What is the recommended approach for HIV (Human Immunodeficiency Virus) serological testing for diagnosis?

HIV Serological Testing for Diagnosis

The recommended approach for HIV diagnosis begins with a fourth-generation HIV antigen/antibody combination assay, followed by an HIV-1/HIV-2 antibody differentiation immunoassay if reactive, and then HIV RNA testing if results are discordant—this modern algorithm is faster and more accurate than the traditional ELISA/Western blot approach. 1

Modern Testing Algorithm (Preferred Approach)

Initial Screening:

• Perform a fourth-generation HIV antigen/antibody combination assay that detects both HIV antibodies and p24 antigen 1
• This test identifies infections earlier than antibody-only tests by detecting the p24 antigen during acute infection 1

If Initial Screen is Reactive:

• Immediately perform an HIV-1/HIV-2 antibody differentiation immunoassay to distinguish between HIV-1 and HIV-2 infection 1
• If the differentiation assay is positive, HIV infection is confirmed 1
• If the differentiation assay is negative (discordant result), perform a qualitative or quantitative nucleic acid amplification test (NAAT/HIV RNA) to rule out acute HIV-1 infection 1

Advantages of this approach: This algorithm does not require Western blot for most cases, reducing cost and turnaround time while maintaining accuracy 1. The fourth-generation assay detects infection earlier than traditional antibody-only tests 1.

Traditional Testing Algorithm (Still Valid)

For settings using traditional methods:

• Begin with enzyme immunoassay (EIA/ELISA) for HIV-1/HIV-2 antibodies 2, 1
• If repeatedly reactive (typically requires two reactive results), perform confirmatory Western blot testing 2, 1
• A positive Western blot confirms HIV infection 1
• A negative Western blot indicates the person is uninfected, unless acute infection is suspected 1
• An indeterminate Western blot requires follow-up testing at 4-6 weeks and consideration of HIV RNA testing 1

Important caveat: Although ELISA and rapid HIV tests are extremely accurate (>98% sensitivity and specificity), false-positive screening results can occur, particularly in patients with autoimmune disorders or pregnancy 2, 3. The Western blot will yield negative results in these false-positive cases 2.

Critical Testing Principles

Never diagnose HIV based on screening test alone:

• All reactive screening tests must be confirmed by Western blot, differentiation assay, or HIV RNA before diagnosis 2, 1
• False-positive screening results can have devastating psychological and social consequences 1
• Informed consent must be obtained before performing HIV testing 1

Window Period Considerations:

• Antibody tests cannot rule out infection that occurred less than 6 months before testing, as HIV antibody is detectable in ≥95% of patients within 6 months of infection 1
• False-negative results can occur during the "window period" of recent infection 1
• If acute HIV infection is suspected based on clinical presentation or high-risk exposure, perform HIV RNA testing even if antibody tests are negative 2, 1

Indeterminate Results Management:

• If HIV-1 Western blot is indeterminate and HIV-2 EIA is not repeatedly reactive, the specimen should be considered indeterminate 2
• Follow-up testing should be performed 6 months later to exclude early HIV-1 infection, especially if risk factors are present 2
• Counsel patients to follow risk-reduction guidelines during the intervening period 2

Special Population Considerations

Infants Born to HIV-Positive Mothers:

• For infants <15-18 months, standard antibody tests are unreliable due to maternal antibody transfer 1
• Definitive diagnosis requires two positive HIV RNA PCR or viral culture tests on separate specimens 1
• Do not use antibody-based testing for diagnosis in this age group 1

HIV-2 Testing Indications:

• Consider HIV-2 testing for persons from endemic regions (West Africa) or their sexual partners 2, 1
• Test for HIV-2 when clinical evidence suggests HIV disease but HIV-1 antibody tests are negative 2, 1
• Test for HIV-2 when HIV-1 Western blot shows unusual indeterminate pattern with gag (p55, p24, p17) plus pol (p66, p51, p32) bands but absent env (gp160, gp120, gp41) bands 2

HIV-2 Testing Algorithm:

• If HIV-1 Western blot is negative or indeterminate, perform HIV-2 EIA 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 2, 1

Post-Diagnosis Requirements

Immediate Actions:

• All persons with positive HIV tests must receive comprehensive medical evaluation or be referred for these services 1
• Obtain CD4 cell count with percentage upon initiation of care to stage disease and guide treatment decisions 2
• Perform HIV viral load testing to assess prognosis and establish baseline for monitoring treatment response 2
• Early intervention is critical even for asymptomatic patients, as newly diagnosed persons may be at different stages of infection 1

Confirmatory Documentation:

• Patients who have no documentation of their HIV serostatus or who were tested anonymously should have an HIV serologic test performed upon initiation of care 2
• This is especially important in asymptomatic patients with normal CD4 counts and undetectable or very low viral loads 2
• Patients may present with misinformation regarding previous test results or may be seeking subsidized services 2

Psychosocial Support:

• Provide behavioral and psychosocial services as an integral part of HIV care 1
• Help patients navigate challenges including accepting diagnosis, coping with stigma, maintaining physical and emotional health, and initiating behavioral changes to prevent HIV transmission 1

Common Pitfalls to Avoid

• Never disclose a positive HIV diagnosis without confirmatory testing—screening tests alone are insufficient 1
• Do not overlook acute HIV infection—if clinical suspicion is high despite negative antibody tests, perform HIV RNA testing 2, 1
• Avoid misinterpreting indeterminate results—these require follow-up testing, not immediate diagnosis 2, 1
• Do not use antibody tests in infants <15-18 months born to HIV-positive mothers—use HIV RNA PCR instead 1
• Remember that CD4 cell counts can be affected by medications and intercurrent illnesses—interpret with caution during acute illness 2