LABORATORY DIAGNOSIS of H.I.V.

Presented by: Nikhil BansalJ.N.M.C.,Wardha

Specific tests for H.I.V infection

1. Antigen detection: p24 antigen

2. Virus isolation

3. Detection of viral nucleic acid

4. Antibody detection

Non Specific tests

1. Total and differential leucocyte count

2. T-lymphocyte subset assays

3. Platelet count

4. IgG and IgA levels

5. Skin Tests for CMI

Specific Tests for HIV infection

1.Antigen Detection:After a single massive infection viral antigen

p24 and RT maybe detected in blood about 2 weeks.

Note: The p24 antigen is the earliest viral marker to appear in the blood.

IgM antibodies appear in about 4-6 weeks to be followed by IgG antibodies.

If the infecting dose is small example in needle-stick injury the process maybe considerably delayed.

The appearance of p24 antigenemia and viremia followed by IgM antibodies response coincides with the acute illness.

The p24 antigen capture assay (ELISA) which uses anti-p24 antibodies as the solid phase and can be used for this.

The test is positive in about 30% cases.

2.Virus isolation

The viruses present in circulation and body fluids, within lymphocytes or free cells.

Virus titres parallel p24 titres, being high soon after infection, low and antibodie-bound during the asymptomatic period, and again high towards the end.

The infectivity being highest in “the early phase and when the person becomes terminally ill”

The virus is present in many parts of the body and can be isolated from the peripheral lymphocytes by the technique of co-cultivation of the patient’s lymphocytes with uninfected lymphocytes in the presence of interleukin-2.

Viral replication can be detected by RT. Note: Because of the risk involved, virus

isolation is to be attempted only in laboratory with adequate containment facilities.

3.Detection of Viral Nucleic Acid

As the most sensitive and specific test, PCR has become the gold standard for diagnosis in all stages of HIV.

DNA PCR

PCR

RNA PCR

A related test, HIV RNA PCR can be used for diagnosis as well as for monitoring the level of viremia.

4.Antibody detection

2-8 weeks to months for the antibodies to appear after infection and during part of this period, the individual maybe highly infectious known as the window period.

Infection can be detected during the window period by the p24 assay.

Antibody testing will have to be done after 2-6 months to ascertain whether infection has occurred or not.

IgM antibodies dissappear in 8-10 weeks while IgG antibodies remain throughout.

TestWindow period

Acute infection

Asymptomatic infection

ARC & AIDS

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p24,RT antigens

Virus isolation

ELISA

Western blot

Specific tests for the laboratory diagnosis of HIV infection

1.Screening test

(a) ELISA

(b) Rapid test

(c) Simple test

2.Supplement tests

(a) Western blot test

(b) Indirect immunofluorescence test

(c) Radio immuno-precipitation assay

Screening Tests

ELISA tests:Direct solid phase antiglobulin ELISA is the

method most commonly used. The antigen is obtained from HIV grown in continuous T-lymphocyte cell line or by recombinant techniques and shouls represent all groups and subtypes of HIV-1 and HIV-2. The antigen is coated on the microtitre wells or other suitable solid surface. The test serum is added. And if the antibody is present, it binds to the antigen.

After washing away the unbound serum, antihuman immunoglobulin linked to a suitable enzyme is added. If test serum contains anti-HIV antibody, a photometrically detectable colour is formed which can be read by special ELISA readers.

Rapid Tests: It has been introduced for the purpose such as cylinder or cassette ELISA, immunochromatographic, coated particle agglutination.

Simple Tests: They take 1-2 hours and do not require expensive equipment.

ELISA test

Supplement tests Western Blot test: The confirmatory test commonly

employed is the Western Blot test. In this test HIV proteins separeated according to their electrophoretic mobility(and molecular weight) by polyacrylamide gel electrophoresis are blotted onto strips of nitrocellulose paper. These strips are reacted with test sera and then with enzyme conjugated antihuman globulin.

A suitable substrate is then added, which produces a prominent colour band where the specific antibody has reacted with the separated viral protein. Bands will be of p24, p31 & gp41, gp120 or gp160.

A positive reaction with proteins representing the three genes gag,pol,env is considered positive even if it shows bands against at least two of the following gene: p24, gp41, gp120/160.

Western Blot test

Indirect Immunofluorescence Test:

HIV infected cells are fixed onto glass slides and then reacted with serum followed by fluorescein conjugated antihuman gamma globulin. In a positive test, apple-green fluorescence appears when examined under fluorescent microscope.

Non-specific tests

Total leucocyte and lymphocyte count to demonstrate leucopenia and a lymphocyte count usually below 2000/mm3.

T cell subset assays. Absolute CD4+T cell count will be usually less then 200/mm3.

Platelet count will show thrombocytopenia.Raised IgA & IgG levels.Diminished CMI as indicated by skin test.Lymph node biopsy showing profound

abnormalities.