Laboratory diagnosis of HIV infection

 

Learning objectives:

1.    Kinetics of immune response in reference to various markers used for laboratory diagnosis

2.    Pre-requisite for HIV testing

3.    NACO (National AIDS Control Organization) strategies for HIV diagnosis in various conditions / situations

4.    Specific tests for HIV infection:

Screening tests (ERS) (antibody detection)

Rapid/ Simple test / ELISA

Supplemental tests (antibody detection)

Western blot assay

Confirmatory tests: Detection of viral RNA / proviral DNA

5.    Tests to measure prognosis / monitoring progress of HIV infection

Kinetics of immune response –:

·       Viremia- Viral entry into the body leads to a transient period of high-level viremia and p24 antigenemia.

·       Window period- 3 to 12 weeks. Virus remains in the blood but antibody does not appear in the blood.

·       Later on Humoral response is evidenced by formation of antibodies against different antigens.

 

Following care should be taken (3Cs) while performing the test for HIV.

·       Consent in written format should be taken before the test is done.

·       Confidentiality of a positive test result is must. 

·       Counselling (Pre-test and Post-test Counselling) should be provided to motivate the individual and induce behavioral changes.

 

NACO STRATEGY FOR HIV DIAGNOSIS

NACO (National AIDS Control Organization, India) has formulated a strategic plan for HIV diagnosis.

Specific tests for HIV infection:

·       Screening tests (ERS) (antibodydetection)

o   Rapid/ Simple test / ELISA

·       Supplemental tests (antibody detection)

o   Western blot assay

·       Confirmatory tests

A)  Screening tests (antibody detection tests):

·       Sample taken : blood in plain vaccute

1.    Rapid/ Simple test

Advantage:

·       Easy to perform and provide quick results (takes < 30 minutes). 

·       Do not require special equipments.

Disadvantage:

·       False positive results

·       Results of a screening test should never be used as the final .It is always subjected to confirmatory tests

          Most commonly used rapid tests in India:

a.     Dot blot assays

b.    Immunochromatography

c.     Dip stick/ comb tests

1.    ELISA

Advantage:

·       Highly sensitive and specific

·       Adaptable to large number of samples  and Cost effective.

Disadvantage:

·       Require special equipments like ELISA reader and washer

·        Require training of laboratory technician. Takes 2-4 hours.

A)  Supplemental test or confirmatory test:

1)   Western blot:

·       Principle: It detects individual antibodies in serum separately against various antigenic fragments of HIV such as, Antibody to gag gene products, pol gene productsand env gene products. The antigen antibody complexes appear as distinct bands on nitrocellulose strip.

·       Sample taken: Blood in plain vaccute

·       WHO criteria i.e. presence of at least two envelope bands (out of gp120, gp160 or gp41) with or without gag or pol bands

·       CDC criteria-presence of any two out of p24, gp 120, gp160, gp41 bands

2)   Viral RNA detection

·       The ‘gold standard’ method for confirmation of HIV diagnosis.

·       Real time RT-PCR- for estimating viral load

·       Sample taken: blood in EDTA(plasma)

·       Uses:

·       For confirmation of diagnosis of HIV/AIDS(can detects even few copies of viral RNA)

·       Diagnosis of HIV during window period (earlier than all available methods :12 days post exposure)

·       Viral load monitoring-monitoring the response to antiretroviral therapy.

Early Infant Diagnosis: Do not perform antibody estimation test . HIV DNA PCR is the test of Choice.

Sample – Blood drawn from heel and taken on filter paper and dried - DBS (dried blood spots)/plasma