The document discusses laboratory diagnosis of HIV infection and its treatment. [1] Several specific tests are used to detect HIV infection including antigen detection, virus isolation, viral nucleic acid detection and antibody detection. [2] Non-specific tests like complete blood count and CD4/CD8 ratio are also used. [3] Opportunistic infections are diagnosed using microscopy, culture and specific tests. HIV treatment involves the use of several classes of antiretroviral drugs that target different stages of the viral lifecycle alone or in combination therapy.

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