HIV Testing and Monitoring

HIV is an enveloped retrovirus with two copies of single-stranded RNA. Its genome carries three major genes with diagnostic value. The gag gene encodes the core structural proteins, including p24, the capsid antigen measured by fourth-generation screening assays. The env gene yields the gp160 precursor, cleaved to gp120 for receptor binding and gp41 for membrane fusion. Recombinant and peptide forms of env and other proteins coat the wells of differentiation immunoassays. The pol gene encodes reverse transcriptase, integrase, and protease, the enzymes that genotypic resistance testing sequences.^{1,21 Abbas AK, et al. Cellular and Molecular Immunology. 2025.2 Coffin J, et al. HIV pathogenesis. 2013.}

Infection requires gp120 to bind the CD4 molecule plus a chemokine coreceptor. R5-tropic strains use CCR5, X4-tropic strains use CXCR4, and dual-tropic strains can use either. The older macrophage-tropic and T-cell-tropic labels do not map cleanly to cell type because CCR5-using virus can also infect CD4 T cells. Reverse transcriptase lacks proofreading, so mutations accumulate rapidly and circulate as a mixture of related variants in one patient. This diversity complicates vaccine design and drives the resistance mutations that genotypic testing reports. CD4 T cells are the primary cellular target, so their loss progressively weakens both humoral and cell-mediated defenses.^{22 Coffin J, et al. HIV pathogenesis. 2013.}

Untreated infection follows a characteristic course. Viral RNA rises to high levels during acute infection, CD4 cells transiently fall, and p24 antigen circulates before antibody appears. Antibody and cytotoxic T-cell responses then partially control replication during a clinically latent period that can last years. As untreated infection advances, viremia increases, CD4 cells decline further, and opportunistic illnesses may develop.^{22 Coffin J, et al. HIV pathogenesis. 2013.} Laboratory markers appear in the same order. Nucleic acid and antigen appear first, antibody follows, and the testing algorithm is designed so the earliest markers can establish the diagnosis.

The recommended sequence for serum and plasma uses three steps. Laboratories report each assay result together with the combined laboratory-algorithm interpretation described in the fourth edition (October 2025) of the Suggested Reporting Language from the Association of Public Health Laboratories (APHL).^{33 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.} A reactive screening result alone is presumptive, so it is never reported as a diagnosis until the supplemental steps are complete.

Step 1: antigen and antibody screening

The initial assay is an HIV-1/HIV-2 antigen/antibody immunoassay, most commonly a fourth-generation laboratory test that detects HIV-1 p24 antigen together with HIV-1 and HIV-2 antibody. A nonreactive result supports the report "HIV-1 antigen and HIV-1/HIV-2 antibodies were not detected. No laboratory evidence of HIV infection." A reactive result is presumptive and requires Step 2.^{33 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.}

Repeat a reactive immunoassay screen in the same run as the manufacturer's instructions direct before you finalize the result, and send a repeatedly reactive screen to supplemental testing. Initial and supplemental tests are chosen to be orthogonal, meaning they use different antigens or different principles, so concurrent nonspecific reactivity becomes unlikely.^{44 Centers for Disease Control and Prevention… Laboratory Testing for the Diagnosis of HIV Infection. 2014.}

Step 2: antibody differentiation

The HIV-1/HIV-2 antibody differentiation immunoassay was developed for this role and distinguishes antibody to HIV-1 from antibody to HIV-2. The Geenius HIV 1/2 Supplemental Assay is a lateral-flow immunochromatographic cassette. The VioOne HIV Profile Supplemental Assay is an enzyme-linked immunosorbent assay (ELISA) read on a microplate. Each assay reports a software-generated conclusion, the Geenius Final Assay Interpretation or the VioOne Results Interpretation, and APHL recommends reporting that combined interpretation and omitting the individual HIV-1 and HIV-2 band or well results.^{33 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.}

In CDC surveillance for the United States during 2010–2017, HIV-2 mono-infection or dual HIV-1 and HIV-2 infection made up about 0.03% of diagnosed HIV infections (113 of 327,700).^{1414 Centers for Disease Control and Prevention. Trends in HIV-2 diagnoses and use of the HIV-1/HIV-2… 2020.} HIV-2 infection is associated particularly with people from West Africa or their partners. Repeat a Geenius HIV-2 Indeterminate result on the same specimen in a new cartridge. A persistent HIV-2 Indeterminate or HIV Indeterminate result requires nucleic acid testing. Supplemental HIV-2 testing, arranged through public health laboratories or CDC, resolves the pattern when HIV-1 RNA is undetectable.^{3,53 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.5 Panel on Antiretroviral Therapy and Medical… Guidelines for the Use of Antiretroviral Agents in Pediatric HIV… 2024.}

Step 3: nucleic acid testing

When the differentiation assay is indeterminate or antibody negative, an HIV nucleic acid amplification test (NAAT) resolves the pattern. A detected HIV-1 RNA result supports the report "Positive for HIV-1. Laboratory evidence of HIV-1 infection consistent with an acute HIV-1 infection," which triggers prompt reporting to the provider and health department. When the differentiation result is HIV antibody negative or HIV-1 Indeterminate and HIV-1 RNA is not detected, the laboratory algorithm interpretation is HIV Negative. When recent exposure is suspected, request a new specimen and repeat the algorithm. A dual HIV-1/HIV-2 diagnostic NAAT can also resolve HIV-2 RNA: HIV-2 RNA detected with HIV-1 RNA not detected supports Acute HIV-2 Positive. Detection of both supports acute HIV-1 and HIV-2 infection.^{33 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.}

When an HIV-1-only NAAT does not detect RNA after a persistent Geenius HIV-2 Indeterminate or HIV Indeterminate result, the interpretation is "HIV-1 Negative, HIV-2 Inconclusive." Refer the specimen for a validated supplemental HIV-2 antibody test or NAAT when one is available. Detection of HIV-2 antibody or HIV-2 RNA supports HIV-2 Positive. If supplemental testing is unavailable or the pattern remains unresolved, request a new specimen in 2 to 4 weeks and repeat the full algorithm. When a dual HIV-1/HIV-2 diagnostic NAAT detects neither RNA, the algorithm interpretation is HIV Negative. When recent exposure is suspected, request a new specimen and repeat testing.^{33 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.}

FDA-approved diagnostic NAATs include the cobas HIV-1/HIV-2 Qualitative test, which detects and differentiates HIV-1 and HIV-2 RNA in serum or plasma, and the dual-claim Aptima HIV-1 Quant Dx and Alinity m HIV-1 assays. The dual-claim assays provide qualitative diagnostic or supplemental results from serum or plasma and quantitative viral-load results from plasma. Exact anticoagulants, input volumes, dilution rules, and result claims remain assay specific. Diagnostic-algorithm testing uses an undiluted specimen unless the instructions for use state otherwise.^{3,63 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.6 Centers for Disease Control and Prevention… Technical Update for HIV Nucleic Acid Tests Approved for… 2023.}

Alternate sequences and PrEP use

The CDC/APHL algorithm uses antibody differentiation as the second step. The NAAT technical update also recognizes sequences that place a diagnostic NAAT second when symptoms coincide with a known or suspected recent exposure or when a person has participated in a vaccine or neutralizing-antibody prevention trial.^{66 Centers for Disease Control and Prevention… Technical Update for HIV Nucleic Acid Tests Approved for… 2023.} In those sequences a negative NAAT is followed by the antibody differentiation assay, and an NAAT result cannot by itself distinguish recent from established infection.^{3,63 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.6 Centers for Disease Control and Prevention… Technical Update for HIV Nucleic Acid Tests Approved for… 2023.}

Antiretroviral drugs taken for treatment or pre-exposure prophylaxis (PrEP) can suppress viremia and delay antibody development, which can limit detection by any single assay. For a patient with a known PrEP history, CDC recommends testing with a laboratory antigen/antibody assay and an HIV RNA assay at the same time, and a negative report may include a comment that antiretroviral drugs can limit detection. When RNA results in the same person fluctuate between detectable and undetectable, call the submitter.^{33 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.}

^{77 Centers for Disease Control and Prevention. HIV testing. 2026.}

A nonreactive result within the window period has limited value, so a patient with a suspected recent exposure is retested with the full algorithm on a later specimen. A single negative NAT does not exclude infection within the window period, because RNA becomes detectable before antibody and can decline again without therapy.^{33 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.}

Each generation of HIV immunoassays shortened the window period by adding an earlier target.

Rapid HIV immunoassays use assay-specific formats and visual endpoints, including lateral-flow lines and flow-through dots, and read in minutes. They are used in point-of-care programs, labor-and-delivery testing for patients of unknown status, and outreach settings. Many professional-use rapid tests are waived under the Clinical Laboratory Improvement Amendments (CLIA) for the specified specimen and procedure. The testing site also needs the appropriate CLIA certificate. The antigen/antibody rapid tests use fingerstick whole blood with a window period of 18 to 90 days. Antibody-only rapid tests use fingerstick blood or oral fluid with a window of 23 to 90 days. Oral-fluid tests also have lower analytical sensitivity than blood assays.^{77 Centers for Disease Control and Prevention. HIV testing. 2026.} Two FDA-approved self-tests are available: the OraQuick oral-fluid antibody test and the INSTI fingerstick whole-blood antibody test.^{7,87 Centers for Disease Control and Prevention. HIV testing. 2026.8 US Food and Drug Administration. INSTI HIV Self Test. 2025.} Every reactive rapid result requires follow-up testing with the serum and plasma algorithm, and a negative rapid result during a suspected recent exposure is repeated with laboratory-based testing.^{3,73 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.7 Centers for Disease Control and Prevention. HIV testing. 2026.}

The 1989 to 2014 screening and confirmation sequence used a screening ELISA followed by Western blot. Western blot was slower, less sensitive than modern screens, confined to reference laboratories, and produced indeterminate patterns that took weeks to resolve, so the current algorithm replaced it with the differentiation immunoassay and NAAT steps.^{44 Centers for Disease Control and Prevention… Laboratory Testing for the Diagnosis of HIV Infection. 2014.}

The revised CDC surveillance case definition places each confirmed case in one of five stages: 0, 1, 2, 3, or unknown. Stage 0 marks early infection, established either from a documented negative or indeterminate test within 180 days of the first confirmed positive result or from an algorithm sequence such as a reactive screen with an indeterminate differentiation assay and detected RNA. Stage 3 corresponds to AIDS and requires either a CD4 measure below the stage 3 threshold or a stage-3-defining opportunistic illness. Surveillance staging is used for population monitoring and is not intended for patient management.^{99 Selik RM, et al. Revised surveillance case definition for HIV infection. 2014.}

^{99 Selik RM, et al. Revised surveillance case definition for HIV infection. 2014.}

The CD4 count takes precedence and the percentage is applied only when the count is unavailable. Children require age-specific thresholds: at younger than 1 year, stage 1 is 1,500 or more cells per µL, stage 2 is 750 to 1,499, and stage 3 is fewer than 750; at ages 1 to 5 years the thresholds are 1,000 or more, 500 to 999, and fewer than 500. A stage-3-defining opportunistic illness, such as Pneumocystis jirovecii pneumonia, places the case in stage 3 at any CD4 value. Stage 0 criteria supersede CD4 staging because a transient CD4 dip during acute infection would otherwise mimic advanced disease.^{99 Selik RM, et al. Revised surveillance case definition for HIV infection. 2014.}

Laboratories report results indicative of HIV infection to the health department in the patient's jurisdiction, with each test result and the laboratory algorithm interpretation. Acute infection interpretations require expedited reporting because transmission risk is high during the viremia of acute infection.^{33 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.}

The two principal monitoring measurements are the CD4 T-cell count by flow cytometry and the plasma HIV-1 RNA viral load by nucleic acid amplification.

CD4 count

Single-platform flow cytometry measures absolute CD4 counts without a separate hematology analyzer by adding calibrated beads and comparing events in the CD4 gate with beads counted in the same tube. Lymphocytes are identified by their bright CD45 fluorescence and low side scatter, and CD3 plus CD4 defines the T-helper population within that gate.^{1010 Mandy FF, et al. Guidelines for performing single-platform absolute CD4+ T-cell… 2003.}

Reports include the absolute CD4 T-cell count and its percentage of total lymphocytes. Some also include a CD4-to-CD8 ratio. In a dual-platform calculation, multiply the white cell count by the lymphocyte fraction, then by the fraction of lymphocytes identified as CD3-positive/CD4-positive T cells. A WBC count of 7,200 cells per µL with 25% lymphocytes gives 7,200 × 0.25 = 1,800 lymphocytes per µL. With 18% CD4 T cells, the absolute CD4 count is 1,800 × 0.18 = 324 cells per µL.^{1010 Mandy FF, et al. Guidelines for performing single-platform absolute CD4+ T-cell… 2003.}

For a confirmed adult HIV case without stage 0 criteria or a stage-3-defining illness, that count meets the CDC surveillance definition of stage 2. If the CD8 count is 810 cells per µL, the CD4-to-CD8 ratio is 324 ÷ 810 = 0.40. The ratio describes the relative counts of the two subsets. It is not a criterion in the CDC surveillance staging table above.^{99 Selik RM, et al. Revised surveillance case definition for HIV infection. 2014.}

HIV-1 RNA viral load

A quantitative HIV-1 RNA assay measures circulating virions in plasma as copies per mL. The baseline value is drawn at entry into care and before therapy starts. Treatment guidelines set how often the treating clinician orders viral load and CD4 testing. A threefold change, 0.5 log10 copies per mL, is the minimal statistically significant change between results. Optimal suppression is a confirmed HIV RNA level below the lower limit of detection, generally fewer than 20 copies per mL, and federal guidance defines virologic failure as the inability to achieve or maintain HIV RNA below 200 copies per mL, a threshold that excludes isolated blips and assay variability. Sustained suppression below 200 copies per mL also prevents sexual transmission, so a suppressed viral load is a public-health measure as well as a treatment endpoint.^{1111 Panel on Antiretroviral Guidelines for… Guidelines for the Use of Antiretroviral Agents in Adults and… 2025.}

CD4 counts supplement the viral load during follow-up. A meaningful change between two counts is roughly 30% of the absolute count, and the absolute value can shift with marrow-suppressive drugs, acute illness, or splenectomy, so the CD4 percentage, which remains stable, is the better parameter in those settings.^{1111 Panel on Antiretroviral Guidelines for… Guidelines for the Use of Antiretroviral Agents in Adults and… 2025.} Run serial specimens for one patient on one platform, because numeric values from different assays are not interchangeable.

Resistance testing

Genotypic resistance testing sequences the reverse-transcriptase, protease, and integrase genes and compares the patient's mutations with a curated list of resistance positions. Current federal guidelines recommend genotypic resistance testing at entry into care and again when virologic failure or an inadequate response to therapy indicates it. Conventional plasma genotyping is recommended for virologic failure with HIV-1 RNA above 200 copies/mL, and low-level specimens may fail to amplify. Integrase-gene testing depends on the treatment and exposure history and the guideline indication.^{1515 Panel on Antiretroviral Guidelines for… Guidelines for the Use of Antiretroviral Agents in Adults and…} An FDA-approved next-generation-sequencing genotyping assay is available for routine use.^{1212 Vela Diagnostics USA, et al. Sentosa SQ HIV-1 Genotyping Assay. 2019.} Phenotypic testing, which grows recombinant virus against graded drug concentrations to derive an inhibitory concentration, remains available through reference laboratories for complex resistance patterns.^{1111 Panel on Antiretroviral Guidelines for… Guidelines for the Use of Antiretroviral Agents in Adults and… 2025.}

Chimeric antigen receptor T-cell therapy and lentiviral gene therapies can give false-positive HIV NAT results because their vectors contain lentiviral long-terminal-repeat sequences. When a patient has received one of these therapies, run confirmatory testing on an alternate platform to separate vector signal from true viremia.^{55 Panel on Antiretroviral Therapy and Medical… Guidelines for the Use of Antiretroviral Agents in Pediatric HIV… 2024.}

Maternal IgG crosses the placenta, so antibody tests, including antigen/antibody combination assays, cannot diagnose HIV infection in an infant younger than 18 months. Diagnosis in this age group uses virologic tests, meaning HIV RNA assays, FDA-approved qualitative HIV-1/HIV-2 RNA tests, or HIV DNA NATs, applied on a fixed schedule for every infant with perinatal exposure.^{1313 Panel on Treatment of HIV During Pregnancy… Recommendations for the Use of Antiretroviral Drugs During… 2026.}

^{1313 Panel on Treatment of HIV During Pregnancy… Recommendations for the Use of Antiretroviral Drugs During… 2026.}

Birth testing is recommended for all infants with perinatal exposure and may be omitted for an infant at low acquisition risk, meaning sustained maternal viral suppression below 50 copies per mL from 20 weeks of gestation through delivery, when follow-up is assured. It is retained for that infant if breastfeeding is planned. Specimens for birth testing are drawn before or immediately after the first antiretroviral dose, and cord blood is never used for diagnostic testing.^{1313 Panel on Treatment of HIV During Pregnancy… Recommendations for the Use of Antiretroviral Drugs During… 2026.}

A positive virologic test is confirmed as soon as possible on a second specimen, because false-positive RNA and DNA results occur. Definitive exclusion of HIV infection in a non-breastfed infant requires two or more negative virologic tests, one at 1 month of age or later and at least 2 to 6 weeks after infant antiretroviral prophylaxis ends, and one at 4 months or later, plus no positive virologic result and no clinical evidence of infection. Two negative antibody tests at 6 months of age or later provide an alternative route to definitive exclusion. For infants who breastfeed, virologic testing continues at the standard age points, at least every 3 months during breastfeeding, and at 4 to 6 weeks and 3 months after breastfeeding ends.^{1313 Panel on Treatment of HIV During Pregnancy… Recommendations for the Use of Antiretroviral Drugs During… 2026.}

A positive antibody or antigen/antibody result after 18 months requires a virologic test to rule out the residual maternal antibody seen in a small share of exposed children whose maternal antibody persists past that age.^{1313 Panel on Treatment of HIV During Pregnancy… Recommendations for the Use of Antiretroviral Drugs During… 2026.}

Serum and plasma specimen requirements are assay specific, and each manufacturer's instructions for use set the specimen type, incubation, and validity criteria. Before you release an HIV result, check that each assay result and the combined algorithm interpretation both appear on the report. Initial tests for HIV-1 and HIV-2 infection of blood and blood components are covered in Product Processing, Storage, Components, and Quality Control.^{33 Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic… 2025.}