How do you interpret HIV labs, including antibody tests, p24 antigen tests, and viral load tests?

How to Interpret HIV Laboratory Tests

Use fourth-generation antigen/antibody combination tests as your primary screening tool, followed by confirmatory HIV-1/HIV-2 differentiation testing for reactive results, and add HIV RNA testing when acute infection is suspected or when results are discordant. 1, 2

Initial Screening Strategy

Fourth-Generation Antigen/Antibody Tests (Preferred)

• Fourth-generation combination assays detect both HIV p24 antigen and HIV-1/HIV-2 antibodies (IgM and IgG), becoming positive 18-45 days post-infection and 4-7 days after detectable virus by nucleic acid testing. 1, 2
• These tests allow earlier diagnosis than antibody-only tests by detecting p24 antigen during the window period before antibody seroconversion occurs. 1
• Laboratory-based fourth-generation tests are more sensitive than rapid point-of-care tests, particularly for acute infection. 2

Antibody-Only Tests (Older Generation)

• Third-generation antibody tests detect HIV IgM and IgG, becoming positive 7-14 days after detectable virus. 1
• At least 95% of infected individuals develop detectable antibodies within 6 months of infection. 2
• Antibody tests cannot definitively rule out infection that occurred less than 6 months before testing. 2

Confirmatory Testing Algorithm

When Screening Test is Reactive

1. Perform HIV-1/HIV-2 antibody differentiation assay to distinguish between HIV-1 and HIV-2 infection. 1
2. If differentiation assay is negative (suggesting possible acute infection or false-positive screening), proceed immediately to HIV RNA testing. 1
3. If differentiation assay is positive, confirm with HIV RNA viral load testing and CD4 count to guide management. 1

Western Blot Interpretation (Traditional Method)

• HIV infection is confirmed by repeatedly reactive EIA plus positive Western blot. 1
• Indeterminate Western blot results (particularly showing gag plus pol bands without env bands) warrant HIV-2 testing and repeat testing in 1 month to distinguish recent seroconversion from false-positive results. 1

HIV RNA Viral Load Testing

Diagnostic Use

• HIV RNA testing is NOT FDA-licensed for diagnosis but is critical for detecting acute infection when antibody/antigen tests are negative or indeterminate. 1
• In acute infection, viral loads are typically >100,000 copies/mL (>5 log₁₀). 1
• Low viral load results (<5,000 copies/mL) may represent false-positives and require confirmation with a second specimen. 1, 3

Monitoring Established Infection

• Report results in both copies/mL (for therapy initiation decisions) and log₁₀ transformation (for monitoring changes). 1
• Changes >0.5 log₁₀ (threefold) represent clinically significant viral load changes beyond normal biological and assay variability. 1
• Use the same assay method consistently, as different assays can differ by >2-fold for the same sample. 1
• Viral load rebound after suppression may indicate drug resistance, poor adherence, drug interactions, or concurrent infections/vaccinations. 1

Critical Pitfalls

• Transient viral load elevations can occur in clinically stable patients; confirm sustained changes before modifying therapy. 1
• Different assays have varying performance with non-B HIV-1 subtypes; Roche Amplicor version 1.0 underdetects subtypes A, E, F, and G. 1
• All current assays have difficulty with HIV-1 group O and do not detect HIV-2. 1

p24 Antigen Testing

Clinical Utility

• p24 antigen appears 14-22 days after infection, before antibody development, making it useful for detecting acute infection. 1
• Free p24 antigen is detectable in 37-95% of patients with CD4 counts 200-500 cells/mm³. 1
• p24 typically becomes undetectable after antibody formation due to immune complex formation with anti-p24 antibodies. 1, 4

Interpretation Challenges

• p24 antigen levels are often suppressed by high antibody levels even when viral RNA is >1,000 copies/mL. 4
• Immune complex dissociation (ICD) methods can increase p24 detection to 75-100% of infected patients. 1
• p24 concentrations may be higher than expected based on RNA levels due to non-virion-associated antigen. 4

Special Testing Scenarios

Acute HIV Infection (Within First Few Weeks)

• Perform HIV RNA testing (nucleic acid test) rather than relying solely on antibody tests, as NAT detects infection 10-14 days after exposure. 2
• Diagnostic NAT can detect acute infection approximately 1 week before fourth-generation antigen/antibody tests. 2
• Typical presentation: high viral load (>100,000 copies/mL), negative or indeterminate antibody tests. 1

Patients on PrEP or Recent Antiretroviral Exposure

• Both laboratory-based antigen/antibody test AND diagnostic NAT are required due to potential viral suppression from antiretrovirals. 2
• PrEP can attenuate or delay HIV seroconversion on all diagnostic assays (antigen, antibody, and nucleic acid detection). 1
• A reactive rapid test in someone on PrEP should be confirmed with combined antibody/antigen testing plus HIV RNA testing. 1

Post-Exposure Testing Timeline

• Initial testing at 4-6 weeks after exposure. 2
• Confirmatory testing at 3 months (12 weeks) after exposure. 2
• Final testing at 6 months in rare cases of delayed seroconversion, particularly with HCV co-infection. 2
• For patients who received post-exposure prophylaxis, final testing should occur 12 weeks after PEP initiation (8 weeks after completion) to account for antiretroviral washout. 2

Neonatal Testing

• Serologic testing is unreliable in neonates due to maternal antibody persistence; use HIV RNA viral load or proviral DNA testing. 1
• Quantitative HIV-1 RNA is as sensitive as qualitative RNA or proviral DNA for diagnosing neonatal infection. 1

HIV-2 Testing Indications

• Test for HIV-2 when patients have epidemiologic risk (from or sexual partners from West Africa, received transfusions in endemic areas). 1
• Test for HIV-2 when Western blot shows unusual indeterminate pattern with gag plus pol bands but no env bands. 1
• 60-91% of HIV-2-infected persons test reactive on HIV-1 whole-virus lysate EIA, but require HIV-2-specific Western blot for confirmation. 1

Quality Assurance Considerations

Essential Report Elements

• Assay method and test kit manufacturer must be noted on all viral load reports. 1
• Reportable ranges as specified in manufacturer's package insert. 1
• Both copies/mL and log₁₀ values for viral load. 1

Items to Exclude from Reports

• Nonstandard abbreviations, jargon, or statements regarding AIDS, counseling, or confidentiality. 1
• Technical details (primer information, optical density, dilution factors, assay numbers). 1
• Statements regarding test sensitivity or specificity. 1