![]() 16 Around 2010, the newer fourth-generation assay replaced third-generation tests by combining the detection of early p24 and HIV antibodies (IgM and IgG). 15 The third-generation assay also detects IgM and IgG against HIV-1, and a diagnosis can be made from 3 weeks post-infection. ![]() First- and second-generation assays detect IgG against HIV-1 infection at approximately 6–10 and 4–6 weeks, respectively. 13, 14Įnzyme immunoassay testing has undergone a progressive evolution over the last 25 years ( Figure 1). A measurable quantifiable signal is produced that indicates the presence or absence of HIV biomarkers. Secondary antibodies conjugated to enzymes detect the captured p24 antigens or HIV antibodies by an enzyme-catalysed reaction of an added substrate. Enzyme-linked immunosorbent assays utilise capture proteins (HIV viral antigen) or capture antibodies, which entrap HIV antibody or HIV antigen, respectively. HIV ELISAs can be categorised into two formats: the indirect ELISA format is used in first- and second-generation HIV assays, and sandwich ELISA format is used in the third-fifth generation HIV assays ( Figure 1). Enzyme-linked immunosorbent assays can detect HIV antibodies and/or p24 proteins. The EIA tests are formatted as enzyme-linked immunosorbent assays (ELISAs). 11 HIV EIA tests detect antibodies produced against HIV and/or p24 HIV antigen. Therefore, an infected person will test negative during the eclipse phase. The eclipse phase of HIV infection is characterised by the inability of laboratory tests to detect HIV. First- and second-generation HIV EIAs employ indirect enzyme-linked immunosorbent assay format in contrast to the sandwich enzyme-linked immunosorbent assay applied in the third-, fourth- and fifth-generation HIV assays. (c) Enzyme immunoassay used in HIV testing utilise enzyme-linked immunosorbent assay design. (b) Technological evolution in HIV assay design has enabled early diagnosis from the point of infection. (a) HIV diagnosis is made by detecting HIV biomarkers: HIV nucleic acid, RNA, HIV-1/2 p24 and HIV antibodies (Immunoglobin M and Immunoglobin G). Laboratory diagnosis of HIV infection in adults. Untreated, the infection evolves into advanced disease (Acquired Immunodeficiency Syndrome ) characterised by opportunistic infections, a rise in HIV RNA levels and a gradual decline in HIV antibodies. Thereafter, the antibody levels plateaux and remain fairly constant. Once antibody has appeared (seroconversion), antibody levels progressively increase over the next months and peak at 5–6 months. 9 The median window period lasts approximately 18 days from infection and generally ranges between 10 and 24 days. The window period is characterised by a lack of antibodies and p24 antigen. HIV is challenging to diagnose in the early ‘window period’ of infection. ![]() Detection of HIV during this early period is important because of the clinical benefit of instituting early antiretroviral therapy. 5, 6 This marks the end of early HIV infection. The viral RNA then decreases to a ‘set point’ level. The appearance of immunoglobulin M (IgM) after approximately 2 weeks is followed closely by immunoglobulin G (IgG) at about 3 to 4 weeks post-infection. Thereafter, an immune response is mounted. 4 Between days 11 and 13 post-infection, the protein (p24) antigen (part of the core protein of HIV) may be detected in newly infected individuals. This rapid replication and seeding of the virus results in very high ribonucleic acid (RNA) levels in blood, which becomes detectable from about 2 weeks post-infection. Infected white blood cells and HIV virions then travel via the bloodstream to organs of the reticuloendothelial system and to lymphoid tissue in the gastrointestinal tract where secondary amplification occurs. This period is known as the eclipse phase (before the virus or antibodies are detected) and lasts approximately 10 days post-infection. The primary viral amplification occurs in regional lymph nodes. HIV biomarkers reflect evolving HIV infectionĭuring the early phase of HIV infection after breach of the mucosal barrier, HIV infects target cells, for example, cluster of differentiation 4 (CD4) T lymphocytes. ![]()
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