What are the confirmatory tests for Human Immunodeficiency Virus (HIV)?

Confirmatory Tests for HIV

The primary confirmatory tests for HIV are Western blot (WB) and immunofluorescence assay (IFA), which must be performed after a positive screening test to definitively diagnose HIV infection. 1

HIV Testing Algorithm

Initial Screening

1. Screening Test: The diagnostic process begins with a sensitive screening test
   • Traditional approach: Enzyme-linked immunosorbent assay (ELISA) or enzyme immunoassay (EIA)
   • Modern approach: 4th generation combination test that detects both HIV-1/HIV-2 antibodies and HIV-1 p24 antigen 2
   • Rapid HIV antibody tests are also available, providing results in 20-60 minutes 1

Confirmatory Testing

1. When screening test is positive/reactive:

   • Confirmation with a more specific supplemental test is mandatory 1
   • Western blot (WB): Detects antibodies to specific HIV antigens
   • Immunofluorescence assay (IFA): Alternative confirmatory method
   • Both tests must be positive to confirm HIV infection 1

2. Modern confirmatory algorithm (CDC/FDA recommended):

   • HIV-1/HIV-2 antibody differentiation immunoassay
   • If negative, proceed to nucleic acid testing (NAT) for HIV-1 RNA 2
   • This algorithm has replaced traditional Western blot in many settings as it can detect early infections more effectively

Special Considerations

Acute HIV Infection

• If acute retroviral syndrome is suspected but antibody tests are negative:
  • Nucleic acid testing (NAT) for HIV RNA should be performed 1, 2
  • A positive NAT confirms acute HIV infection before antibodies develop

Indeterminate Western Blot Results

• Indeterminate results may occur in:
  • Early seroconversion
  • Advanced HIV disease/AIDS (some patients may not produce antibodies to specific HIV antigens) 3
  • Pregnant women (higher rates of indeterminate results - up to 16.67%) 4
• Management of indeterminate results:
  • Retest in 1 month to distinguish between recent seroconversion and negative status 1
  • Consider HIV RNA testing to resolve indeterminate status 4
  • Most common indeterminate WB patterns: single p24 band, or gp160 and p24 bands 4

HIV-2 Testing

• Routine testing for HIV-2 is not recommended in the US except in:
  • Blood centers
  • Persons from HIV-2 endemic regions (parts of West Africa, Angola, Mozambique, Portugal, France)
  • Sexual partners of persons from endemic regions
  • Cases with clinical evidence of HIV disease but negative HIV-1 tests 1

Pediatric HIV Testing

• For infants <18 months: HIV nucleic acid amplification tests (detecting HIV DNA or RNA) are required for diagnosis due to the presence of maternal antibodies 1
• Definitive determination of HIV infection in infants should be based on:
  • Laboratory evidence of HIV in blood or tissues by culture, nucleic acid, or antigen detection
  • Or presence of antibody with compatible immunologic profile and clinical course 1

Test Performance

• 4th generation combination tests offer earlier detection (4-7 days after infection) compared to antibody-only tests (7-14 days) 2
• Modern 4th generation tests demonstrate excellent performance with sensitivity of 100% and specificity of 99.99% 2
• Rapid HIV tests have shown high sensitivity (100%) and specificity (99.9%) with positive predictive value of 90% in some studies 1

Common Pitfalls

• False-negative results may occur during the "window period" (time between infection and detectable antibodies)
• HIV antibody tests cannot rule out infection that occurred less than 6 months before testing 1
• Some end-stage HIV/AIDS patients may produce insufficient antibodies, leading to indeterminate or negative results on some immunoassays 3
• Relying solely on commercial line immunoassays without nucleic acid testing may delay diagnosis in difficult cases 3

Remember that informed consent must be obtained before HIV testing, and positive test results require appropriate medical and psychosocial evaluation and monitoring services 1.