ALL ABOUT HIV

1. Laboratory Diagnosis of
HIV Infection & its
Treatment.
Presented By :-
JITENDRA KUMAR PANDEY
PG, Medical Student
MGM MEDICAL COLLEGE, MUMBAI

2. Laboratory Diagnosis of HIV Infection.
Specific tests for HIV infection :
1) Ag. detection :- p24 Ag
2) Virus isolation :- virus culture
3) Viral nucleic acid detection :- PCR
4) Ab. Detection :- Anti -HIV antibody detection (IgM, IgG )
Non-specific tests :
1) TLC, DLC :-
2) T-lymphocyte subset assays :-
3) Platelet count :-
4) IgG & IgA level :-
5) Skin test for CMI :-
Tests for opportunistic infections & tumour :

3. A) Specific tests for HIV infection
1) Antigen detection :-
 The virus Ag. p24 & Reverse transcriptase (RT) detection in
blood.
 p24 is the earliest virus marker.
 With seroconversion, Ab become detectable & p24 Ag
disappear during long asymptomatic phase.
 p24 antigenemia reappears with the onset of clinical
disease.

4. 2) Virus isolation :-
 Virus is not routinely isolated.
 HIV is Present in blood, body fluids, within CD4
Lymphocytes.
 Patients lymphocytes are co-cultivated with uninfected
human lymphocytes in presence of IL-2.
 Virus replication is detected by RT activity & presence of
viral Ag.

5. 3) Viral nucleic acid detection :-
 Detected by PCR.
 Useful for diagnosis in window period.
 2 type of PCR been used, DNA PCR & RNA PCR.
 DNA PCR – proviral DNA is amplified.
 RNA PCR – for diagnosis & monitoring level of viraemia.
 Highly sensitive & specific test.
 Costly, indicated only when other methods give inconclusive
result.

6. 4) Antibody detection :-
 Simple & most commonly used technique.
 IgM Abs appears 1st usually in 3-4 weeks followed by IgG
Abs.
 IgM disappear in 8-10 weeks.
 Detection of HIV infection is made by detecting serum Abs to
viral proteins i.e. core (p24) or envelope (gp120 & gp41)
 2 types of serological test
i.e. 1. Screening tests &
2. Confirmatory tests.

7. Screening tests (E/R/S) :-
a) ELISA:- b) Rapid tests:- c) Simple tests:-
- Dot blot assay. - Based on the
- Particle agglutination. principle of
- HIV spot. ELISA.
- Comb test.
Confirmatory tests:-
a) Western blot test.
b) Indirect immunofluorescence test.
c) Radio immunoprecipitation assay

8. Screening tests:-
a) ELISA:-
Specimens to be collected for Antibody detection:-
• Blood / Serum / Plasma
• Saliva / Urine
 Good screening test.
 Highly sensitive & specific test.
 Direct solid phase ELISA is used.
 HIV Ag is prepared from HIV grown in the continuous cell line
or by recombinant technique.
 HIV viral Ag is coated on surface of microtitre wells.

9. HIV 1 / 2 ELISA procedure flow chart :
HIV viral Ag coated microtitre wells is taken
↓
Test serum is added
↓
Unbound serum is washed
↓
Anti-human goat immunoglobulin linked to a suitable enzyme is added
↓
Colour forming substrate is added
↓
Photometrically detectable colour is formed in positive test
↓
Add stop solution
↓
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 Pos. for HIV 1 / 2 Abs.

10. Fig. A :- ELISA kit . Fig. B :- Microtitre wells.
Fig. C :- ELISA washer Fig. D :- ELISA Reader.

11. b) Rapid tests:-
 Quick (30 minutes)
 Easy to perform
 No sophisticated instruments are required.
 Eg. Comb test, HIV spot test (Tri-dot),
Dot-blot assay etc.
Disadvantages:
 Tedious,
if large no. samples have to be tested at one time.

12. Principle of HIV tri-dot

13. c) Simple tests:-
 Simple, requires 1-2 hrs
 Easy to perform
 No sophisticated instruments are required.
 Based on the principle of ELISA.
 Eg. Particle agglutination test.
 Less sensitive than ELISA.

14. Confirmatory tests
a) Western blot:-
 Detection of HIV viral proteins.
 HIV proteins are separated by PAGE & blotted
onto nitrocellulose paper strip.
 Test serum is allowed to react with the strip
(Abs to HIV proteins if present combines with HIV
fragments).
 Strip is washed & treated with enzyme-conjugated
anti-human gamma globulin.
 Suitable substrate is added – produce color band
on the strip.
 Position of color band on the strip indicates the Ag
with which Abs had reacted.
 Abs to p24 (gag gene, core protein), p31 (pol gene,
reverse transcriptase) & gp41, gp120 or gp160 (env
gene, env protein) is commonly detected.
 Positive – if at least 2 bands appear against any 2 proteins.

15. b) Indirect immunofluorescence test:-
 HIV infected cells are fixed onto a clean glass slides & then reacted with
serum followed by fluorescein conjugate anti-human gamma globulin.
 Apple green fluorescence appear in the positive test under fluorescent
microscope.
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).

16. B) Non-specific Tests :-
1) TLC & DLC :-
leucopenia with lymphocytopenia.
2) T-lymphocyte subset assay :-
 Normal CD4:CD8 T-cell is 2:1
 Reversed to 0.5:1 in AIDS
 CD4 lymphocytes count is < 200/mm3
3) Platelet count :-
 Thrombocytopenia.
4) IgG & IgA level in Blood :-
 Both are raised.
5) Skin test for CMI :-
 CMI is diminished.

17. C) Tests for opportunistic infections
& tumour:-
Opportunistic infections :-
 Diagnosed by microscopy & culture.
 Tuberculosis, Salmonellosis, CMV, Herpes simplex, Variicella-zooster,
EB virus, Candidiasis, Cryptococcosis, Aspergillosis, Histoplasmosis,
Toxoplasmosis, cryptosporodiosis, Isosporosis, etc.
Malignancy / Tumour :-
 Kaposi's sarcoma, B-cell lymphoma,
Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma etc.

18. HIV Treatment

19. Anti-HIV Drugs
1. Nucleoside reverse transcriptase inhibitors:
2. Non-Nucleoside reverse transcriptase inhibitors:
3. Protease inhibitors:
4. Fusion inhibitors:
5. Highly active antiretroviral therapy (HAART):

20. 1) Nucleoside reverse transcriptase inhibitors
(NRTIs) :
 Azidothymidine (AZT)
 Dideoxycytidine (ddc)
 Dideoxyinosine (ddi)
 Abacavir (ABC)
 Lamivudine (3TC)
 Stavudine (d4T)
MOA :-
NRTIs block the reverse transcriptase,
an enzyme HIV needs to make copies of itself.

21. 2) Non-nucleoside reverse transcriptase
inhibitors (NNRTIs) :
 Nevirapine
 Delavirdine
 Efavirenz
MOA :-
NNRTIs bind to the RT and alter reverse transcriptase,
an enzyme HIV needs to make copies of itself.

22. 3) Protease inhibitors (PIs):
 Saquinavir (Invirase)
 Ritonavir (Norvir)
 Indinavir
 Nelfinavir
MOA :-
PIs block the HIV protease,
an enzyme HIV needs to make copies of itself.

23. 4) Fusion inhibitors:
 Enfuvirtide
MOA :-
Fusion inhibitors block the HIV from entering the CD4 cells
of he immune system.

24. 5) Highly active antiretroviral therapy (HAART):
Combination of -
 Indinavir / Azidothymidine / Lamivudine.
 Ritonavir / Azidothymidine / Lamivudine.
 Nelfinavir / Azidothymidine / Lamivudine.
 Nevirapine / Azidothymidine / Dideoxyinosine.
 Nevirapine / Indinavir / Lamivudine.

25.  In the current guidelines Azidothymidine (AZT) is recommended for the
treatment of asymptomatic / mild-symptomatic people with CD4 count < 500
& for the treatment of infected pregnant women's to reduce the transmission
of virus to fetus.
 Apart from antiretroviral therapy other measures in treatment of AIDS
includes – treatment & prophylaxis of opportunistic infections & tumors.

26. Mode of actions of anti-HIV drugs

27. Mode of actions of anti-HIV drugs

28. HIV patient after treatment