HIV Lab Tests: From Screening to Confirmation
🔍 Case: a 25 year old male presents with sore throat and fevers x 2 weeks. History of multiple sexual partners, no known high-risk exposures.
What is our differential? For sub-acute/chronic fevers and flu like symptoms I like to think of 3 major mechanisms: infectious, autoimmune/inflammatory, and malignant.
One key differential is HIV. Why? First, the acute stage of HIV illness can present with fevers, flu-like symptoms (sore throat, lymphadenopathy, myalgias), rash; and this patient reports having multiple sexual partners. With these clues alone, our pre-test probability is at least intermediate. Second, it is a must-not miss diagnosis.
What is HIV?
Human immune deficiency virus is a sexually transmitted and blood-borne infection (exposure to vaginal fluid, semen, rectal fluid, blood, and human milk from a person with HIV) (1).
What is the natural progression of HIV?
Remember the 3 stages of infection (1).
Acute: 40-90% of patients experience non-specific symptoms - e.g. sore throat, fever, lymphadenopathy, rash, myalgias. Symptoms manifest ~ 2-6 weeks post exposure, lasting 10-15 days. Tip: can see hematologic disturbances (lymphopenia, thrombocytopenia) on CBC.
Asymptomatic (latent) stage: HIV infects + replicates in CD4 T lymphocytes. By 10 year post infection, ~50% have advanced HIV, ~30% have milder symptoms, <20% asymptomatic if untreated. Levels of CD4 counts are correlated with possible clinical manifestations.
Acquired immunodeficiency syndrome (AIDs): HIV + AND one or more clinical illnesses that characterize AIDs (e.g. opportunistic infections, primary neurologic diseases, malignancy).
🔍 Back to the case: since HIV is one of our must-not-miss diagnoses with this presentation, what lab test do we start with?
💡 Tip: remember to ask for patient consent to order HIV testing!
BOTTOM LINE: Patients with HIV can present during the acute stage of illness, latent stage (asymptomatic) or if it has progressed to AIDs (HIV positive and clinical illnesses that characterize AIDs).
Lab Tests for HIV: What do we Order?
All labs performing HIV screening in Canada use fourth generation (combination) HIV tests, which detect both HIV antibodies and the HIV p24 antigen (2) – this is commonly done in the US too (varies by state).
How accurate is the p24 antigen/HIV antibody test?
This combination test has improved sensitivity in early infection, and nearly 100% sensitivity and specificity for chronic infection (meaning there is a risk of false negative results during acute stage of illness).
Some people will have a positive (reactive) result as early as 15-20 days after HIV exposure, by 3 weeks about 65-70% will have a reactive result, by 6 weeks >99%; BUT the window period can be up to 12 weeks (2,3).
Clinical application: if we test too early, we may get a false negative result. If you have an intermediate-high pre-test probability for HIV infection, consider re-testing your patient up to 12 weeks post-exposure.
BOTTOM LINE: p24 antigen/HIV antibody lab test is commonly used as a first line test for suspected HIV - it has a window period of up to 12 weeks (will detect most cases of HIV at ~6 weeks, but can be up to 12 weeks).
Is Rapid HIV testing (point of care) accurate?
Rapid HIV tests require just a few drops of blood from a finger prick, with results available within minutes. However, these tests only detect HIV antibodies, meaning they have a longer “window period” — typically between 20 days and 12 weeks after exposure (2).
If a rapid test is reactive (positive), the result must be confirmed with a laboratory-based p24 antigen/HIV antibody test. If the result is negative, health professionals recommend repeat testing up to 12 weeks after the potential exposure to ensure accuracy (2).
What is the HIV PCR Test?
The HIV PCR test measures viral load by detecting HIV’s genetic material (RNA or DNA) directly in the blood. It is especially useful when an early diagnosis is needed—for example:
Detecting acute HIV infection within the first few weeks after a high-risk exposure
Testing infants born to mothers with HIV, since maternal antibodies can affect standard tests
Clarifying uncertain or indeterminate results from p24 antigen/HIV antibody testing
BOTTOM LINE:
The HIV PCR test detects the virus itself, not just antibodies, making it the go-to test for early diagnosis, infant testing, and resolving unclear results.
Rapid point-of-care HIV tests give fast results, but always confirm and re-test if risk is high
What happens if the p24 antigen/HIV antibody test returns reactive (positive?)
If the result is reactive or indeterminate, a confirmatory test is performed using an HIV-1/HIV-2 antibody differentiation assay. This test looks for antibodies to at least two distinct HIV proteins. The confirmatory test is highly accurate, with a specificity greater than 99.9%, meaning the chance of a false positive result is extremely low (4).
What happens if the p24 antigen/HIV antibody test returns non-reactive (negative), but I’m still concerned about infection?
If your risk of exposure is considered intermediate or high, it’s reasonable to repeat the test. HIV antibodies typically become detectable within about 3 weeks, and by 12 weeks after exposure, nearly all infections will be picked up. Retesting provides reassurance and ensures nothing is missed (2).
🔍 Back to the case: results of p24 antigen/HIV antibody test = NON-REACTIVE. p24 antigen/HIV antibody test is ordered again at 6 weeks post-exposure, as we are worried about an initial false-negative result. Result = REACTIVE (positive) to HIV 1/2.
What are next steps if we receive a positive HIV test?
If a patient receives a positive HIV result in primary care, it must be reported to public health (mandatory). The patient should then be referred to an infectious disease or HIV specialist to initiate antiretroviral therapy as soon as possible. In some practices, primary care providers who are trained may begin treatment right away.
What should we do while waiting for specialist consultation?
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References
Government of Canada. 2025. HIV and AIDs: For health Professionals. Available form https://www.canada.ca/en/public-health/services/diseases/hiv-aids/health-professionals.html
Government of Canada. 2025. Approach to HIV Screening. Available from https://www.canada.ca/en/public-health/services/publications/diseases-conditions/hiv-factsheet-types-screening-tests.html
Ontario Guidelines for Providers Offering HIV Testing. 2020. Available from https://hivtestingontario.ca/ontario-guidelines-for-providers-offering-hiv-testing/
Public Health Ontario. 2025. HIV – Diagnostic Serology. Available from https://www.publichealthontario.ca/en/Laboratory-Services/Test-Information-Index/HIV-Diagnostic-Serology