HIV-1/2 Antigen and Antibodies, Fourth Generation, With Reflexes
HIV-1/2 Antigen and Antibodies, Fourth Generation, With Reflexes
This test is used to screen for and diagnose HIV-1/HIV-2 infection (including acute infection) and to differentiate HIV-1 from HIV-2 infection.
Test Summary
HIV-1/2 Antigen and Antibodies, Fourth Generation, With Reflexes
Test code: 91431
Clinical use
- Screen for and diagnose HIV-1 and/or HIV-2 infection
- Differentiate HIV-1 from HIV-2 infection
Clinical background
An estimated 1.2 million people in the United States were living with HIV infection in 2022, of whom about 13% did not know they were infected.1 Another 31,800 people were newly infected in 2022.1 Identification and treatment of patients with HIV infection can lead to substantial decreases in transmission, morbidity, and death.2 The US Preventive Services Task Force and Centers for Disease Control and Prevention (CDC) thus recommend voluntary, opt-out HIV screening for everyone 15 to 65 years of age and for people who are pregnant.2,3 More frequent screening and screening younger children or older adults may be appropriate based on individual risk assessments.2,3 Detecting HIV infection during the acute phase (before seroconversion) is important because this phase is marked by high viral load and contributes disproportionately to transmission.3,4 In addition, HIV testing is recommended immediately before starting pre-exposure prophylaxis (PrEP) for HIV and regularly thereafter because taking PrEP during acute HIV infection may induce resistance to antiretroviral drugs.5
The CDC recommends an HIV diagnostic testing algorithm based on fourth-generation antigen/antibody (Ag/Ab) combination assays (Figure).4,6 The first step in the algorithm is a fourth-generation Ag/Ab assay, which simultaneously detects HIV-1 p24 antigen and HIV-1/2 antibodies. Because the p24 antigen is detectable before seroconversion, this type of assay can detect HIV-1 in the acute phase of infection.4 The inclusion of HIV-1 and HIV-2 antibodies allows detection after seroconversion, when the p24 antigen becomes undetectable. Fourth-generation Ag/Ab assays have >99.7% sensitivity and >99.5% specificity for HIV infection4 and can identify most (>80%) acute infections that would otherwise require nucleic acid testing for detection.7 The fourth-generation assay used by Quest Diagnostics requires repeat testing of reactive (positive) results before reflexing to supplemental testing.
To confirm repeatedly reactive results from fourth-generation Ag/Ab assays, the CDC recommends using an HIV-1/HIV-2 antibody differentiation assay as a supplemental test.4,6 Differentiating HIV-1 and HIV-2 can have treatment implications, as HIV-2 does not respond to some antiretroviral agents.4 However, HIV-1/HIV-2 antibody assays may yield negative results during acute infection before seroconversion. Thus, HIV-1/HIV-2 RNA testing is used as the last step in the algorithm to resolve infection status for people with repeatedly reactive HIV Ag/Ab results but negative or indeterminate HIV-1/HIV-2 antibody results.4,6 In accordance with the latest guidance from the CDC,6 results for HIV-1 and HIV-2 RNA are individually reported.
Individuals suitable for testing
- Pregnant individuals, including those who are in labor or delivery
- Individuals 15 to 65 years of age
- Individuals at increased risk of HIV infection
- Children ≥2 years of age with suspected HIV infection
For testing options for individuals starting or taking PrEP, see HIV Pre-Exposure Prophylaxis (PrEP): Laboratory Testing | Test Guide | Quest Diagnostics.
Methods
- Chemiluminescent microparticle immunoassay specific for
- HIV-1 p24 antigen
- HIV-1 (groups M and O) and HIV-2 antibodies
- Specimens with repeatedly reactive screening results (indicating the presence of p24 antigen and/or HIV-1/HIV-2 antibodies) are reflexed to the supplemental HIV-1/HIV-2 antibody differentiation test (an immunochromatography test).
- Specimens with negative or indeterminate HIV-1/HIV-2 antibody differentiation results are reflexed to the HIV-1 and HIV-2 RNA, Qualitative Real-Time PCR test. See Figure for reflex pathway and reporting.
Reflex tests are performed at an additional charge and are associated with an additional CPT code.
Interpretive information
Interpretative information for the reflex pathway is depicted in the Figure.
References
- Estimated HIV incidence and prevalence in the United States, 2018–2022. HIV Surveillance Supplemental Report 2024;29(No. 1). Centers for Disease Control and Prevention. Published May 2024. Accessed June 9, 2025. https://www.cdc.gov/hiv-data/nhss/estimated-hiv-incidence-and-prevalence.html
- US Preventive Services Task Force, Owens DK, Davidson KW, et al. Screening for HIV infection. JAMA. 2019;321(23):2326-2336. doi:10.1001/jama.2019.6587
- Workowski KA, Bachmann LH, Chan PA, et al. Sexually transmitted infections treatment guidelines, 2021. MMWR Recomm Rep. 2021;70(4):1-187. doi:10.15585/mmwr.rr7004a1
- Branson BM, Owen SM, Wesolowski LG, et al. Laboratory testing for the diagnosis of HIV infection: updated recommendations. Centers for Disease Control and Prevention. Published June 27, 2014. Accessed June 5, 2025. https://stacks.cdc.gov/view/cdc/23447
- US Public Health Service: preexposure prophylaxis for the prevention of HIV infection in the United States—2021 update: a clinical practice guideline. Centers for Disease Control and Prevention. Published December 2021. Accessed June 5, 2025. https://stacks.cdc.gov/view/cdc/112360
- National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Association of Public Health Laboratories. Technical update for HIV nucleic acid tests approved for diagnostic purposes. Centers for Disease Control and Prevention. Published May 16, 2023. Accessed June 6, 2025. https://stacks.cdc.gov/view/cdc/129018
- Pandori MW, Hackett J, Louie B, et al. Assessment of the ability of a fourth-generation immunoassay for human immunodeficiency virus (HIV) antibody and p24 antigen to detect both acute and recent HIV infections in a high-risk setting. J Clin Microbiol. 2009;47(8):2639-2642. doi:10.1128/jcm.00119-09
Content reviewed 06/2025
This test is used to screen for and diagnose HIV-1/HIV-2 infection (including acute infection) and to differentiate HIV-1 from HIV-2 infection.
Test Summary
HIV-1/2 Antigen and Antibodies, Fourth Generation, With Reflexes
Test code: 91431
Clinical use
- Screen for and diagnose HIV-1 and/or HIV-2 infection
- Differentiate HIV-1 from HIV-2 infection
Clinical background
An estimated 1.2 million people in the United States were living with HIV infection in 2022, of whom about 13% did not know they were infected.1 Another 31,800 people were newly infected in 2022.1 Identification and treatment of patients with HIV infection can lead to substantial decreases in transmission, morbidity, and death.2 The US Preventive Services Task Force and Centers for Disease Control and Prevention (CDC) thus recommend voluntary, opt-out HIV screening for everyone 15 to 65 years of age and for people who are pregnant.2,3 More frequent screening and screening younger children or older adults may be appropriate based on individual risk assessments.2,3 Detecting HIV infection during the acute phase (before seroconversion) is important because this phase is marked by high viral load and contributes disproportionately to transmission.3,4 In addition, HIV testing is recommended immediately before starting pre-exposure prophylaxis (PrEP) for HIV and regularly thereafter because taking PrEP during acute HIV infection may induce resistance to antiretroviral drugs.5
The CDC recommends an HIV diagnostic testing algorithm based on fourth-generation antigen/antibody (Ag/Ab) combination assays (Figure).4,6 The first step in the algorithm is a fourth-generation Ag/Ab assay, which simultaneously detects HIV-1 p24 antigen and HIV-1/2 antibodies. Because the p24 antigen is detectable before seroconversion, this type of assay can detect HIV-1 in the acute phase of infection.4 The inclusion of HIV-1 and HIV-2 antibodies allows detection after seroconversion, when the p24 antigen becomes undetectable. Fourth-generation Ag/Ab assays have >99.7% sensitivity and >99.5% specificity for HIV infection4 and can identify most (>80%) acute infections that would otherwise require nucleic acid testing for detection.7 The fourth-generation assay used by Quest Diagnostics requires repeat testing of reactive (positive) results before reflexing to supplemental testing.
To confirm repeatedly reactive results from fourth-generation Ag/Ab assays, the CDC recommends using an HIV-1/HIV-2 antibody differentiation assay as a supplemental test.4,6 Differentiating HIV-1 and HIV-2 can have treatment implications, as HIV-2 does not respond to some antiretroviral agents.4 However, HIV-1/HIV-2 antibody assays may yield negative results during acute infection before seroconversion. Thus, HIV-1/HIV-2 RNA testing is used as the last step in the algorithm to resolve infection status for people with repeatedly reactive HIV Ag/Ab results but negative or indeterminate HIV-1/HIV-2 antibody results.4,6 In accordance with the latest guidance from the CDC,6 results for HIV-1 and HIV-2 RNA are individually reported.
Individuals suitable for testing
- Pregnant individuals, including those who are in labor or delivery
- Individuals 15 to 65 years of age
- Individuals at increased risk of HIV infection
- Children ≥2 years of age with suspected HIV infection
For testing options for individuals starting or taking PrEP, see HIV Pre-Exposure Prophylaxis (PrEP): Laboratory Testing | Test Guide | Quest Diagnostics.
Methods
- Chemiluminescent microparticle immunoassay specific for
- HIV-1 p24 antigen
- HIV-1 (groups M and O) and HIV-2 antibodies
- Specimens with repeatedly reactive screening results (indicating the presence of p24 antigen and/or HIV-1/HIV-2 antibodies) are reflexed to the supplemental HIV-1/HIV-2 antibody differentiation test (an immunochromatography test).
- Specimens with negative or indeterminate HIV-1/HIV-2 antibody differentiation results are reflexed to the HIV-1 and HIV-2 RNA, Qualitative Real-Time PCR test. See Figure for reflex pathway and reporting.
Reflex tests are performed at an additional charge and are associated with an additional CPT code.
Interpretive information
Interpretative information for the reflex pathway is depicted in the Figure.
References
- Estimated HIV incidence and prevalence in the United States, 2018–2022. HIV Surveillance Supplemental Report 2024;29(No. 1). Centers for Disease Control and Prevention. Published May 2024. Accessed June 9, 2025. https://www.cdc.gov/hiv-data/nhss/estimated-hiv-incidence-and-prevalence.html
- US Preventive Services Task Force, Owens DK, Davidson KW, et al. Screening for HIV infection. JAMA. 2019;321(23):2326-2336. doi:10.1001/jama.2019.6587
- Workowski KA, Bachmann LH, Chan PA, et al. Sexually transmitted infections treatment guidelines, 2021. MMWR Recomm Rep. 2021;70(4):1-187. doi:10.15585/mmwr.rr7004a1
- Branson BM, Owen SM, Wesolowski LG, et al. Laboratory testing for the diagnosis of HIV infection: updated recommendations. Centers for Disease Control and Prevention. Published June 27, 2014. Accessed June 5, 2025. https://stacks.cdc.gov/view/cdc/23447
- US Public Health Service: preexposure prophylaxis for the prevention of HIV infection in the United States—2021 update: a clinical practice guideline. Centers for Disease Control and Prevention. Published December 2021. Accessed June 5, 2025. https://stacks.cdc.gov/view/cdc/112360
- National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Association of Public Health Laboratories. Technical update for HIV nucleic acid tests approved for diagnostic purposes. Centers for Disease Control and Prevention. Published May 16, 2023. Accessed June 6, 2025. https://stacks.cdc.gov/view/cdc/129018
- Pandori MW, Hackett J, Louie B, et al. Assessment of the ability of a fourth-generation immunoassay for human immunodeficiency virus (HIV) antibody and p24 antigen to detect both acute and recent HIV infections in a high-risk setting. J Clin Microbiol. 2009;47(8):2639-2642. doi:10.1128/jcm.00119-09
Content reviewed 06/2025