HIV Diagnostics and Laboratory Investigation

From Harrison's Principles of Internal Medicine, 22nd Edition (2025)

Overview

• 1985: Routine antibody screening for blood donors in the US
• 1996: p24 antigen capture assay added to blood bank screening
• 2002: Nucleic acid testing (NAT) licensed for routine blood donor screening

• Antibody testing alone: ~22 days
• p24 antigen testing: ~16 days
• NAT: ~12 days

1. Diagnosis of HIV Infection

A. Fourth-Generation EIA/ELISA (Recommended Initial Test)

• Detection of antibodies to HIV-1 and HIV-2
• Detection of p24 antigen of HIV

• Sensitivity: >99.5%
• Results scored as: Positive (highly reactive), Negative (nonreactive), or Indeterminate (partially reactive)
• Uses both natural and recombinant antigens, continuously updated

• Antibodies to class II antigens (post-pregnancy, transfusion, transplantation)
• Autoantibodies
• Hepatic disease
• Recent influenza vaccination
• Acute viral infections
• Administration of an HIV vaccine

B. Confirmatory Testing Algorithm (Per CDC, Fig. 208-31)

1. Start: Fourth-generation HIV-1/2 antigen-antibody immunoassay
   • If negative: HIV ruled out (retest only if recent exposure within 3 months)
   • If indeterminate or positive: Repeat the test
2. Repeat negative on two occasions: Initial reading was technical error - patient is negative
3. Repeat indeterminate or positive: Proceed to HIV-1/HIV-2 antibody differentiation immunoassay (e.g., Bio-Rad Geenius)
   • Positive for HIV-1 and/or HIV-2: Diagnosis confirmed
   • Negative or indeterminate: Proceed to HIV-1 RNA NAT (nucleic acid testing)
4. NAT positive + negative antibody test: Indicates acute HIV infection (early infection before antibodies develop)

Important note: The Western blot, previously used as a confirmatory test, is no longer recommended for this purpose. A positive fourth-generation assay confirmed by a second HIV-1- or HIV-2-specific immunoassay or plasma HIV RNA level is now adequate for diagnosis.

2. Nucleic Acid Testing (NAT) / HIV RNA

1. Diagnosis - especially in the acute/early window period when antibodies are absent
2. Monitoring disease progression and treatment response

Key viral load benchmarks:

• Elite nonprogressors: HIV RNA levels <50 copies/mL without antiretroviral therapy (ART)
• Long-term nonprogressors: Very low HIV RNA, little/no CD4+ T cell decline over years
• High HIV RNA levels = faster progression to symptomatic disease

3. Laboratory Monitoring of HIV Infection

A. CD4+ T Lymphocyte Count

• The average rate of CD4+ T cell decline in untreated patients is ~50/µL per year
• AIDS is diagnosed in individuals age ≥6 years with HIV + CD4+ T cell count <200/µL

B. Plasma HIV RNA (Viral Load) Monitoring

• Directly correlates with rate of disease progression
• Used to guide initiation of ART and to assess treatment response
• Patients with high HIV RNA levels progress to symptomatic disease faster than those with low levels
• Used alongside CD4+ count for comprehensive disease monitoring

4. Laboratory Investigation of Specific HIV-Associated Conditions

Pneumocystis jirovecii Pneumonia (PCP)

• Routine lab evaluation is of limited diagnostic help
• Mild leukocytosis (may be absent if prior neutropenia)
• Elevated LDH - common finding
• Arterial blood gases: Hypoxemia, decreased PaO2, increased alveolar-arterial (a-a) gradient - important for diagnosis AND staging severity
• Definitive diagnosis: Demonstration of organism in induced sputum, BAL, transbronchial biopsy, or open-lung biopsy
• PCR can detect P. jirovecii DNA in specimens where histology fails

5. Summary of Key Laboratory Tests in HIV Management