Laboratory diagnosis of HIV

Laboratory diagnosis of HIV by Dr Nikhil Bansal

1. LABORATORY DIAGNOSIS
Of H.I.V.
Presented by: NIkHIL BANSAL
J.N.M.C.,WARDHA

2. SpeCIfIC TeSTS fOR H.I.V INfeCTION
1.Antigen detection: p24 antigen
2.Virus isolation
3.Detection of viral nucleic acid
4.Antibody detection

3. 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

4. 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.

5. 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.

6. 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”

7. 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.

8. 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

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

10. 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.

11. Test
Window
period
Acute
infection
Asymptomatic
infection
ARC & AIDS
+
+ +
+
+
+
+
+
+
+
+
+
-
-
±
-
-
p24,RT
antigens
Virus
isolation
ELISA
Western
blot
Specific tests for the laboratory diagnosis of HIV infection

12. 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

13. 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.

14. 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.

15. ELISA test

16. 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.

17. 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.

18. Western Blot test

19. 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.

20. 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.