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 :

1) Ag. detection :- p24 Ag1) Ag. detection :- p24 Ag

2) Virus isolation :- virus culture2) Virus isolation :- virus culture

3) Viral nucleic acid detection :- PCR3) Viral nucleic acid detection :- PCR

4) Ab. Detection :- Anti -HIV antibody detection (IgM, IgG )4) Ab. Detection :- Anti -HIV antibody detection (IgM, IgG )

Non-specific tests :Non-specific tests :

1) TLC, DLC :-1) TLC, DLC :-

2) T-lymphocyte subset assays :-2) T-lymphocyte subset assays :-

3) Platelet count :-3) Platelet count :-

4) IgG & IgA level :-4) IgG & IgA level :-

5) Skin test for CMI :-5) Skin test for CMI :-

Tests for opportunistic infections & tumour :Tests for opportunistic infections & tumour :

A) Specific tests for HIV infectionA) Specific tests for HIV infection1) Antigen detection :-1) Antigen detection :-

The virus Ag. p24 & Reverse transcriptase (RT) detection in The virus Ag. p24 & Reverse transcriptase (RT) detection in

blood.blood.

p24 is the earliest virus marker.p24 is the earliest virus marker.

With seroconversion, Ab become detectable & p24 Ag With seroconversion, Ab become detectable & p24 Ag

disappear during long asymptomatic phase.disappear during long asymptomatic phase.

p24 antigenemia reappears with the onset of clinical p24 antigenemia reappears with the onset of clinical

disease.disease.

2) Virus isolation :- 2) Virus isolation :-

Virus is not routinely isolated.Virus is not routinely isolated.

HIV is Present in blood, body fluids, within CD4 HIV is Present in blood, body fluids, within CD4

Lymphocytes.Lymphocytes.

Patients lymphocytes are co-cultivated with uninfected Patients lymphocytes are co-cultivated with uninfected

human lymphocytes in presence of IL-2.human lymphocytes in presence of IL-2.

Virus replication is detected by RT activity & presence of Virus replication is detected by RT activity & presence of

viral Ag.viral Ag.

3) Viral nucleic acid detection :-3) Viral nucleic acid detection :-

Detected by PCR. Detected by PCR.

Useful for diagnosis in window period.Useful for diagnosis in window period.

2 type of PCR been used, DNA PCR & RNA PCR.2 type of PCR been used, DNA PCR & RNA PCR.

DNA PCR – proviral DNA is amplified.DNA PCR – proviral DNA is amplified.

RNA PCR – for diagnosis & monitoring level of viraemia.RNA PCR – for diagnosis & monitoring level of viraemia.

Highly sensitive & specific test.Highly sensitive & specific test.

Costly, indicated only when other methods give inconclusive Costly, indicated only when other methods give inconclusive

result.result.

4) Antibody detection :-4) Antibody detection :-

Simple & most commonly used technique.Simple & most commonly used technique.

IgM Abs appears 1IgM Abs appears 1stst usually in 3-4 weeks followed by IgG usually in 3-4 weeks followed by IgG

Abs.Abs.

IgM disappear in 8-10 weeks.IgM disappear in 8-10 weeks.

Detection of HIV infection is made by detecting serum Abs to Detection of HIV infection is made by detecting serum Abs to

viral proteins i.e. core (p24) or envelope (gp120 & gp41)viral proteins i.e. core (p24) or envelope (gp120 & gp41)

2 types of serological test 2 types of serological test

i.e. 1. Screening tests &i.e. 1. Screening tests &

2. Confirmatory tests.2. Confirmatory tests.

Screening tests (E/R/S) :-Screening tests (E/R/S) :-

a)a) ELISA:- b) Rapid tests:- c) Simple tests:-ELISA:- b) Rapid tests:- c) Simple tests:-

- Dot blot assay. - Based on the - Dot blot assay. - Based on the

- Particle agglutination. principle of- Particle agglutination. principle of

- HIV spot. ELISA.- HIV spot. ELISA.

- Comb test.- Comb test.

Confirmatory tests:-Confirmatory tests:-

a)a) Western blot test.Western blot test.

b)b) Indirect immunofluorescence test.Indirect immunofluorescence test.

c)c) Radio immunoprecipitation assayRadio immunoprecipitation assay

Screening tests:-Screening tests:-a) ELISA:-a) ELISA:-

Specimens to be collected for Antibody detection:-Specimens to be collected for Antibody detection:-

• • Blood / Serum / Plasma Blood / Serum / Plasma

• • Saliva / Urine Saliva / Urine

Good screening test.Good screening test.

Highly sensitive & specific test.Highly sensitive & specific test.

Direct solid phase ELISA is used.Direct solid phase ELISA is used.

HIV Ag is prepared from HIV grown in the continuous cell line HIV Ag is prepared from HIV grown in the continuous cell line

or by recombinant technique.or by recombinant technique.

HIV viral Ag is coated on surface of microtitre wells.HIV viral Ag is coated on surface of microtitre wells.

HIV 1 / 2 ELISA procedure flow chart :HIV 1 / 2 ELISA procedure flow chart :

HIV viral Ag coated microtitre wells is takenHIV viral Ag coated microtitre wells is taken

↓↓

Test serum is addedTest serum is added

↓↓

Unbound serum is washedUnbound serum is washed

↓↓

Anti-human goat immunoglobulin linked to a suitable enzyme is addedAnti-human goat immunoglobulin linked to a suitable enzyme is added

↓↓

Colour forming substrate is addedColour forming substrate is added

↓↓

Photometrically detectable colour is formed in positive testPhotometrically detectable colour is formed in positive test

↓↓

Add stop solutionAdd stop solution

↓↓

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.

Eg. Particle agglutination test.Eg. Particle agglutination test.

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

3) Platelet count :-3) Platelet count :- Thrombocytopenia.Thrombocytopenia.

4) IgG & IgA level in Blood :-4) IgG & IgA level in Blood :- Both are raised.Both are raised.

5) Skin test for CMI :-5) Skin test for CMI :-

CMI is diminished.CMI is diminished.

C) Tests for opportunistic infections C) Tests for opportunistic infections & tumour:-& tumour:-

Opportunistic infections :-Opportunistic infections :-

Diagnosed by microscopy & culture.Diagnosed by microscopy & culture.

Tuberculosis, Salmonellosis, CMV, Herpes simplex, Variicella-zooster,Tuberculosis, Salmonellosis, CMV, Herpes simplex, Variicella-zooster,

EB virus, Candidiasis, Cryptococcosis, Aspergillosis, Histoplasmosis, EB virus, Candidiasis, Cryptococcosis, Aspergillosis, Histoplasmosis,

Toxoplasmosis, cryptosporodiosis, Isosporosis, etc.Toxoplasmosis, cryptosporodiosis, Isosporosis, etc.

Malignancy / Tumour :-Malignancy / Tumour :-

Kaposi's sarcoma, B-cell lymphoma, Kaposi's sarcoma, B-cell lymphoma,

Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma etc.Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma etc.

HIV TreatmentHIV Treatment

Anti-HIV DrugsAnti-HIV Drugs1.1. Nucleoside reverse transcriptase inhibitors:Nucleoside reverse transcriptase inhibitors:

2.2. Non-Nucleoside reverse transcriptase inhibitors:Non-Nucleoside reverse transcriptase inhibitors:

3.3. Protease inhibitors:Protease inhibitors:

4.4. Fusion inhibitors:Fusion inhibitors:

5.5. Highly active antiretroviral therapy (HAART):Highly active antiretroviral therapy (HAART):

1) Nucleoside reverse transcriptase inhibitors1) Nucleoside reverse transcriptase inhibitors(NRTIs) :(NRTIs) :

Azidothymidine (AZT)Azidothymidine (AZT)

Dideoxycytidine (ddc)Dideoxycytidine (ddc)

Dideoxyinosine (ddi)Dideoxyinosine (ddi)

Abacavir (ABC)Abacavir (ABC)

Lamivudine (3TC)Lamivudine (3TC)

Stavudine (d4T)Stavudine (d4T)

MOA :-MOA :-

NRTIs block the reverse transcriptase,NRTIs block the reverse transcriptase,

an enzyme HIV needs to make copies of itself. an enzyme HIV needs to make copies of itself.

2) Non-nucleoside reverse transcriptase inhibitors 2) Non-nucleoside reverse transcriptase inhibitors (NNRTIs) :(NNRTIs) :

NevirapineNevirapine

DelavirdineDelavirdine

EfavirenzEfavirenz

MOA :- MOA :-

NNRTIs bind to the RT and alter reverse transcriptase,NNRTIs bind to the RT and alter reverse transcriptase,

an enzyme HIV needs to make copies of itself. an enzyme HIV needs to make copies of itself.

3) Protease inhibitors (PIs):3) Protease inhibitors (PIs):

Saquinavir (Invirase)Saquinavir (Invirase)

Ritonavir (Norvir)Ritonavir (Norvir)

IndinavirIndinavir

NelfinavirNelfinavir

MOA :- MOA :-

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):

Combination of -Combination of -

Indinavir / Azidothymidine / Lamivudine.Indinavir / Azidothymidine / Lamivudine.

Ritonavir / Azidothymidine / Lamivudine.Ritonavir / Azidothymidine / Lamivudine.

Nelfinavir / Azidothymidine / Lamivudine.Nelfinavir / Azidothymidine / Lamivudine.

Nevirapine / Azidothymidine / Dideoxyinosine.Nevirapine / Azidothymidine / Dideoxyinosine.

Nevirapine / Indinavir / Lamivudine.Nevirapine / Indinavir / Lamivudine.

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