Laboratory Diagnosis of HIV Infection & its Treatment. Presented By :- JITENDRA KUMAR PANDEY PG, Medical Student MGM MEDICAL COLLEGE, MUMBAI
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 :
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.
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.
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.
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.
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
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.
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.
Fig. A :- ELISA kit . Fig. B :- Microtitre wells.Fig. C :- ELISA washer Fig. D :- ELISA Reader.
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.
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.
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.
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).
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.
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.
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.
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.
4) Fusion inhibitors: Enfuvirtide MOA :- Fusion inhibitors block the HIV from entering the CD4 cells of he immune system.
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.