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Laboratory Diagnosis of HIV Infection

Laboratory diagnosis of HIV infection involves specific tests like ELISA, rapid tests, Western blot, and viral load tests as well as non-specific tests like CD4 count. Diagnosis in the window period or for infants relies on antigen detection and viral load testing. Monitoring involves regular CD4 and viral load testing in response to antiretroviral therapy.

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6K views26 pages

Laboratory Diagnosis of HIV Infection

Laboratory diagnosis of HIV infection involves specific tests like ELISA, rapid tests, Western blot, and viral load tests as well as non-specific tests like CD4 count. Diagnosis in the window period or for infants relies on antigen detection and viral load testing. Monitoring involves regular CD4 and viral load testing in response to antiretroviral therapy.

Uploaded by

12. Akshit Atwal
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Laboratory diagnosis of HIV

infection
Laboratory Diagnosis
• 3Cs are very important -
• Consent
• Confidentiality
• Counselling.

The laboratory tests are divided in to:-


Specific tests
Non specific tests.
Laboratory Diagnosis
• Specific tests :
• 1. Screening tests (antibody detection)–
• a. ELISA
• b. Rapid/Simple test
• 2.Supplemental tests (antibody detection)–
• a. Western Blot assay
• b. Line Immunoassay (LIA)
• 3.Confirmatory tests :
• p24 antigen detection (after12-26 days of infection)
• Viral culture
• HIV RNA detection (after10-14 days of infection)
• HIV DNA detection- for diagnosis of paediatric infection
• Non-Specific tests :
• A. Total and differential leucocyte count
• B. T-lymphocyte subset assays (altered CD4:CD8 T cell
ratio)
• C. Platelet count
• D. IgG and IgA levels
• E. Skin tests for CMI
1. Screening tests (antibody detection)–
• a. ELISA :
• most commonly performed screening test at blood
banks and tertiary care sites.
• sensitive, specific & cost effective.
• 3rd generation ELISA : uses recombinant/synthetic
peptides as antigen to detect HIV antibodies.
• 4th generation ELISA : detects both HIV antibodies &
p24 antigen by using combination of
recombinant/synthetic peptides
• b. Rapid/Simple test :
• These tests are most commonly used in India.
• Require less than 30 minutes.
• No requirement of special equipment.

• Work on various principles such as-


• 1. Dot blot assays (Immunoconcentration /flow
through method) eg.- Tridot test
• 2. Immunochromatography (ICT, lateral flow assay)
• 3. Particle agglutination assays
• 4. Dip stick/Comb tests (Enzyme immune assay-
based tests)
Tridot test
Immunochromatography (ICT, lateral flow assay)
Dip stick/Comb test

A. B.
2.Supplemental tests (antibody detection)–
• Highly specific antibody detection tests
• Expensive
• Labor intensive.
• A. Western Blot :
• Most commonly used test
• Recommended by NACO.
• Works on principle of immunoblot technique.
• It detects individual antibodies in serum separately against
various antigenic fragments of HIV.
• - antibody to gag gene products (core protein-
p55,p40,p24,p18)
• -antibody to pol gene products (reverse transcriptase-p65/66,
p55/51, p31
• -antibody to env gene (envelope protein-gp120, gp160,gp41)

• Result- presence of at least two envelope bands (out of gp120,


gp160 or gp41) with or without gag or pol bands.
• presence of any two; out of p24,gp120, gp160 & gp41 bands.
Western Blot Test for HIV:
• B. Indirect immunofluorescence test :
• HIV infected cells are fixed on to glass slide
• Reacted with serum followed by fluorescein
conjugated antihuman gamma globulin.
• Positive result – apple green fluorescence appear
when examined under fluorescent microscope
Detection of p24 Core Antigen
• The p24 antigen becomes detectable after 12-26 days of infection and
lasts for 3-4 weeks.
• It is elevated during the late advanced stage of AIDS.
• P24 Ag is detected by 4th generation ELISA.
• It is less sensitive .

• Uses :
• Confirmation of diagnosis of HIV/AIDS
• Diagnosis of HIV during the window period
• To diagnose the last stage of HIV/AIDS (immune collapse) or CNS disease
• Diagnosis of HIV in infants
• Monitoring the progress of HIV infection
• To resolve equivocal western blot results.
Viral RNA detection
• It is the gold standard method for confirmation of HIV
diagnosis.
• Most sensitive and specific method.
• Viral RNA can be detected by polymerase chain reaction (PCR).
• RT-PCR is used for estimating viral load.
• Most important tool for monitoring the response to
antiretroviral therapy.
• RT-PCR can successfully differentiate between HIV-1 and HIV-2.
• Best tool for diagnosis of HIV during window period.
• Detects HIV earlier than all available methods (10-14 days after
post exposure )
Isolation of Virus
• Isolation is time consuming, expensive.
• Takes longer time (6weeks or more), not sensitive.
• Co-cultivation is the method used for isolation.
• PBMCs (Peripheral blood mononuclear cells)
obtained from patient are co cultured alonwith the
PBMCs from healthy donor
• Followed by detection of viral RNA or Ag in culture
suspension.
Non-Specific tests :

• CD4 T cell count:


• Carried out by flow cytometry
• Useful in assessing the risk of opportunistic
infections.
• Initiation of antiretroviral therapy.
• Monitoring the response to ART.
• Abnormal proteins – neoprotein and beta 2-
macroglobulin are elevted.
NACO Strategy for HIV Diagnosis :
Monitoring of HIV :
• Following tools are available for monitoring
the response to antiretroviral therapy -
• CD4 T cell count
• HIV RNA load
• P24 antigen detection
• Neoprotein and β2 macroglobulin level.
Diagnosis of Paediatric HIV Infection :
• All maternal antibodies would disappear by 18 months.
• IgG assays can be performed after 18 months of birth
•
• various methods for diagnosis of paediatric HIV
infection are as follows :-
• HIV DNA PCR
• HIV RNA detection
• P24 antigen detection
• IgG ELISA (only after 18 months of age)
HIV DNA PCR
• Most recommended method for diagnosis of
paediatric HIV.
• Baby is tested for HIV DNA PCR at 6 weeks by
DBS(Dry Blood Spot) collection.
• If found positive, then reconfirmed by a repeat HIV
DNA PCR.
• In Positive result – baby is initiated on a lifelong ART.
Diagnosis of HIV in window period :
• Window period: initial time between the exposure of
individual to HIV and appearance of detectable level of
antibodies in serum.

• During window period antibodies are detectable after 3


to 12 weeks of infection (not useful)
• Therefore,
• P24 antigen detection (30% cases can be detected by
4th generation ELISA) (12-26 days)
• HIV RNA detection by RT-PCR 10-14 days)
Treatment of HIV Infection
(ART Guideline, NACO 2018)
• TREAT ALL
• Highly active antiretroviral therapy (HAART)
• TLE regimen- Tenofovir + Lamivudine + Efavirenz
(HIV-1 infection)
• TL + LR regimen- Tenofovir-Lamivudine + Lopinavir-
Ritonavir (HIV1/2 co-infection & post exposure
prophylaxis for healthcare workers )

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