Which screening assays are used in HIV testing?

Updated: Sep 19, 2018
  • Author: David J Cennimo, MD, FAAP, FACP, AAHIVS; Chief Editor: Michael Stuart Bronze, MD  more...
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Answer

The available screening assays rely on the detection of antibodies, the p24 antigen, or the viral nucleic acid. Human immunodeficiency virus (HIV) antibodies can be detected with enzyme-linked immunosorbent assay (ELISA) or enzyme immunoassay (EIA), particle agglutination, and chemiluminescent immunoassay (CIA). The source for antibody testing for EIA can be the whole blood, serum, plasma, saliva, or urine.

Currently, a fourth-generation EIA assay that detects both antibodies and the p24 antigen (HIV 1/2 antigen/antibody combination immunoassay) is available and the recommended screening platform for HIV testing. [22, 18]

EIA is the assay most widely used for the initial evaluation of established HIV-1 or HIV-2 infection. In an attempt to improve both sensitivity and specificity, 4 generations of EIAs have been produced since their introduction two decades ago.

The first-generation EIA relies on the detection of antibodies directed against a coated well with whole-cell lysate of infected cells. The second-generation EIA substituted the whole-cell lysate for recombinant-produced HIV antigens. Third-generation EIAs differ from the two previous generations. In this case, antibodies are detected through the “antigen sandwich” technique, in which the enzyme is linked to the antigen rather than to the antibody. This technique detects both immunoglobulin G (IgG) and immunoglobulin M (IgM), therefore allowing earlier antibody identification.

In the fourth-generation EIA, the wells are coated with both p24 antibodies and HIV-1 antigens. Host-derived p24 antigens or antibodies directed at these molecules are detected using an enzyme-labeled antibody.

Antibody response to HIV is detected at approximately 3–6 weeks after infection, depending on the generation of the EIA being used. [23] The detection of the p24 antigen by the fourth-generation assays shortens the window by 4.4–4.8 days compared to third-generation assays. [24] Individuals who test negative on the initial evaluation should undergo repeat antibody testing in 3 months in case they had not seroconverted at the initial evaluation.

Sequence of appearance of laboratory markers for H Sequence of appearance of laboratory markers for HIV-1 infection. Note that units for vertical axis are not noted because their magnitude differs for RNA, p24 antigen, and antibody. Courtesy of the CDC (Centers for Disease Control and Prevention and Association of Public Health Laboratories. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. Online at: http://stacks.cdc.gov/view/cdc/23447. Published June 27, 2014. Accessed Oct 24, 2016.)

According to the Association of Public Health Laboratories and the CDC [18] , a positive HIV-1 antigen/antibody and a second HIV-1/HIV-2 antibody-based differentiation test should be performed. A positive test confirms established HIV infection (i.e. the recommendations for Western blot have been removed). If the confirmatory HIV antibody testing is negative, the original combination test may have detected p24 anigen and not antibody. In this case, a nucleic acid amplification test (NAT) is performed. If the NAT result is positive, acute HIV infection is present. If negative, HIV -1 infection is ruled out. Note there are no NAT tests available for HIV-2 at this time.

Recommended laboratory HIV testing algorithm for s Recommended laboratory HIV testing algorithm for serum or plasma specimens. Courtesy of the CDC (Centers for Disease Control and Prevention and Association of Public Health Laboratories. 2018 Quick reference guide: Recommended laboratory HIV testing algorithm for serum or plasma specimens. Online at: https://stacks.cdc.gov/view/cdc/50872. Published January 2018. Accessed Sept 10, 2018.)
Guidance for Reporting Results from the HIV Labora Guidance for Reporting Results from the HIV Laboratory Eiagnostic Testing Algorithm for Serum and Plasma Specimens. Courtesy of the CDC (Centers for Disease Control and Prevention and Association of Public Health Laboratories. 2018 Quick reference guide: Recommended laboratory HIV testing algorithm for serum or plasma specimens. Online at: https://stacks.cdc.gov/view/cdc/50872. Published January 2018. Accessed Sept 10, 2018.)

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