The CDC reports that at least 50% of the new U.S. HIV cases are transmitted by acutely infected individuals who are unaware that they are infected. "The prevalence of high-risk sexual behavior is reduced substantially after people become aware they are HIV+."• Therefore, HIV testing is integral to preventing the transmission of HIV. HIV infection can be diagnosed by serologic tests that detect antibodies against HIV-1 and HIV-2 and by virologic tests that can detect HIV antigens or ribonucleic acid (RNA).

The three main functions of HIV testing are (1) screening of donated blood and organs, (2) epidemiological surveillance of HIV prevalence or trends and (3) diagnosis of infection in individuals.

1. American blood banks employ two level testing:
   • 4th generation combination test that detects HIV p24 antigen and HIV IgM and IgG antibodies. This test shortens the detection period to 15–20 days.
   • HIV nucleic acid amplification technology (NAT) further reduces transfusion risk of HIV transmission to about one in two million. While the risk of HIV infection via transfusion has been nearly eliminated a slight risk remains due to the "testing window".
   
   Not all organ procurement organizations use the same testing standard as blood banks. A recent survey of organ procurement organizations (OPO) found that 52% of OPOs report always performing HIV NAT on deceased donors, whereas 24% of OPOs never perform HIV NAT. This may have resulted in a portion of the 200 unexpected transmissions of HIV, HBV, and HCV through transplants between 2007 and 2010.
2. HIV/AIDS surveillance enhances efforts to prevent HIV transmission, improve allocation of resources for treatment services, assist in evaluating the impact of public health interventions and tracking the rise of viral variants including: HIV-1 groups M,N,O,P, HIV-2.
3. HIV infected individuals present the greatest risk of transmission in the acute HIV infection (AHI) phase. During AHI, virus concentration rapidly increases in plasma and sexual fluids. The AHI phase begins 2-4 weeks after infection and continues until immune system rebounds as evidenced by a decline in HIV plasma concentration and increase in CD4⁺ counts. It is estimated that 50% of patients will seek medical care during AHI for symptoms resembling mononucleosis or influenza. The remainder of persons may have few to no symptoms.
   

The FDA considers the sensitivity and a specificity of HIV tests when granting approval. A test with high sensitivity is one that can detect minute amounts of HIV antigen or antibodies and thus has a low level of false negative responses. A test with high specificity produces very few false positives. There are two main types of HIV tests approved for use in America. The majority of tests administered are enzyme-linked immunosorbent assays (ELISA or EIA), that detect HIV antibodies in the serum or plasma but some tests can use whole blood, dried bloodspots, oral transudate or urine. Tests that detect HIV nucleic acids like RNA and DNA are used on pooled samples by blood banks and in individual patients when antibodies may be very low, when antibody tests are indeterminate or when maternal HIV antibodies may cause a false positive in neonatal testing.

• Enzyme-linked immunosorbent assay (ELISA or EIA) is often the first test used to screen for HIV. 3rd EIAs technology begins with synthetic HIV proteins adhered to the bottom of assay wells. The wells are flooded with diluted patient plasma. Patient plasma antibodies produced in response to HIV infection bind to the HIV proteins at the bottom of the wells. Secondary antibodies and signalling enzyme are added that binds to the HIV antigen/patient antibody complex allowing it to be identified. If the test is negative a follow-up test may be done later if the patient is high risk or if testing could have occurred before adequate levels of antibodies were produced (window period).
  • 4th generation EIA tests can identify p24 antigen or HIV antibody, but does not distinguish between them. The 4th generation EIA test has a shorter window period than 3rd generation EIA tests and can be used as a supplemental test instead of a Western Blot or NAT.
  • 3rd generation EIA tests like 4th Gen. tests are IgM based.
    
• Western Blot was once the "gold standard" for HIV testing due to antibody specificity. The western blot detects antibodies to multiple specific HIV proteins. A western blot is considered negative if no antibodies bind to any of the specific test HIV proteins including: gp 160, gp120, p68, p55, gp41, gp41, p40, p34, p24, p17.
  
  
• Rapid or point-of-care tests provide several advantages over laboratory based EIA and Western Blot including: results within an hour, simple procedure, oral test available. A positive Rapid Antibody Tests should be confirmed with a 4th generation EIA or a Western Blot. A negative Rapid test does not rule out HIV infection. It can take several months after HIV infection for the antibody response to reach detectable levels but most people reach a detectable level after six weeks.
  
  

  	Specimen Type	Sensitivity	Specificity	Approved for HIV-2	Read time
  OraQuick In-Home HIV-1/2 Test (OTC)	Oral fluid	92%	99.8%	Yes	20-40
  OraQuick ADVANCE Rapid HIV-1/2 Antibody Test	Oral fluid	99.3%	99.8%	Yes	20-40 minutes
  	Whole blood (venipuncture or finger stick	99.6%	100%
  	Plasma	99.6%	99.9%
  Uni-Gold Recombigen HIV	Whole blood (finger stick or venipuncture)	100%	99.7	No	10-12 minutes
  	Serum & Plasma	100%	99.8%
  Reveal G-3 Rapid HIV-1 Antibody Test	Serum	99.8%	99.1%	No	Read immediately
  	Plasma	99.8%	98.6%
  MultiSpot HIV-1/HIV-2 Rapid Test	Serum	100%	99.93%	Yes - differentiates between HIV-1 and HIV-2	Read immediately or up to 24 hrs
  	Plasma	100%	99.91%
  Clearview HIV 1/2 STAT-PAK	Whole blood (venipuncture or finger stick	99.7%	99.9%	Yes	15-20 minutes
  	Serum & Plasma	99.7%	99.9%
  Clearview COMPLETE HIV 1/2	Whole blood (venipuncture or finger stick	99.7%	99.9%	Yes	15-20 minutes
  	Serum & Plasma	99.7%	99.9%

Tests used to stage the disease or to evaluate the effectiveness of treatment include:

CD4 t-cell count

CD4⁺ cells aka Helper T cells are a subset of lymphocytes. CD4⁺ lymphocytes aren't phagocytic or cytotoxic, rather they activate other immune cells. When a specialized cell like an epithelial Langerhans cell captures an antigen, it migrates to the lymphatic system. In the lymphatic system the dendritic cell presents a processed portion of the antigen to a Helper T cells. The dendritic cell/antigen complex activates Helper T cells to secrete chemicals called lymphokines. These lymphokines stimulate cytotoxic T cells and B cells to grow and divide, attract neutrophils, and enhance the ability of macrophages to engulf and destroy microbes. HIV progressively destroys CD4⁺ cells as a result of viral reproduction. Loss of Helper T cells derails the immune response.

Viral load

The viral load indicates the number of copies of HIV in the blood. Viral load in conjunction with CD4 results provide an indication of disease stage and the effectiveness of treatment. The higher viral load the greater the risk for immune system damage that in turns leaves the body at risk for opportunistic infections.

HIV viral load may be measured by assays such as:

• PCR (polymerase chain reaction) method uses an enzyme to multiply the HIV in the blood sample. Then a chemical reaction marks the virus. The markers are measured and used to calculate the amount of virus. Roche and Abbott produce this type of test.
• NAT (nucleic acid amplification technology) method amplifies viral proteins to derive a count.
• bDNA (branched DNA) method combines a material that gives off light with the sample. This material connects with the HIV particles. The amount of light is measured and converted to a viral count.

A non-detectable result does not mean that there is no virus present; just that it was not detected. The newer versions of tests can now detect a load as few as 5 copies and as high as 1.5 million. A level of 55,000 requires antiretroviral treatment. Antiretroviral treatment is considered to be working if viral load drops by 90% within 8 weeks and continues to drop to less than 50 copies within 6 months. Different tests that give slightly different results. Therefore, when possible, people should be tested consistently with the same type of test.

HIV Phenotypic and Genotypic Testing

HIV's rapid mutation rate, rapid reproductive rate, and enormous population size eventually results in antiretroviral resistance. Phenotypic susceptibility testing assesses the ability of specific drugs or drug combinations to inhibit viral replication in cultured cells. Genotypic testing assesses the genetic composition of HIV variants in infected individuals to determine precisely which resistance mutations they carry.