Laboratory Diagnosis Laboratory Diagnosis of HIV Infection & of HIV Infection & its Treatment. its Treatment. Presented By Presented By :- :- JITENDRA KUMAR PANDEY JITENDRA KUMAR PANDEY PG, Medical Student PG, Medical Student MGM MEDICAL COLLEGE, MUMBAI MGM MEDICAL COLLEGE, MUMBAI
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Laboratory Diagnosis of Laboratory Diagnosis of HIV Infection & its HIV Infection & its
Treatment.Treatment.
Presented By Presented By :-:-JITENDRA KUMAR PANDEYJITENDRA KUMAR PANDEY
PG, Medical StudentPG, Medical StudentMGM MEDICAL COLLEGE, MUMBAIMGM MEDICAL COLLEGE, MUMBAI
Laboratory Diagnosis of HIV Infection.Laboratory Diagnosis of HIV Infection.
Specific tests for HIV infection :Specific tests for HIV infection :
HIV viral Ag coated microtitre wells is takenHIV viral Ag coated microtitre wells is taken
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Test serum is addedTest serum is added
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Unbound serum is washedUnbound serum is washed
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Anti-human goat immunoglobulin linked to a suitable enzyme is addedAnti-human goat immunoglobulin linked to a suitable enzyme is added
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Colour forming substrate is addedColour forming substrate is added
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Photometrically detectable colour is formed in positive testPhotometrically detectable colour is formed in positive test
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Add stop solutionAdd stop solution
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Absorbance of these is read by ELISA reader.Absorbance of these is read by ELISA reader.
Absorbance value < cut-off value are considered Neg. for HIV 1 / 2 Abs.Absorbance value < cut-off value are considered Neg. for HIV 1 / 2 Abs. Absorbance value ≥ cut-off value are considered Pos. for HIV 1 / 2 Abs.Absorbance value ≥ cut-off value are considered Pos. for HIV 1 / 2 Abs.
Fig. A :- ELISA kit . Fig. B :- Microtitre wells.Fig. A :- ELISA kit . Fig. B :- Microtitre wells.
Fig. C :- ELISA washerFig. C :- ELISA washer Fig. D :- ELISA Reader.
b) Rapid tests:- b) Rapid tests:-
Quick (30 minutes) Quick (30 minutes)
Easy to performEasy to perform
No sophisticated instruments are required.No sophisticated instruments are required.
Eg. Comb test, HIV spot test (Tri-dot),Eg. Comb test, HIV spot test (Tri-dot),
Dot-blot assay etc.Dot-blot assay etc.
Disadvantages:Disadvantages: Tedious,Tedious,
if large no. samples have to be tested at one time.if large no. samples have to be tested at one time.
Principle of HIV tri-dotPrinciple of HIV tri-dot
c) Simple tests:- c) Simple tests:-
Simple, requires 1-2 hrsSimple, requires 1-2 hrs
Easy to performEasy to perform
No sophisticated instruments are required.No sophisticated instruments are required.
Based on the principle of ELISA.Based on the principle of ELISA.
Less sensitive than ELISA.Less sensitive than ELISA.
Confirmatory testsConfirmatory testsa) Western blot:-a) Western blot:- Detection of HIV viral proteins.Detection of HIV viral proteins. HIV proteins are separated by PAGE & blottedHIV proteins are separated by PAGE & blotted
onto nitrocellulose paper strip.onto nitrocellulose paper strip. Test serum is allowed to react with the stripTest serum is allowed to react with the strip
(Abs to HIV proteins if present combines with HIV(Abs to HIV proteins if present combines with HIV
fragments).fragments). Strip is washed & treated with enzyme-conjugatedStrip is washed & treated with enzyme-conjugated
anti-human gamma globulin.anti-human gamma globulin. Suitable substrate is added – produce color bandSuitable substrate is added – produce color band
on the strip.on the strip. Position of color band on the strip indicates the Ag Position of color band on the strip indicates the Ag
with which Abs had reacted.with which Abs had reacted. Abs to p24 (gag gene, core protein), p31 (pol gene,Abs to p24 (gag gene, core protein), p31 (pol gene,
reverse transcriptase) & gp41, gp120 or gp160 (env reverse transcriptase) & gp41, gp120 or gp160 (env
gene, env protein) is commonly detected.gene, env protein) is commonly detected. Positive – if at least 2 bands appear against any 2 proteins.Positive – if at least 2 bands appear against any 2 proteins.
b) Indirect immunofluorescence test:-b) Indirect immunofluorescence test:-
HIV infected cells are fixed onto a clean glass slides & then reacted with HIV infected cells are fixed onto a clean glass slides & then reacted with
serum followed by fluorescein conjugate anti-human gamma globulin.serum followed by fluorescein conjugate anti-human gamma globulin.
Apple green fluorescence appear in the positive test under fluorescent Apple green fluorescence appear in the positive test under fluorescent
microscope.microscope.
Figure:- Cells infected with HIV virus and stained by fluorescent antibody test.Figure:- Cells infected with HIV virus and stained by fluorescent antibody test.
Note: Bright apple-green fluorescent foci in 80–90% cells (B) in comparison to un-infected cells with no fluorescent foci (A). Note: Bright apple-green fluorescent foci in 80–90% cells (B) in comparison to un-infected cells with no fluorescent foci (A).
B) Non-specific Tests :-B) Non-specific Tests :-1) TLC & DLC :-1) TLC & DLC :-leucopenia with lymphocytopenia.leucopenia with lymphocytopenia.
2) T-lymphocyte subset assay :-2) T-lymphocyte subset assay :- Normal CD4:CD8 T-cell is 2:1Normal CD4:CD8 T-cell is 2:1
Reversed to 0.5:1 in AIDSReversed to 0.5:1 in AIDS
CD4 lymphocytes count is < 200/mm3CD4 lymphocytes count is < 200/mm3
PIs block the HIV protease,PIs block the HIV protease,
an enzyme HIV needs to make copies of itself. an enzyme HIV needs to make copies of itself.
4) Fusion inhibitors:4) Fusion inhibitors:
Enfuvirtide Enfuvirtide
MOA :- MOA :-
Fusion inhibitors block the HIV from entering the CD4 Fusion inhibitors block the HIV from entering the CD4 cells cells of he immune system. of he immune system.
5) Highly active antiretroviral therapy (HAART):5) Highly active antiretroviral therapy (HAART):
In the current guidelines Azidothymidine (AZT) is recommended for the In the current guidelines Azidothymidine (AZT) is recommended for the treatment of asymptomatic / mild-symptomatic people with CD4 count < 500 treatment of asymptomatic / mild-symptomatic people with CD4 count < 500 & for the treatment of infected pregnant women's to reduce the transmission & for the treatment of infected pregnant women's to reduce the transmission of virus to fetus.of virus to fetus.
Apart from antiretroviral therapy other measures in treatment of AIDS Apart from antiretroviral therapy other measures in treatment of AIDS includes – treatment & prophylaxis of opportunistic infections & tumors.includes – treatment & prophylaxis of opportunistic infections & tumors.
Mode of actions of anti-HIV drugsMode of actions of anti-HIV drugs
Mode of actions of anti-HIV drugsMode of actions of anti-HIV drugs
HIV patient after treatmentHIV patient after treatment