Investigation and Diagnosis of HIV

Who needs HIV testing ?
- sexually active
* ≥3 sexual partners in the last 12 months
-a male who has had sex with another male
-Drug addicts who take drugs by injection
*sharing needles and/or other equipment.
-STD patients
-Pregnant woman / woman who wants to make sure she is not infected by HIV before getting pregnant
-Patients who received a blood transfusion prior to 1985, or a sexual partner received a transfusion and later tested positive for HIV
-Health care worker with direct exposure to blood on the job

HIV Antibody Testing :
-Detects HIV antibodies in blood or other body fluids
-can be detected about 3 to 8 weeks after exposure to the virus
-Sample collected for testing:
*Blood
>inserting a needle into a vein in the arm
*Oral sample
>swab completely once around the outer part of the upper and lower gums
*Urine
-Screening tests available :
*ELISA
*Confirmatory test : Western Blot
*Home collection kit
-If you are tested too soon, result may be negative despite the fact that you are infected (false negative).
*may be tested using a p24 antigen test that can detect actual viral protein in the blood 1 to 3-4 weeks after exposure or an HIV RNA test (viral load) 
>detects the presence of the virus

ELISA - (Enzyme-linked Immunosorbent Assay Test)
-A common serological test
-For the presence of particular antigens or antibodies (Serum)
-Direct and indirect
*Direct : Employs monoclonal antibodies to detect the presence of particular antigens in a sample
*Indirect :To determine the presence of specific antibody (eg.HIV antibodies)

Indirect ELISA :
Appropriate antigen is first absorbed to the walls of a microtiter plate.
If serum contains antibodies against the antigens – bind to antigens (form complex)
Rinsing removes any antibodies that do not specifically attach to antigen
Addition of an antibody-enzyme conjugate
*Enzyme-conjugated anti-human IgG antibodies as reporter.
Enzyme-conjugated antibody will bind to the complex
Sample is rinsed again to remove any unbound antibodies
Substrate for the enzyme is added
Colour change – indicates the presence of antibodies that react against the antigens

-If test is taken right after the exposure and the result is negative
(False positive)
-If result is negative after “window period”
(HIV negative)
-If result is positive
*Confirmatory test is required
> WESTERN BLOT TEST

Western Blot Test :
HIV proteins are extracted.
Proteins are electrophoresed into a gel.
Proteins migrate through the gel when they are separated based upon size and charge. smaller proteins migrate through the gel faster than larger proteins.
Sufficiently separated proteins can be transferred to a solid membrane for WB analysis.
electric current is applied to the gel
All sites on the membrane which do not contain blotted protein from the gel can then be non-specifically "blocked"
*so that antibody (serum) will not non-specifically bind to them
*causing a false positive result.
membrane is cut into strips
*to facilitate testing of a large number of samples for antibodies directed against the blotted protein (antigen).
Procedure continues similar to ELISA.

Home Collection Kit :
-Approved by the US Food and Drug Administration (FDA)
-Take a sample of your blood from a finger at home and mail it to a testing center
-Hear your results later over the phone, along with appropriate counseling.

HIV Viral Load :
-the amount (viral load) of RNA (nucleic acid) of the human immunodeficiency virus (HIV) in blood.
-It is measured during the “window period” before the antibodies present
-Sample is collected from blood
*drawn by needle from a vein in your arm
-Can be measured after the therapy has started
*To evaluate whether the therapy is effective
-Test result:
*tests are reported as the number of HIV copies in a milliliter (copies/mL) of blood
*high viral load: from 5,000 to 10,000 copies/mL
*low viral load: between 40 to 500 copies/mL
>risk of disease progression is low.
-Tests available :
*p24 antigen count testing
>Followed by CD4 count
*Polymerase Chain Reaction (PCR)

p24 Antigen Count Test :
-p24 is a protein "shell" on the surface of HIV.
-help diagnose HIV viral protein in blood during early HIV infection.
*before HIV antibody is produced
-Result is positive : 1 week to 3-4 weeks after infection with HIV.
-Not detected until about 1 week after infection with HIV
*the virus haven’t multiplied to sufficient numbers to be detected.
-About 3-8 weeks after initial exposure, antibodies are produced.
*results of the p24 test will usually be negative although the person may be infected with HIV.
*antibodies bind to the p24 protein
>P24 protein no longer be detected in the blood.
 *HIV antibody test is used
-Later in the course of HIV, p24 protein become detectable if the disease is untreated.

CD4 Count :
-measurement of how many functional CD4 T-cells are circulating in the blood.
-CD4 cells - host cells that aid HIV in replication.
*HIV attaches to the CD4 cells
*allowing the virus to enter and infect the CD4 cells
*Damage the CD4 cells
-The lower the absolute CD4 count, the weaker the immune system.
-is measured by a simple blood test
-is reported as the number of CD4 cells per cubic millimeter of blood.
-Normal value :
*600 to 1200 cells per cubic millimter of blood
-Between 600 and 350 :
*In an HIV+ person, this range is considered "very good".
-Between 350 and 200 :
*The immune system is weakened
*HIV+ person may be at increased risk for infection and illness.
-Less that 200 :
*The immune system is severely weakened
*HIV+ person is at a much greater risk of opportunistic infections.