Introduction

An estimated 15% of patients living with HIV in the U.S. remain unaware of their status. HIV testing should be integrated across all levels of the healthcare system to facilitate early diagnosis. Approximately 40% of new HIV infections are transmitted by individuals living with undiagnosed HIV. Early detection reduces the risk of HIV-related complications and lowers the likelihood of transmission.[1][2]

Who to Screen

HIV testing should be considered during every clinical encounter, regardless of specialty, and all healthcare providers should be familiar with current screening recommendations.[3][4][5][6] Routine screening is recommended for all individuals aged 13 to 64 years in healthcare settings, without an upper age limit for those with ongoing risk factors. A 1-time test is sufficient for most individuals at low risk. However, certain populations require more frequent testing. These groups include men who have sex with men, particularly those aged 13 to 24 years, and individuals who inject drugs, exchange sex for money or food, engage in sexual activity with partners of unknown HIV status, and have partners who live with HIV, inject drugs, or are bisexual.

Patients should be screened for HIV if they present with clinical signs or symptoms suggestive of HIV infection. Acute retroviral syndrome may occur 2 to 4 weeks after transmission and often presents as a constellation of nonspecific symptoms, such as fever, sore throat, and rash. Patients reporting possible HIV exposure should also be tested, regardless of symptomatology. These patients include individuals who request sexually transmitted infection testing, sustain occupational needle stick injuries or significant mucous membrane exposures, or have suspected or confirmed sexual or percutaneous exposures, such as needle sharing for substance use.

In cases of possible HIV exposure, repeated testing is recommended during the 4- to 6-week period following exposure, with a final test at 12 weeks. Postexposure prophylaxis (PEP) should be considered for these patients and initiated as soon as possible, ideally within 24 hours and no later than 72 hours after exposure. The recommended PEP regimen includes a 2nd-generation integrase inhibitor in combination with tenofovir and either emtricitabine or lamivudine, administered for a total of 28 days.

Patients initiating preexposure prophylaxis (PrEP) should undergo HIV testing within 7 days of starting therapy, and again every 2 to 6 months while receiving this treatment. HIV testing methods and frequency during PrEP vary with the route and agent used. See below for further discussion.

Pregnant women should be screened for HIV early in pregnancy.[7] Early detection facilitates timely interventions to reduce the risk of perinatal transmission.

Specimen Requirements and Procedure

HIV testing may be conducted using various specimen types, such as serum, plasma, whole blood (venous or capillary), dried blood spots, and oral fluid. Selection depends on the testing method, clinical context, target population, and logistical considerations. Blood-derived specimens, including serum, plasma, and dried blood spots, are generally preferred due to their higher concentrations of HIV antibodies and compatibility with additional testing, such as screening for syphilis, hepatitis coinfections, or antiretroviral drug resistance. However, these specimens require invasive collection procedures, trained personnel, and appropriate biohazard waste disposal.[8]

Maintaining sample quality demands strict adherence to collection, processing, labeling, and storage protocols, including the use of sterile tubes, proper clotting and centrifugation times, unique specimen identification, and cold chain management when necessary. Additionally, accurate documentation and strict confidentiality are essential for both clinical diagnosis and public health surveillance.

Diagnostic Tests

Screening Procedure

The current standard of care for diagnosing HIV in clinical settings is the serum-based HIV 4th-generation test, which detects both antibodies and antigen. Only antibody tests were routinely used before the 2014 recommendation by the U.S. Centers for Disease Control and Prevention (CDC). The 4th-generation assay detects antibodies to HIV-1 and HIV-2, as well as the p24 antigen, enabling earlier identification of infection following exposure. The p24 antigen may be detected as early as 14 days postexposure.

A reflex HIV RNA test is required to confirm or exclude infection if the antigen is positive while the antibody remains negative. A confirmatory test is performed to differentiate between HIV-1 and HIV-2 antibodies when both the antigen and antibody are positive. This ability to distinguish HIV-1 from HIV-2 is a feature unique to 4th-generation testing and was not available in earlier test versions.

In cases of suspected acute HIV infection occurring fewer than 14 days after exposure, an HIV RNA test should be performed as the initial diagnostic test. Depending on the sensitivity of the RNA assay, viral RNA may be detectable as early as 5 to 10 days posttransmission. HIV RNA testing is also warranted when 4th-generation test results are indeterminate.

The 4th-generation HIV test offers significant advantages over earlier-generation assays, primarily due to its ability to detect infection at an earlier stage and differentiate between HIV-1 and HIV-2—an important distinction with therapeutic implications. In the Vaginal and Oral Interventions to Control the Epidemic (VOICE) study, 4th-generation testing identified 28% of HIV infections that were missed by 3rd-generation tests.

False-negative results in HIV screening typically occur when testing fails to detect early infection. The use of 4th-generation assays reduces this risk, although very early infections may still evade detection. False-positive rates for both 3rd- and 4th-generation tests remain extremely low. Data indicate that 3rd-generation testing, when followed by a confirmatory Western blot, has a false-positive rate as low as 0.0004% to 0.0007%.

Fourth-generation assays can yield false-positive antigen-antibody results, recognized as invalid when both the HIV-1/2 antibody test and confirmatory RNA test are negative. A reactive antigen-antibody screen must be followed by antibody and RNA testing to establish a definitive positive or negative result. Partial results from a 4-generation assay, such as those caused by insufficient sample volume, are invalid and should not be interpreted (see Image. HIV Diagnostic Algorithm.). (Source: CDC, 2018).

Point-of-care testing for HIV continues to advance. At present, a single-use, rapid assay is available that detects both HIV-1 p24 antigen and antibodies to HIV-1/2. This fingerstick-based test is waived under the Clinical Laboratory Improvement Amendments (CLIA) and can identify infection earlier than antibody-only rapid tests. However, the sensitivity of this assessment during early infection is lower than that of the recommended laboratory-based, 4th-generation serum assay. Several antibody-only point-of-care tests are also available but not routinely recommended in clinical care. These diagnostic tools may have utility in specific circumstances, such as occupational exposures or undocumented prenatal HIV screening at the time of labor.[9]

In nonclinical settings, oral swab tests continue to be primarily based on rapid antibody tests based on enzyme-linked immunosorbent assay (ELISA) technology, performed as rapid point-of-care diagnostics. Positive results must be confirmed with a serum-based Western blot. A key advantage of rapid tests is their applicability in settings outside traditional healthcare facilities, such as community health fair venues, faith-based organizations, and HIV service centers. Results are typically available within 20 minutes, reducing the likelihood of loss to follow-up. ELISA tests detect only antibodies and can identify infection approximately 3 weeks after transmission.[10]

Testing Procedures

The CDC first recommended routine HIV screening in 2006 for individuals aged 13 to 64 years. This approach replaced risk-based assessment and counseling, which were both time-intensive and unreliable for accurately estimating a patient’s true risk. Routine screening improves case detection by identifying individuals who may not disclose risk factors or recognize their own vulnerability to HIV infection. Although state laws vary, no U.S. state requires signed consent for HIV testing. Patients must be verbally informed that testing will be conducted and given the opportunity to decline. HIV testing in clinical settings is commonly implemented through opt-out screening.

PrEP provides clinicians and patients with highly effective strategies for preventing HIV acquisition. Any patient requesting the treatment is a suitable candidate for prescription, irrespective of documented risk factors. HIV testing requirements differ by regimen. The standard oral regimen consists of daily emtricitabine with tenofovir, with testing every 3 months. Long-acting injectable cabotegravir is administered intramuscularly every 2 months, with testing before each dose. Injectable lenacapavir is administered subcutaneously every 6 months, with testing before each administration.

For injectable PrEP, HIV RNA testing should accompany 4th-generation antigen-antibody testing at baseline and at each follow-up visit. Fourth-generation testing alone may be insufficient for detecting acute infection acquired during injectable PrEP use. Prompt identification of seroconversion is essential to prevent viral resistance, which could limit future treatment options. (Source: CDC, 2021)

Interfering Factors

A negative HIV test result should prompt an assessment of whether the patient may be within the window period—the interval following HIV transmission during which the antigen or antibody remains undetectable. In cases of suspected acute infection within the first 2 weeks postexposure, HIV RNA testing may be performed to detect early viral replication. Alternatively, the patient may undergo serial 4th-generation testing at 1 and 3 months following the potential exposure. Although most individuals will test positive within 3 weeks using 4th-generation assays, some may not seroconvert for up to 3 months. Accordingly, every negative result should be accompanied by an explanation of the window period and a plan for follow-up testing, if warranted. A test conducted more than 3 months after the last at-risk exposure is considered conclusive for ruling out infection.

Prevention and risk-reduction strategies should be addressed regardless of test results. Individuals in the window period may carry a high viral load, increasing the likelihood of HIV transmission to others. Providers should assess the patient's risk profile and offer tailored guidance on reducing transmission, such as avoiding needle sharing, using condoms consistently, and modifying sexual practices. All patients who test negative for HIV should be evaluated for PrEP, which involves the daily use of emtricitabine with tenofovir to prevent HIV transmission.[11][12]

When a patient tests positive for HIV, the result should be delivered in a nonjudgmental, calm, and confidential environment that allows sufficient time for counseling and emotional support. Disclosure must remain confidential unless the patient requests the presence of another individual. After the result is shared, the provider should allow space for the patient's emotional response, which may include fear, sadness, shame, anger, or shock, regardless of whether the diagnosis was expected. Positive test results should be given to patients in person. However, results may be provided by phone when delays arise.[13]

Following this initial response, the provider should offer basic education on HIV, emphasizing that it is a chronic, manageable condition. The distinction between HIV and AIDS should be clearly explained. Patients should be encouraged to ask questions about the disease process. If specific questions cannot be answered, the provider should acknowledge this limitation and facilitate a referral to a knowledgeable clinician.

Patients should also be informed of available support services, including peer counseling and social work resources, and asked whether they would like to engage with these programs. Before the conclusion of the visit, the provider should arrange follow-up with an HIV specialist and, when appropriate, initiate referral to an HIV-focused support organization.

Assessing the patient’s emotional and physical safety during the results visit is essential. The diagnosis of HIV can be overwhelming, even when anticipated. The presence of at least 1 trusted individual for emotional support must be ensured, and the risk of domestic violence should be assessed. Any history of mental health conditions should be explored, and the need for referral to a therapist or psychiatrist must be determined. Screening for suicidal or homicidal ideation should be completed as part of this assessment.

Risk-reduction counseling must be emphasized. In most states, partner notification services are available and may be conducted anonymously. The patient should be asked whether they would like to participate in partner notification. The importance of condom use, cessation of needle sharing, and safe sexual practices must be reinforced. Viral load is often high during acute infection, increasing the likelihood of transmission to others. The patient’s understanding of HIV transmission should be assessed, particularly regarding casual contact and potential concerns about household or familial transmission.

Several major organizations now advocate for same-day treatment initiation, a strategy referred to as “test and treat.” Although implementation may vary and remains the subject of ongoing discussion, the approach is based on evidence that earlier initiation of antiretroviral therapy reduces both transmission risk and the likelihood of HIV-related complications. Same-day treatment should be considered based on the clinical setting, the provider's expertise, and the availability of resources.

Results, Reporting, and Critical Findings

Effective HIV management requires prompt coordination of follow-up care after diagnosis. CDC data indicate that among an estimated 1 million adults living with HIV in the U.S., only 57% are receiving treatment, adhering to antiretroviral therapy, and achieving viral suppression. In 2022, 87% of adults with HIV had received a diagnosis, 66% had accessed care, 47% were retained in care, and 57% achieved sustained adherence with resultant viral suppression. (Source: CDC, 2025)

The HIV care continuum illustrates opportunities for intervention at each step and serves as a tool to motivate healthcare providers to improve patient outcomes. HIV management can occur in various settings, including primary care practices, infectious disease specialty clinics, federally qualified health centers, and dedicated HIV care facilities. Any practice offering HIV screening should establish a formal relationship with a treatment provider and implement a process for confirming access to care following diagnosis.

Clinical Significance

In the U.S., approximately 15% of individuals living with HIV are unaware of their status and account for up to 40% of new transmissions. In 2015, the estimated median delay between infection and diagnosis was 3 years. Among individuals who are newly diagnosed, 2/3 had not undergone HIV testing in the 12 months prior, despite having seen a healthcare provider during that period. These findings underscore the importance of routine HIV screening in all healthcare settings.

Opt-out testing simplifies the process and increases uptake. All individuals aged 13 to 65 years should undergo at least 1 lifetime HIV screening, regardless of perceived risk. Patients with ongoing risk factors should be screened as often as every 3 to 6 months. The recommended 4th-generation HIV test detects both HIV antibodies and p24 antigen, allowing for identification as early as 14 days posttransmission. An HIV RNA viral load test should be performed in cases where acute infection is suspected, ie, within 2 weeks of possible exposure.

Early diagnosis reduces HIV-related morbidity and mortality and limits further transmission. Viral load is often significantly elevated during acute infection, increasing the likelihood of transmission to partners. Risk-reduction counseling at the time of diagnosis, coupled with immediate initiation of antiretroviral therapy, further decreases the risk of transmission.

The HIV care continuum highlights the ongoing need to expand routine screening and ensure timely linkage to care to improve national HIV outcomes.[14] Strengthening each step in the continuum not only improves individual patient outcomes but also contributes to broader public health goals.

Quality Control and Lab Safety

A robust and comprehensive quality management system forms the foundation for ensuring the accuracy, reliability, and consistency of HIV testing services. This system encompasses multiple interdependent components, including organizational structure, personnel training and competency, equipment calibration and maintenance, procurement and inventory control, standardization of testing procedures, precise documentation, data oversight, occurrence reporting, internal and external evaluations, and continuous quality improvement activities.

At the core of this framework are quality assurance measures, which are structured, proactive efforts designed to ensure that all testing procedures consistently produce valid and reliable results. Two essential components of quality assurance in HIV diagnostics are internal quality control (IQC) and external quality assessment (EQA).[15][16] 

IQC procedures are routinely performed by laboratory personnel at the site level and are embedded in daily workflows. Best practices require the use of control materials—samples with known HIV-positive and HIV-negative status—tested alongside patient specimens to confirm assay performance. IQC is mandatory for each new lot or shipment of test kits and reagents, after significant events such as prolonged storage, temperature excursions, or transport delays, at the beginning of each testing session, and at scheduled intervals during extended testing runs. Modern automated immunoassay analyzers often feature integrated IQC monitoring and alert systems, enabling real-time surveillance and prompt corrective actions in response to deviations.[17][18]

IQC data are visualized on Levey-Jennings charts to detect trends and identify random or systematic errors. Westgard rules, often applied automatically through laboratory software, flag out-of-control results and initiate corrective measures. Any unexpected control result warrants immediate investigation, including retesting, reagent verification, or equipment recalibration, with every step carefully documented. Ongoing analysis of IQC findings supports continuous quality improvement and helps maintain high standards of precision and reliability in HIV testing.[19]

EQA provides an impartial and systematic evaluation of a laboratory’s testing performance, serving as an essential complement to IQC measures. A key component of EQA is proficiency testing, in which participating laboratories receive blinded specimens that are tested as part of routine operations, with results submitted to an external agency for assessment. While proficiency testing is often used interchangeably with EQA, it is technically a subset of the broader EQA framework. EQA encompasses a wider range of strategies beyond proficiency testing, including the retesting of previously analyzed clinical specimens, interlaboratory comparisons, and periodic on-site supervisory assessments aimed at evaluating compliance with standard protocols and overall quality systems.[20][21]

The results obtained through EQA offer laboratories objective and standardized feedback, enabling them to identify analytical errors, procedural inconsistencies, and areas for improvement. This insight is critical for guiding targeted corrective actions, such as staff retraining, process refinement, and increased supervisory oversight.

In the context of HIV testing, where diagnostic accuracy directly impacts patient management, surveillance, and public health strategies, EQA plays a pivotal role in ensuring consistent, reliable, and high-quality results across all testing sites. Furthermore, participation in EQA programs is strongly recommended by global health authorities and accreditation bodies, such as the World Health Organization and the International Organization for Standardization 15189, as a cornerstone of laboratory quality assurance and continual performance improvement.[22][23]

Laboratory safety is a foundational component of HIV testing, encompassing all testing modalities, from rapid diagnostic tests to automated immunoassays and molecular techniques. Given the infectious nature of HIV, rigorous biosafety protocols are required to protect laboratory personnel from occupational exposure and preserve the integrity of test results. These measures include consistent use of personal protective equipment, strict adherence to standard precautions when handling blood or other potentially infectious materials, and proper disposal of biohazardous waste. Work surfaces and equipment must be routinely disinfected, and procedures involving open specimens should be conducted in designated areas or within biosafety cabinets when necessary.

All staff must be trained in the management of accidental exposures, including needlestick injuries and specimen spills, and the safe operation of HIV testing platforms. Regular competency evaluations, safety drills, and scheduled equipment maintenance reinforce a culture of safety. Robust laboratory safety practices are critical not only for protecting personnel but also for ensuring the accuracy and reliability of HIV test results.[24][25]

Enhancing Healthcare Team Outcomes

The healthcare system must integrate routine HIV screening across all care settings to identify undiagnosed HIV cases. Screening should not be confined to primary care or limited to traditionally high-risk populations, such as individuals residing in areas of high prevalence or receiving prenatal care. Instead, HIV testing should be normalized and extended to specialty practices, including dermatology, otolaryngology, and general surgery, particularly for patients who may not routinely engage with primary care.

Emergency department-based screening is increasingly recognized as a critical component of HIV detection. Nontargeted HIV testing in this setting has demonstrated improved acceptance, particularly when the timing and method of test delivery align with the patient’s clinical workflow.[26] Combined HIV and hepatitis C virus screening strategies have also proven both effective and efficient in increasing case identification.[27]

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Disclosure: Katie Huynh declares no relevant financial relationships with ineligible companies.

Disclosure: Muhammad Zubair declares no relevant financial relationships with ineligible companies.

Disclosure: Chadi Kahwaji declares no relevant financial relationships with ineligible companies.