HIV Confirmatory Testing
The recommended confirmatory test for HIV infection is the Western blot or an HIV-1/HIV-2 antibody differentiation immunoassay, depending on which testing algorithm is used. 1, 2
Modern Testing Algorithm (Preferred Approach)
The CDC and Association of Public Health Laboratories now recommend a newer algorithm that does not require Western blot for most cases: 1
- Initial screening: Fourth-generation HIV antigen/antibody combination assay (detects both HIV antibodies and p24 antigen) 1, 3
- If reactive: Perform an HIV-1/HIV-2 antibody differentiation immunoassay to distinguish between HIV-1 and HIV-2 1
- If differentiation assay is positive: Confirms HIV infection; proceed to viral load and CD4 testing for management 1
- If differentiation assay is negative: Perform qualitative or quantitative nucleic acid amplification test (NAAT/HIV RNA) to rule out acute HIV-1 infection 1, 4
This modern algorithm allows earlier detection of infection (4-7 days after detectable virus) compared to older methods. 1
Traditional Testing Algorithm (Still Valid)
The traditional approach remains acceptable and uses: 1, 2
- Initial screening: Enzyme immunoassay (EIA/ELISA) for HIV-1/HIV-2 antibodies 1, 5
- If repeatedly reactive: Confirmatory testing with Western blot 1, 2, 5
- Positive Western blot: Confirms HIV infection 1
- Negative Western blot: Person is considered uninfected (unless acute infection suspected) 1
- Indeterminate Western blot: Requires follow-up testing at 4-6 weeks and consideration of HIV RNA testing 2, 6
Alternative Confirmatory Methods
Immunofluorescence assay (IFA) can be used as an alternative supplemental test, with positive and negative results interpreted similarly to Western blot. 1 However, indeterminate IFA results (both infected and uninfected cells fluoresce) require Western blot testing for resolution. 1
Critical Pitfalls to Avoid
Never diagnose HIV based on screening test alone: All reactive screening tests must be confirmed before diagnosis. 2, 5 False-positive screening results do occur, and misdiagnosis has devastating psychological and social consequences. 1
Window period limitations: Antibody tests cannot rule out infection within 6 months of exposure, as HIV antibody is detectable in ≥95% of patients only after 6 months. 2 Fourth-generation tests reduce this window but don't eliminate it entirely. 1
Discordant results require RNA testing: When rapid tests and laboratory tests disagree, perform HIV RNA (NAAT) immediately to rule out acute infection or false-positive results. 4
Indeterminate Western blots are common in pregnancy: Nonspecific reactions producing indeterminate results occur more frequently in pregnant or parous women, though overall rates remain low (<1 in 4,000). 1 These typically represent false-positives rather than true infection. 1
Retesting recommended for all positive results: Consider obtaining a second specimen for confirmatory testing in persons with positive Western blot results to exclude specimen mislabeling or laboratory error. 1
Special Populations
Infants <15-18 months born to HIV-positive mothers: Standard antibody tests are unreliable due to maternal antibody transfer. 1, 2 Definitive diagnosis requires two positive HIV RNA PCR or viral culture tests on separate specimens. 1, 2
HIV-2 testing considerations: Test for HIV-2 in persons from endemic regions (West Africa), their sexual partners, or when clinical evidence suggests HIV disease but HIV-1 tests are negative. 2 If HIV-2 EIA is repeatedly reactive with negative/indeterminate HIV-1 Western blot, send specimen to state public health laboratory for HIV-2 supplemental testing. 1
Post-Confirmation Requirements
Once HIV infection is confirmed: 2
- Provide comprehensive medical evaluation or referral immediately 2
- Order baseline CD4 count and viral load for staging and treatment decisions 2
- Initiate behavioral and psychosocial support services as integral components of care 2
- Begin antiretroviral therapy discussions regardless of symptom status, as early intervention improves outcomes 2