If an HIV enzyme-linked immunosorbent assay (ELISA) is positive, what confirmatory test(s) should be performed?

HIV Confirmatory Testing After Positive ELISA

Yes, a positive HIV ELISA must always be confirmed with supplemental testing—specifically a Western blot or HIV-1/HIV-2 antibody differentiation immunoassay—before making a definitive diagnosis. 1

Modern Testing Algorithm (Preferred Approach)

The CDC recommends a streamlined algorithm that does not require Western blot in most cases:

Step 1: Initial Screening

• Begin with a fourth-generation HIV antigen/antibody combination assay (detects both HIV antibodies and p24 antigen) 1, 2
• This test can detect infection approximately 2 weeks earlier than antibody-only tests 2

Step 2: If Screening Test is Reactive

• Perform an HIV-1/HIV-2 antibody differentiation immunoassay to distinguish between HIV-1 and HIV-2 1
• A positive differentiation assay confirms HIV infection—no Western blot needed 1

Step 3: If Differentiation Assay is Negative or Indeterminate

• Perform a nucleic acid amplification test (NAAT/HIV RNA) to rule out acute HIV-1 infection 1
• This detects virus during the window period before antibodies fully develop 1

Traditional Testing Algorithm (Still Valid)

Confirmatory Testing with Western Blot

• If using the traditional approach, a repeatedly reactive ELISA must be confirmed with Western blot 3
• A positive Western blot confirms HIV infection 3
• A negative Western blot indicates the person is uninfected (unless acute infection is suspected) 1

Indeterminate Western Blot Results

• If Western blot is indeterminate and HIV-2 EIA is not repeatedly reactive, the specimen is considered indeterminate 3
• Follow-up testing should occur 4-6 weeks later to exclude early HIV-1 infection 1
• Consider HIV RNA testing to detect acute infection 1

Alternative Confirmatory Methods

• Immunofluorescence assay (IFA) can substitute for Western blot 3
• Positive and negative IFA results are interpreted the same as Western blot 3
• Indeterminate IFA results require Western blot testing for resolution 1

Critical Pitfalls to Avoid

Never Diagnose Without Confirmation

• Never disclose a positive HIV diagnosis based on screening test alone—false positives can occur with devastating psychological and social consequences 1, 2
• All reactive screening tests must be confirmed before diagnosis 1

Window Period Considerations

• Antibody tests cannot rule out infection that occurred less than 6 months before testing 1, 4
• At least 95% of infected individuals develop detectable antibodies within 6 months 4
• For suspected acute infection (within first few weeks), perform NAAT rather than relying solely on antibody tests 4

Retesting Requirements

• Consider retesting with a second specimen for persons with positive Western blot results at first testing 3
• This helps exclude contamination or laboratory error 5

Special Population Considerations

HIV-2 Testing

• Consider HIV-2 testing in persons from endemic regions (West Africa) or their sexual partners 1, 2
• Test for HIV-2 when clinical evidence suggests HIV disease but HIV-1 tests are negative 1, 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 3

Infants Born to HIV-Positive Mothers

• Standard antibody tests are unreliable in infants <15-18 months due to maternal antibody transfer 1, 2
• Definitive diagnosis requires two positive HIV RNA PCR or viral culture tests on separate specimens 1, 2

Patients on PrEP or Antiretrovirals

• PrEP use can attenuate or delay HIV seroconversion in diagnostic assays 1
• Both laboratory-based Ag/Ab test AND diagnostic NAAT are required for follow-up testing due to potential viral suppression 6