HIV Testing and Monitoring

HIV infection is diagnosed by a sequence of laboratory tests, where each step answers the question the previous one raised. A fourth-generation antigen/antibody assay closes most of the window period, an antibody differentiation assay identifies the viral type, and a nucleic acid test resolves the patterns that antibody methods cannot. After diagnosis, the two principal monitoring measurements, the CD4 T-cell count and the plasma HIV-1 RNA level, place the infection in a surveillance stage and track the response to antiretroviral therapy.

Viral structure and diagnostic targets

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.¹,²

Infection requires gp120 to bind the CD4 molecule plus a chemokine co-receptor: CCR5 for macrophage-tropic strains and CXCR4 for T-cell-tropic strains. 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.²

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. In advanced infection, viremia returns, CD4 cells fall further, and opportunistic illnesses develop.² Laboratory testing follows the same timeline: nucleic acid and antigen appear first, antibody follows, and the testing sequence is designed so the earliest markers carry the diagnosis.

The recommended testing algorithm

The recommended sequence for serum and plasma uses three steps, and laboratories report each assay result together with a combined interpretation, the laboratory algorithm interpretation, drawn from the APHL Suggested Reporting Language fourth edition (October 2025).³ A reactive screening result alone is presumptive; the diagnosis requires the supplemental steps that follow.

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 2.³

An immunoassay screen is repeated within the same run according to the manufacturer’s instructions before the result is finalized, and a repeatedly reactive screen proceeds 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.⁴

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 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 while omitting the individual HIV-1 and HIV-2 band or well results.³

HIV-2 infection is rare in the United States, under 0.03 percent of reported infections, and concentrates in people from West Africa or their partners. A Geenius result of HIV-2 Indeterminate is repeated on the same specimen in a new cartridge; an HIV-2 or HIV Indeterminate that persists proceeds to nucleic acid testing, and supplemental HIV-2 testing, arranged through public health laboratories or CDC, resolves the pattern when HIV-1 RNA is undetectable.³,⁵

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 HIV-1 RNA is “not detected,” the interpretation is that HIV antibodies were not confirmed and HIV-1 RNA was not detected; the report recommends repeat testing with a new specimen in 2 to 4 weeks when recent exposure is suspected.³

FDA-approved diagnostic NAATs include the cobas HIV-1/HIV-2 Qualitative test, which detects and differentiates HIV-1 and HIV-2 RNA, and the dual-claim Aptima HIV-1 Quant Dx and Alinity m HIV-1 assays, which provide diagnostic qualitative results and quantitative viral loads. Plasma is accepted for both qualitative and quantitative results, while serum is acceptable only for the qualitative claim, and diagnostic testing requires undiluted specimens.³,⁶

Alternate sequences and PrEP use

CDC and APHL kept antibody differentiation as the second step, yet the NAAT technical update 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.⁶ 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.³,⁶

Antiretroviral drugs taken for treatment or pre-exposure prophylaxis (PrEP) can suppress viremia and delay antibody development, which limits what any single assay shows. 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 carry the comment that antiretroviral drugs can limit detection. Fluctuating detectable and undetectable RNA results in the same person warrant a call to the submitter.³

Window periods

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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 negative NAT cannot close the window alone, because RNA becomes detectable before antibody and can decline again without therapy.³

Assay generations

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

The fifth-generation multiplex design reports the individual p24, HIV-1 antibody, and HIV-2 antibody signals from one specimen, and APHL cautions that its complex result format requires assay-specific interpretation.³

Rapid tests and self-tests

Rapid tests are immunochromatographic immunoassays that produce a visual line in minutes. They serve point-of-care programs, labor-and-delivery testing for patients of unknown status, and outreach settings, and most carry CLIA Certificates of Waiver. The antigen/antibody rapid tests use fingerstick whole blood with a window period of 18 to 90 days, while 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.⁷ Two FDA-approved self-tests are available: an oral-fluid antibody test and a fingerstick antibody test.⁷ 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.³,⁷

The 1989 to 2014 screening and confirmation sequence used a first-generation 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.⁴

Staging and surveillance reporting

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. The surveillance staging supports population monitoring and is not a patient-management tool.⁸

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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 500.⁸ A stage-3-defining opportunistic illness, such as Pneumocystis jirovecii pneumonia, places the case in stage 3 at any CD4 value, while stage 0 criteria supersede CD4 staging because a transient CD4 dip during acute infection would otherwise mimic advanced disease.⁸

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 viremia during that period carries a high transmission risk.³

Monitoring

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.⁹

Most reports include the absolute CD4 count, the CD4 percentage of total lymphocytes, and the CD4-to-CD8 ratio. When a hematology analyzer supplies the differential, the absolute count can be calculated: multiply the white cell count by the lymphocyte fraction, then multiply that product by the CD4 percentage. A WBC count of 7,200 cells per µL with 25 percent lymphocytes gives 1,800 lymphocytes per µL; 18 percent of those lymphocytes expressing CD4 gives 324 cells per µL, which falls in stage 2 for an adult. A CD8 count of 810 gives a CD4-to-CD8 ratio of 324 divided by 810, or 0.40, the inverted ratio typical of progressive disease, where a normal ratio is roughly 2:1.⁹

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. Federal treatment guidance for adults and adolescents directs a viral load measurement when therapy begins and again within 4 to 8 weeks, with repeat measurements every 4 to 8 weeks until the result falls below the assay’s limit of detection; on a stable suppressive regimen, monitoring repeats every 3 to 4 months, and an adherent patient whose viral load has been suppressed for more than a year with stable clinical status may extend the interval to 6 months.¹⁰ 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.¹⁰ Sustained suppression below 200 copies per mL also prevents sexual transmission, which makes laboratory suppression a public-health measure as well as a treatment endpoint.¹¹

CD4 counts support the viral load during follow-up. The count is measured at entry into care, repeated 3 months after therapy starts for people who began with counts below 300 cells per µL, and repeated every 3 to 4 months during the first 1 to 2 years of suppression at counts below 300 cells per µL; at 300 cells per µL or higher with viral suppression, the interval extends to 6 months, and after 1 to 2 years of suppression CD4 testing becomes optional unless clinical status changes.¹¹ A meaningful change between two counts is roughly 30 percent 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.¹¹ Serial results for one patient stay 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. Baseline genotypic testing before therapy starts is the standard of practice in current federal guidelines, and sequencing is repeated when viral load rebounds above assay detection limits.¹⁰ An FDA-approved next-generation-sequencing genotyping assay serves routine use.¹¹ Phenotypic testing, which grows recombinant virus against graded drug concentrations to derive an inhibitory concentration, remains available through reference laboratories for complex resistance patterns.¹⁰

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; laboratories serving those patients run confirmatory testing on an alternate platform to distinguish vector signal from true viremia.¹²

Testing in infants under 18 months

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.¹³

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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.¹³

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 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.¹³ 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.¹³

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.¹³

Quality and reporting notes

Serum and plasma specimen requirements are assay specific, and each manufacturer’s instructions for use governs specimen type, incubation, and validity criteria. Initial tests for HIV-1 and HIV-2 infection of blood and blood components are covered in Product Processing, Storage, Components, and Quality Control.³

References

1. Abbas AK, Lichtman AH, Pillai S, Henrickson S. Cellular and Molecular Immunology. 11th ed. Elsevier; 2025.
2. Coffin J, Swanstrom R. HIV pathogenesis: dynamics and genetics of viral populations and infected cells. Cold Spring Harb Perspect Med. 2020;10(1):a038489. doi:10.1101/cshperspect.a038489.
3. Association of Public Health Laboratories. Suggested Reporting Language for the HIV Laboratory Diagnostic Testing Algorithm, Fourth Edition. October 2025. Accessed August 31, 2026.
4. Centers for Disease Control and Prevention and Association of Public Health Laboratories. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. June 27, 2014. doi:10.15620/cdc.23447.
5. Panel on Antiretroviral Therapy and Medical Management of Children Living With HIV. Guidelines for the Use of Antiretroviral Agents in Pediatric HIV Infection. Updated December 27, 2024. Accessed August 31, 2026.
6. Centers for Disease Control and Prevention and Association of Public Health Laboratories. Technical Update for HIV Nucleic Acid Tests Approved for Diagnostic Purposes. May 16, 2023. Accessed August 31, 2026.
7. Centers for Disease Control and Prevention. HIV testing. Updated August 17, 2026. Accessed August 31, 2026.
8. Selik RM, Mokotoff ED, Branson B, Owen SM, Whitmore S, Hall HI. Revised surveillance case definition for HIV infection: United States, 2014. MMWR Recomm Rep. 2014;63(RR-03):1-10.
9. Mandy FF, Nicholson JKA, McDougal JS. Guidelines for performing single-platform absolute CD4+ T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. MMWR Recomm Rep. 2003;52(RR-2):1-13.
10. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents with HIV: plasma HIV-1 RNA (viral load) and CD4 count monitoring; drug-resistance testing. Updated September 25, 2025. Accessed August 31, 2026.
11. Vela Diagnostics USA, Inc. Sentosa SQ HIV-1 Genotyping Assay: Summary of Safety and Effectiveness. Approved November 5, 2019. Accessed August 31, 2026.
12. Panel on Antiretroviral Therapy and Medical Management of Children Living With HIV. Guidelines for the Use of Antiretroviral Agents in Pediatric HIV Infection. Updated December 27, 2024. Accessed August 31, 2026.
13. Panel on Treatment of HIV During Pregnancy and Prevention of Perinatal Transmission. Recommendations for the Use of Antiretroviral Drugs During Pregnancy and Interventions to Reduce Perinatal HIV Transmission in the United States. Updated June 25, 2026. Accessed August 31, 2026.