Immunodeficiency Virus 1 - Testing


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Human Immunodeficiency Virus 1
: testing


Current testing algorithm

HIV testing in the USA is generally based on a combination rapid tests performed in point-of-care (POC) facilities (usually screening tests) and laboratory-based in vitro diagnostics (usually confirmatory tests). The reactive (i.e. positive) screening test, whether a POC rapid test or laboratory-based enzyme immunoassay (EIA), requires a positive confirmatory test before patient is considered infected. This algorithm has not changed significantly since 1985. However, there have been dramatic increases in the sensitivity and specificity.

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Enzyme Immunoassays (EIAs)

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Test results

Test's properties

Sensitivity: test 100% sensitive if it exhibits positive (reactive) results for all infected patients i.e produces no false negatives. However, false-positives are allowed.

Specificity: test has 100% specificity if it shows negative results for all healthy patients, i.e. produces no false positives. However, the test can miss infected specimens.

In reality test cannot be 100% sensitive and 100% specific at the same time.

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Window period

Window period (or transient state) is defined as time that passes from a reference event to onset of detectability of a specific marker. Duration of window period depends on sensitivity of the test and specifics of patient's immune response.

Seroconversion is an event when pathogen-specific antibodies that were developing as a result of infection become detectable by tests.

Examples of window period:

For a patient usually the first window period is most important. Current CDC guidelines concerning this window period:

Most people will develop detectable antibodies within 2 to 8 weeks (the average is 25 days). Even so, there is a chance that some individuals will take longer to develop detectable antibodies. Therefore, if the initial negative HIV test was conducted within the first 3 months after possible exposure, repeat testing should be considered >3 months after the exposure occurred to account for the possibility of a false-negative result. Ninety-seven percent of persons will develop antibodies in the first 3 months following the time of their infection. In very rare cases, it can take up to 6 months to develop antibodies to HIV.

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Rapid EIA HIV tests

Rapid HIV tests are single-use EIAs that contain all necessary reagents and yield results in less than 30 min. The tests that use whole blood or oral fluid can be performed in non-laboratory settings. Tests that require serum or plasma samples must be performed in laboratory. Patients learn their test result during the same office visit and thus access effective treatment earlier, with significant benefits for long-term survival and quality of life.

Ora-Quick Advance Rapid HIV-1/2 Antibody Test (OraSure Technologies) oral fluid or whole blood; results are read within 20-40 min.
Reveal G3 Rapid HIV-1 Antibody Test (MedMira Laboratories)serum or plasma specimen; results are read immediately
Uni-Gold Recombinogen HIV (Trinity Biotech)whole blood, serum, or plasma; results in 10 min.
Multispot HIV-1/HIV-2 Rapid Test (Bio-Rad Laboratories)plasma or serum; results are read in 15 min. after preparation of specimen
Clearview HIV 1/2 Stat-Pak Assay (Chembio Diagnostic Systems)whole blood, serum or plasma; results are read in 15-20 min.
Sure Check HIV 1/2 Assay (Chembio Diagnostic Systems)whole blood, serum or plasma; results are read in 15-20 min.

Sensitivity of these tests (possibility of false-negatives) ranges from 99.6 (98.5-99.9%) for Ora-Quick Advance Rapid HIV-1/2 Antibody Test to 100% for Multispot HIV-1/HIV-2 Rapid Test. Specificity (possibility of false-positives) ranges from 99.7% (99.0-100) for Uni-Gold Recombinogen HIV to 100% (99.7-100) for Ora-Quick Advance Rapid HIV-1/2 Antibody Test.

A positive rapid test results are preliminary and require confirmation with a supplemental confirmatory test, usually Western blot or indirect immunofluorescence assay. If the confirmatory test result is negative or indeterminate, follow-up testing after 1 month is recommended.

A recognized problem in US screening programs is the loss of contact with individuals between an initial positive test result and the subsequent confirmatory test, particularly when confirmation is laboratory-based and turnaround time measured in days (2-6 days for Western blot). For example, results from the State of New York showed that at least 25% individuals with positive rapid tests didn't return for confirmatory test results. Similar statistics were reported in other states.

These data suggest that second rapid test preferably on the same day would be useful in the resolution of a false-positive initial test. It was shown that 6-22% of reactive results obtained by first rapid test were negative on second rapid test and later by Western blot.

False-positive HIV serologic screens can be caused by recent influenza vaccination, incidental viral infections, autoimmune disease, renal failure, cystic fibrosis, multiple pregnancies, blood transfusions, liver diseases, intravenous substance abuse, hemodialysis, or vaccination against hepatitis B and rabies.

Because false-positive results in HIV serologic rapid screening tests are not rare, two different rapid tests (developed by different companies) would alleviate psychological distress of patients who were initially tested reactive but later found not infected.

A negative result of a rapid test is conclusive and generally requires no follow-up testing. However, because results of these antibody tests may be negative before seroconversion, an individual with a possible recent exposure to HIV should be retested within 3 months.

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Nucleic acid amplification tests (NAAT)

These tests detect viral RNA in plasma at concentration of 50 copies/ml or more. In retrospective study of archived plasma samples, NAAT detects HIV infection ~1 week before the p24 antigen tests and ~12 days before a sensitive, third generation antibody test. These tests play valuable role in identifying very early infection, sometimes within first 1-2 weeks after exposure, when neither antibodies nor p24 are not yet detectable.

To offset costs of RNA testing, pooled RNA screening of antibody-negative specimens was developed. With pooled testing, 100 Ab-negative specimens can be screened with just one RNA test by creating 10 miniature pools with 10 specimens each and then combining the 10 miniature pools into a master pool for screening. If the master pool is found positive, it is deconstructed to determine which of the miniature pools was positive and the component specimens of that pool are then tested to identify positive sample(s).

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Virological and Serological Events following infection


HIV-1 markers' dynamics


After infection, virus start replicating in proximity of inoculation site. Following second to third week after exposure, high titer systemic viremia occurs. Thus, HIV genomic RNA is present before the patient has developed detectable anti-HIV antibodies and is therefore a powerful marker of recent infection. In most cases, HIV RNA remains detectable throughout the course of untreated infection, albeit at levels much lower than in the acute infection. However, if the infection remains untreated, RNA levels rise again at the final stage of the disease.

A protein component of the virus core, p24 antigen, is usually detectable within a few days after onset viremia. As the patient's immune system initiates a response, levels of both the virus and p24 fall. The p24 usually becomes undetectable until the degradation of immune system associated with AIDS, typically about 10 years later. Thus, p24 antigen is a marker of recent as well as late infection. As a marker of recent infection in the absence of antibodies p24 can be unreliable (possibility of false-positives) and short-lived (1-2 weeks).

The initial immune response is typically heralded by a virus-specific IgM antibody response. This IgM response is variable in duration and intensity, generally peaking within 1-2 weeks, falling to background 1-2 weeks later. Contemporaneously, the long-lived high-titer IgG response develops. A gradual increase in anti-HIV titer occurs over several months as antibodies to various viral antigens develop.

Avidity is a synergistic, strong binding of antigen to antibody.

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Western blot

The major antibody specificities detected in HIV-1 Western blot analysis include:

GAG proteinsHIV-1=p18,p24,p40,p55
POL proteinsHIV-1=p31,p51,p65
ENV glycoproteinsHIV-1=gp41,gp120,gp60
HIV-2=gp34,gp34 trimer (gp105),gp120, gp140

To be considered positive for HIV-1 test must show reactivity to at least two of the major bands: gp120/gp160, gp41 or p24.

To be considered positive for HIV-2 test must show reactivity to two env bands or p26 and one env band.

An indeterminate Western blot result can be caused by a weak titer of anti-HIV antibodies (as seen in early seroconversion), advanced AIDS, infection with unusual HIV type, or past experimental HIV vaccines. It can also be caused by the presence of antibodies cross-reactive against HIV antigens (incidental viral infection; vaccination against influenza, hepatitis, or rabies; or HTLV infection) or reactivity to nonviral components of the Western blot (various autoimmune disorders, multiple pregnancies, and multiple blood transfusions).

An indeterminate Western blot result should be followed up with quantitative NAAT if early seroconversion is suspected, with a repeat immunoassay and Western blot analysis performed in 2-4 weeks.

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Various types of HIV-specific laboratory tests used in research, diagnostics and treatment

  1. Screening assays for HIV antibodies
    • HIV IgG
    • HIV IgM/IgG antibody assays
    • HIV antibody/antigen (p24) assays
  2. Confirmatory assays
    • Western blot
    • indirect immunofluorescence assay
  3. Nucleic acid testing
    • Quantitative HIV RNA plasma load
    • HIV DNA on peripheral blood mononuclear cells (PBMC) testing
    • Nucleic acid testing to screen blood donations
  4. Guiding therapy
    • Antiretroviral drug resistance genotyping
    • Co-receptor usage
    • Pharmacogenomic assays
  5. Epidemiological and forensic studies
    • HIV subtyping
    • HIV sequencing and phylogenetic analysis
    • Detuned HIV antibody assays
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