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  1. 1. Laboratory Diagnosis of HIV Infection & its Treatment. Presented By :- JITENDRA KUMAR PANDEY PG, Medical Student MGM MEDICAL COLLEGE, MUMBAI
  2. 2. Laboratory Diagnosis of HIV Infection.Specific tests for HIV infection :1) Ag. detection :- p24 Ag2) Virus isolation :- virus culture3) Viral nucleic acid detection :- PCR4) 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. 3. A) Specific tests for HIV infection1) 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. 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. 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. 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. 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. 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. 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. 10. Fig. A :- ELISA kit . Fig. B :- Microtitre wells.Fig. C :- ELISA washer Fig. D :- ELISA Reader.
  11. 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. 12. Principle of HIV tri-dot
  13. 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. 14. Confirmatory testsa) 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. 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. 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/mm33) Platelet count :- Thrombocytopenia.4) IgG & IgA level in Blood :- Both are raised.5) Skin test for CMI :- CMI is diminished.
  17. 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 :- Kaposis sarcoma, B-cell lymphoma, Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma etc.
  18. 18. HIV Treatment
  19. 19. Anti-HIV Drugs1. Nucleoside reverse transcriptase inhibitors:2. Non-Nucleoside reverse transcriptase inhibitors:3. Protease inhibitors:4. Fusion inhibitors:5. Highly active antiretroviral therapy (HAART):
  20. 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. 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. 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. 23. 4) Fusion inhibitors: Enfuvirtide MOA :- Fusion inhibitors block the HIV from entering the CD4 cells of he immune system.
  24. 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. 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 womens 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. 26. Mode of actions of anti-HIV drugs
  27. 27. Mode of actions of anti-HIV drugs
  28. 28. HIV patient after treatment