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Subject: Human Immunodeficiency Virus (HIV-1) Confirmatory Western Blot Assay
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The Western blot (WB) assay is a method in which individual proteins of an HIV-1 lysate or recombinant HIV proteins allow the determination of antigenic specificity of the antibodies in the patients' serum. This is considered a confirmation test for HIV serology.
Western blot assays are specific for different HIV species and subtypes.
HIV-1 antibodies present in the specimen bind to HIV-1 antigens (p15, p17, p24, p31, gp41, p51, p66, p55, gp120, gp160).
Alternative confirmation testing algorithms have been described, including use of a second, different EIA, IFA, line immunoassay, or nucleic acid amplification test.
The Western blot is used for confirmation of repeatedly reactive HIV antibody screen or rapid HIV antibody results.
Western blots specific for HIV-2 or HIV-1 subtypes other than M should be considered for patients at epidemiologic risk for such infections, or when HIV-1 WB gives indeterminate or negative results. HIV-2 is most commonly acquired in West Africa.
Positive result: As established by CDC, the interpretive criterion for HIV-1 is defined by the presence of any two of the following bands: p24, gp41, and gp120/160.
Indeterminate results: HIV infection is neither confirmed nor ruled out; additional testing is required.
Indeterminate results may be caused by factors related to HIV infection, including a weak titer of anti–HIV-1 antibodies (e.g., early seroconversion), advanced AIDS, or infection with HIV-2 or HIV-1 subtype O. Nonspecific causes include autoantibodies, hemodialysis, hypergammaglobulinemia, or recent vaccination.
Additional testing for indeterminate results may include HIV-1 RNA testing, WB for HIV-2 or HIV-1 subtype O, or repeat HIV serology and WB in 4–6 weeks.
Negative result: Negative results do not exclude the diagnosis of HIV infection; further testing is required.
Additional testing for negative results may include HIV-1 RNA testing, WB for HIV-2 or HIV-1 subtype O, or repeat HIV serology and WB in 4–6 weeks.
Persistent reactivity of the antibody screening assay on repeat testing with negative confirmation by WB pattern suggests nonspecific reactivity and the absence of HIV infection.
HIV WB testing should be ordered only on sera that are repeatedly reactive by HIV screening EIA or rapid HIV antibody tests.
Patients with HIV-2 infection may give indeterminate results for HIV-1 Western blots. For HIV-2 WB test, no single standard can currently be applied to all tests. The CDC recommends that each test be interpreted by the criteria suggested by the kit manufacturer.
HIV-2 cross-reacts with HIV-1 in serologic tests. A positive screen test for HIV 1 and 2 antibodies with a repeated negative or indeterminate HIV-1 WB suggests positive HIV-2 infection and need to be confirmed HIV-2–specific test.