HIV Screening: Overview

Who Should Be Screened

• Clinicians do not consistently ask about risk factors
• Patients may under-report risk behaviors
• Not all HIV-infected patients have identifiable risk factors

The Standard Diagnostic Algorithm (CDC Recommended)

Step 1 - Initial Screen: 4th Generation Antigen/Antibody Combination Immunoassay (ELISA)

• Detects: HIV-1/HIV-2 antibodies (IgM + IgG) AND the p24 capsid antigen simultaneously
• Window period: ~16-18 days from infection (rapid point-of-care version: ~19-20 days)
• This is the current standard of care recommended by the CDC
• Some manufacturers require repeating the test if initially reactive

Step 2 - Confirmatory: HIV-1/HIV-2 Antibody Differentiation Immunoassay

• Performed on any specimen reactive on the initial screen
• Differentiates between HIV-1 and HIV-2 antibodies
• Window period: ~32-36 days

Step 3 - Reflex: Nucleic Acid Test (NAT/NAAT) for HIV-1 RNA

• Used when the initial 4th gen screen is positive but the differentiation assay is negative or indeterminate
• This resolves early acute infections where antibodies haven't yet formed
• Shortest window period: RNA detectable at ~10-12 days (qualitative) or ~6-10 days (quantitative viral load assay)
• Dermatology 5th Ed (Elsevier); Red Book 2021; Quick Compendium of Clinical Pathology 5th ed

Test Types and Window Periods

• Dermatology 5th Ed, Table 78.4

5th Generation Tests (Emerging)

• Simultaneously detect and differentiate p24 antigen vs. HIV-1 antibodies vs. HIV-2 antibodies as three separate results
• Theoretical advantage: a p24-positive/antibody-negative result indicates very early infection
• FDA-cleared for initial screening but not yet widely used for confirmation
• Quick Compendium of Clinical Pathology; Jawetz Medical Microbiology 28th Ed

Western Blot (Historical Confirmatory Test)

Special Populations

• Maternal antibodies can persist for months, making antibody-based tests unreliable
• Use HIV DNA PCR (proviral DNA PCR) for early diagnosis in neonates
• Test at birth, at 2 weeks, at 4-6 weeks, and at 4-6 months

• Order HIV RNA (viral load/NAT) directly - antibody tests will be falsely negative
• Acute retroviral syndrome occurs 7-14 days after acquisition and lasts 5-7 days; many patients are not ill enough to seek care

• Antibody assays may also be falsely negative due to immune exhaustion - use virologic (NAT) testing

Other Diagnostic Tests (Monitoring, Not Primary Screening)

Point-of-Care and Home Tests

• Rapid tests (e.g., OraQuick): results in <30 minutes from oral fluid or fingerstick blood; slightly longer window period (~19-20 days for 4th gen rapid)
• At-home mail-in tests: fingerprick sample sent to lab, results next day
• Point-of-care tests have a longer window period than lab-based assays and should be confirmed if reactive

Consent

Triple Testing in HIV - Two Meanings

Meaning 1: The 3-Step (Triple) HIV Laboratory Diagnostic Algorithm

Step 1 - 4th Generation Antigen/Antibody Immunoassay (Initial Screen)

• Detects HIV-1/HIV-2 antibodies (IgM + IgG) AND HIV-1 p24 antigen simultaneously
• Highly sensitive - designed to catch all true positives including acute infection
• Window period: ~16-18 days
• If non-reactive → report as HIV negative (no further testing needed)
• If reactive → proceed to Step 2

Step 2 - HIV-1/HIV-2 Antibody Differentiation Immunoassay (Confirmatory)

• Differentiates between HIV-1 antibodies vs. HIV-2 antibodies separately
• If reactive for HIV-1 or HIV-2 → confirmed HIV diagnosis; initiate ART referral
• If reactive but undifferentiated (both HIV-1 + HIV-2) → repeat differentiation test
• If non-reactive or indeterminate → proceed to Step 3
• Window period: ~32-36 days

Step 3 - Nucleic Acid Test (NAT/NAAT) for HIV-1 RNA (Reflex)

• Used specifically when Step 1 is reactive but Step 2 is negative/indeterminate
• This scenario = acute HIV infection (virus present, antibodies not yet formed)
• If NAT positive → confirmed acute HIV-1 infection
• If NAT negative → false positive on Step 1; report as HIV negative
• Window period: RNA detectable at ~10-12 days
• Turnaround time for the combined Step 1+2 assay is ~60 minutes; NAT can take a few days
• Tintinalli's Emergency Medicine; Goldman-Cecil Medicine; Red Book 2021

Step 1: 4th gen Ag/Ab EIA
     |
     ├─ Non-reactive → HIV NEGATIVE
     |
     └─ Reactive ↓
         Step 2: HIV-1/HIV-2 Differentiation Assay
              |
              ├─ Reactive (HIV-1 or HIV-2) → CONFIRMED HIV
              |
              └─ Negative/Indeterminate ↓
                  Step 3: HIV-1 RNA NAT
                       |
                       ├─ Positive → ACUTE HIV INFECTION
                       └─ Negative → FALSE POSITIVE (HIV negative)

Meaning 2: Triple Elimination Screening (Antenatal / Pregnancy)

Why Triple?

• HIV: ~15-45% transmission to infant
• Hepatitis B: ~70-90% transmission if mother is HBeAg positive
• Syphilis: can cause congenital syphilis, miscarriage, or stillbirth

WHO Prequalification (July 2025 - Very Recent)

• Determine™ HIV Early Detect (HIV-1/2 Ag/Ab)
• Determine™ HBsAg 2
• Determine™ Syphilis TP

Antenatal Protocol (NHS/WHO Standard)

• Offered at the booking appointment (ideally before 10 weeks gestation)
• A single blood draw from the arm
• Testing should be as early as possible so treatment can begin promptly
• If positive: specialist midwife referral, further confirmatory workup, and care planning
• Opt-out approach encouraged (patient informed but must actively decline)
• Repeat testing in 3rd trimester for high-risk women
• NHS Antenatal Screening; WHO 2025 prequalification notice

Cost-Effectiveness

Key Takeaway