourth-generation laboratory tests are highly accurate. The key measures of accuracy are sensitivity (the percentage of correctly positive results when HIV is actually present) and specificity (the percentage of correctly negative results when HIV is not present).
In terms of sensitivity, a review by the Centers for Disease Control and Prevention (CDC) identified four studies of two different assays, with sensitivity consistently above 99.7% for established infections. A Health Protection Agency evaluation found that nine out of ten tests evaluated had a sensitivity of 100%, while a French evaluation reported that ten out of twelve tests achieved the same sensitivity. The lowest sensitivity observed was 99.8%.
Similar results were noted for specificity. Both tests examined by the CDC had a specificity of 99.5% or above. All tests in the Health Protection Agency evaluation demonstrated a specificity of 99.7% or higher, and the French study indicated that all tests produced after the year 2000 had a specificity of 99.8% or above.
Like any screening test, a reactive result (a preliminary positive result) must be verified with confirmatory tests. The laboratory should re-test the sample using a different assay before communicating the preliminary results. A new sample should then be taken and tested to rule out any clerical errors, such as the mixing up of two patients’ samples.
The British HIV Association states that test results should typically be available within 48 hours. The main delay in obtaining results is due to samples being analyzed in batches, which may require several days for a lab to accumulate enough samples. The actual process of conducting the test usually takes only a few hours.
Fourth-generation tests are highly sensitive and specific, making them more reliable for detecting early infection compared to rapid tests. They are appropriate when immediate results are not necessary and when individuals are likely to return for their results. Since testing is conducted by a central lab, implementing quality control programs is easier.