Laboratory diagnosis of HIV Infection

Tests to identify HIV infection can be divided into different categories: virus cultivation, antigen detection and viral genome amplification (PCR).

Sample Collection and Transport

  • For standard HIV-1/HIV-2 antibody testing a single tube (10 mL) of whole blood is sufficient.
  • Specimens can be stored at room temperature up to 3 days, at 4°C for up to 7 days.
  • For longer storage periods, the serum or plasma must be separated from the clot or cells and stored at -20°C.
  • Specimens for PCR generally need to be processed within 48 h of collection as viral DNA denatures over time and becomes undetectable.
Virus cultivation
Virus can be isolated from infected persons in most phases of the infection. Peripheral blood mononuclear cells (PBMC) can be co-cultivated with activated PBMC from HIV-negative donors in the presence of IL-2. A positive result is recognized by appearance of virus antigen (p 24) or reverse transcriptase activity in the culture medium. Virus cultivation is not done as the part of routine laboratory diagnosis method. 
Course of HIV Infection
Image 1: Course of HIV Infection
Antigen/Antibody detection
Antibodies usually become detectable from 3 to 12 weeks after infection. As a rule, an infected person remains antibody-positive for life, but antibody titres often fall in patients with AIDS (see image 1).
 The most widely applied tests are the indirect and the competitive ELISA, using mostly a mixture of viral antigens. It is recommended that confirmatory tests are carried out to exclude the possibility of false positive results. These are either variations of ELISA tests or Western blot analysis of antibody specificity.
  1. Point of Care tests (POCT) for HIV
    These tests provide rapid, on-site HIV results in a format that is relatively easy to perform.

    Rapid tests for the diagnosis of HIV infection
                                                                                          Rapid tests for the diagnosis of HIV infection
  2. ELISA for HIV diagnosis
    It is a commonly used as a screening assay for many infectious diseases, including HIV.  It is highly sensitive test but false positives can be seen. Current ‘window period’ (the time from exposure to seroconversion) for HIV is less than three weeks in most cases.
    All HIV diagnostic laboratories must confirm repeated EIA screen-positive results ( done by using different part of the viral antigen ) by a confirmatory assay, usually with Western blot.
    Specimens that screen positive in the first assay but negative in the second assay should still be considered for confirmatory testing if the patient is symptomatic or high risk.
  3. p24 antigen testing
    p24 antigen tests are also enzyme immuno-assay  (EIA) based. Antibody is used to capture the disrupted p24 antigen from patient serum.
    P24 antigen test is useful

    • for specimens from patients that are high risk and symptomatic but HIV EIA-negative (for Ab testing), or
    • for specimens that are EIA-positive but Western blot-negative or –indeterminate
    • for confirmation of neonatal HIV infection
  4. Western blot Test
    Western blot is a confirmatory diagnosis of HIV infection. Western blot is a highly specific immunoblot that allows for the visualization of antibodies to the structural polypeptides of HIV.
Western Blot Test for HIV diagnosis
Western Blot Test for HIV diagnosis

Viral genome amplification (PCR)

The PCR technique represents a major advance in the diagnosis of HIV infection. This powerful technique can amplify target DNA present in minute amounts. PCR is useful when Ab testing may be insufficient to determine whether a patient is infected
  • in the diagnosis of HIV infection in infants born to infected mothers (presence of maternal IgG antibodies excludes serological testing during the first few months after birth)
  • resolving indeterminate Western blot results and
  • testing immunocompromised individuals who may not mount an antibody response.
 Quantitative determination of plasma viremia (virion RNA) by reverse transcription PCR (RT-PCR) has become a major tool to follow the progression of HIV infection in untreated patients and to monitor the effects of antiviral chemotherapy in patients. It is used in conjunction with CD4 counts.
CD4+ lymphocyte count
A hallmark of chronic HIV infection is the depletion of CD4+ lymphocytes and loss of these cells is closely associated with acquisition of the characteristic opportunistic infections. The monitoring of CD4+ lymphocyte count is therefore an important determinant for clinical staging, initiation of antiviral therapy and Pneumocystis jiroveci pneumonia prophylaxis.

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