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detection of virus-specific antibodies

HIV TESTING

detection of viral material

UNAIDS main objectives


screening of donated blood epidemiological surveillance of HIV prevalence or trends diagnosis of infection in individuals.

Screening assays

DIAGNOSIS
Tests to Confirm HIV Infection

Who?
Mandatory Muslim brides Prisoners-drug addicts Entrance test-Pusat Serenti Recommended pregnant women Persons at high risk for HIV infection

WHO policy on HIV Testing for children and young infants


Orphans and vulnerable children (including street children) Children before adoption Infants who have been abandoned Children who have been sexually abuse Children living in child-headed household Children living in custodial guardians

Where?
Available in all government clinics and hospitals FOR FREE Available in private clinics and hospitals.

HIV Screening Assays

Immunologic Principle
particle agglutination, immunodot (dipstick), immunofiltration (flow-through device), immunochromatography (lateralflow)

FDA-Approved Rapid HIV Antibody Screening Tests February 2008


OraQuick ADVANCE Rapid HIV-1/2 Antibody Test Uni-Gold Recombigen HIV

Reveal G-3 Rapid HIV-1 Antibody Test

MultiSpotHIV-1/HIV-2 Rapid Test

ClearviewCOMPLETE HIV 1/2

ClearviewHIV 1/2 STAT-PAK

Rapid Test

Rapid Test

Rapid Test

Rapid test in Malaysia


Results of this test can be obtained in less than 15 minutes. This test had to go through the scrutiny of the Malaysian Institute of Medical Research before being accepted for use at health clinics. The Rapid Test has a sensitivity and specificity of between 99.8% and 99.9%.

Advantage Good for testing 1 to 100 specimens at a time Requires minimal equipment and reagents Can be performed in a clinic (onsite testing) Highly skilled staff not required Very easy to interpret test results Results in < 45 minutes Test kits can be stored at room temperature (increased stability)

Limitations Not good for testing >100 specimens at a time The QA/QC is performed at multiple sites: requires more control May cost more perindividual test than EIA Choice of testing strategy may require multiple specimens Interreader variability may provide inconsistent results with some assay formats (e.g.,particle agglutination)

Tests to Confirm HIV Infection

Methodology
-electrophoretic technique to separate HIV antigens derived from a lysate of virus grown in culture -("blotted") to nitrocellulose paper -paper is cut into thin strips (each with the full distribution of viral protein antigen bands) -incubated with a 1:50 or 1:100 dilution of a test sample or a control -Washed~incubated with a labeled (tagged) antihuman globulin -enzyme (horseradish peroxidase or alkaline phosphatase) react with antigen-antibody complex -band formed

Classification of HIV-antibody status

Positive

Negative

Indeterminate

Positive
For positive classification CDC guidelines-which require reactivity to at least 2 of the following antigens p24, gp41, gp120/160

Negative
For negative classification Absence of all bands Exception for WHO -that results also can be reported as negative if there is only a very weak p17 band

Indeterminate
For indeterminate classification -is reactivity to 1 or more antigens, but not fulfilling the criteria for positivity -indeterminate results show only weak reactions to the Gag proteins (mostly p17, p24 and/or p55)

Indeterminate WB result
- WHO recommends retesting persons after 2 weeks, -while other organizations suggest waiting 1-6 months before retesting

Retest result

negative or the band profiles do not progress

progress serologically (more bands or greater intensity of bands) or converts to positive (seroconversion)

* individuals who have received vaccination for HIV (eg, subunit gp160) may be misidentified as positive based on reactions to the envelope antigens alone

Cause
hypergammaglobulinemia, the presence of cross-reactive antibodies, infection by HIV-2, infection by an unknown (but related retrovirus)

* autoimmune diseases (eg, systemic lupus erythematosus) can cause falsepositive HIV tests

Limitations
Nonquantitative Training It is complex to administer and may produce indeterminate results if a person has a transitory infection with another virus.

the initial and second tests must be of different principle (bead vs microtiter) and/or use a different antigen source (lysate vs recombinant or synthetic peptide).

HIV Diagnosis Dilemmas


1) indeterminate Western blot results; 2) Minimally Reactive Western Blot Results 3) inconsistent results when repeating specimens or testing follow-up specimens; 4) the occurrence of technical errors; 5) false-negative results due to HIV Group O viruses; 6) laboratory diagnosis of HIV infection in the newborn.

Indeterminate Western Blot Results


Solutions -Retesting- follow-up specimen in 1-3 months - IFA, PCR, viral culture, or other antigen assays

Minimally Reactive Western Blot Results


In these cases, it is important to note on the report form that "on rare occasions, this profile has been found in persons who are not infected, and submission of a new specimen in several weeks is recommended."

inconsistent results
Due-mislabeling or technical errors.

Solution Investigation through quality assurance monitoring

Technical Errors
Solution -dedicated supervisory review mechanism -Outline the quality assurance measures

False-Negative Results for HIV Group O


Cause infected by HIV Group O viruses

Solution Health care providers can be vigilant by inquiring as to the geographic origin of persons tested, or their contact with persons from these areas of Africa

Diagnosis in the Newborn

HIV test use for managing therapy

Viral Load

Viral Load technique


The PCR (polymerase chain reaction) method uses an enzyme to multiply the HIV in the blood sample. Then a chemical reaction marks the virus. The markers are measured and used to calculate the amount of virus. Roche and Abbott produce this type of test. The bDNA (branched DNA) method combines a material that gives off light with the sample. This material connects with the HIV particles. The amount of light is measured and converted to a viral count. Bayer produces this test. The NASBA (nucleic acid sequence based amplification) method amplifies viral proteins to derive a count. It is manufactured by bioMerieux.

Summary
Malaysia Rapid Test Western Blot *PCR for infants and children

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