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FeedbackHIV type 1 (HIV-1) and type 2 (HIV-2) are the two etiologic agents of AIDS. Although pre- and postexposure treatments have become available to avoid contracting HIV, infection rates remain high among at-risk populations, and HIV continues to be a major public health problem.
Screening, both population screening and more frequent screening in those at risk, is recommended in asymptomatic individuals because the early detection of disease is critical for improving treatment outcomes. Diagnostic testing includes “fourth generation testing” that detects both HIV antigens and antibodies, HIV-1/HIV-2 antibody differentiation testing, and nucleic acid amplification testing (NAAT). For more information, see Screening and Diagnostic Testing.
Antiretroviral drug resistance testing is recommended before the start of antiretroviral therapy (ART) for HIV to guide treatment selection and whenever treatment failures occur. For more information, see Drug Resistance Tests.
CD4 lymphocyte count and viral load testing can be used to monitor treatment response. For more information, see Monitoring.
Quick Answers for Clinicians
The length of time after exposure before HIV infection can be detected (window period) depends on the type of test used. Nucleic acid amplification testing (NAAT) can identify HIV 10-33 days after infection. Fourth generation antigen/antibody tests can detect HIV 18-45 days after infection. Antibody-only tests require additional time to yield reliable results (23-90 days after exposure) because the patient must mount an immune response to the HIV infection before antibodies can be detected. NAAT is not generally recommended for initial screening in asymptomatic patients. However, NAAT is recommended in certain cases, such as in patients who present with symptoms of HIV infection with known recent exposure, in infants born to individuals with HIV, and for monitoring of disease (see Laboratory Testing).
If a rapid test result is negative in a patient with a possible exposure, testing should be repeated after the window period to confirm the negative result. In general, repeat testing should be considered in high-risk patients if clinically indicated.
When an individual contracts HIV, the virus is integrated into the genome of the CD4 cells, some of which survive the infection. The HIV DNA in those CD4 cells is referred to as archived proviral DNA, and it can be detected in whole blood even when the viral load is too low to be detected by standard plasma nucleic acid amplification testing (NAAT). Thus, genotyping proviral, or archived, DNA may be useful in detecting drug resistance in patients whose plasma HIV RNA concentrations are too low to be detected. Testing for proviral DNA may assist in diagnosing infants with perinatal exposure because proviral DNA is less likely to be affected by maternal use of antiretrovirals (ARVs).
HIV-infected individuals with resistant viral strains may pass those strains on to others. Transmitted drug resistance is linked to impaired virologic response to initial antiretroviral therapy (ART) regimens. Ten to 17% of individuals with HIV in high-income countries who have not yet been treated with ART have resistance to at least one ART drug, which underscores the importance of resistance testing in treatment-naive patients before therapy initiation.
Antiretroviral therapy (ART) should begin within 7 days of an HIV diagnosis, and as early as the day of diagnosis if immediate treatment can be coordinated. The start of treatment should not be postponed until resistance test results are received; the regimen can be adjusted if necessary after results are in. Several studies have shown improved viral suppression outcomes in patients who begin treatment promptly, although this favorable outcome may not continue after the first year. The rapid treatment recommendation applies to all patients with HIV who do not have opportunistic infections (including pregnant patients and those with malignancies), even if the HIV diagnosis is uncertain because of discrepant test results. Even in patients with opportunistic infections, starting ART within 2 weeks of HIV diagnosis is often recommended. However, initiation of ART may be delayed for patients with tuberculosis and CD4 cell counts of ≥50/µL (ART should be initiated within 2-8 weeks after the start of tuberculosis therapy) and for patients with cryptococcal meningitis (ART should be initiated 4-6 weeks after the initiation of antifungal therapy).
In patients who experience virologic failure during antiretroviral therapy (ART), resistance testing is most informative if performed while the patient is still receiving the regimen or within 4 weeks of therapy cessation to ensure that the resistant virus will be detected if present. Acquired drug resistance in HIV occurs during therapy as a result of drug pressure (selection). When an ART regimen is discontinued or interrupted, the HIV population within a patient may revert to primarily wild type within 4-6 weeks. The drug-resistant virus may still be present, but at concentrations below the limit of detection of genotypic or phenotypic tests. However, even low concentrations of drug-resistant virus can lead to treatment failure when ART is reinitiated.
Genotypic testing for drug resistance historically has been performed using Sanger sequencing. However, as next generation sequencing (NGS) has become more accessible, it has become a viable method for detecting drug-resistant variants. NGS offers several advantages over Sanger sequencing. Sanger sequencing cannot detect resistant variants at frequencies below approximately 20%, whereas NGS can identify these variants at lower concentrations (typically shown to be 5-10%). Additionally, NGS allows for the simultaneous assessment of resistance to protease inhibitor (PI), nucleoside reverse transcriptase inhibitor (NRTI), non-NRTI (NNRTI), and integrase strand transfer inhibitor (INSTI) drug classes, whereas Sanger sequencing requires a separate test to detect INSTI resistance. Finally, NGS can more easily detect and interpret insertions and deletions in the HIV genome that often confound Sanger tests.
Indications for Testing
Recommendations for laboratory testing to screen for HIV vary based on the patient and recommending organization. Screening at least once is recommended in all individuals; more frequent screening is recommended in individuals at high risk and those who are pregnant. Groups at greatest risk for HIV include men who have sex with men (MSM); individuals with HIV-positive sexual partners; intravenous drug users; individuals with other sexually transmitted infections; and individuals who have had multiple sex partners since their last HIV test or have had partners with HIV risk factors or an unknown sexual history. Additionally, screening is also appropriate in individuals considering pre- or postexposure prophylaxis.
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CDCa (endorsed by AAFP, ACOG, ACP, IDSA) |
All individuals 13-64 yrs of age (testing recommended at least once) Patients at high risk for HIV (annual screening recommended) Pregnant individuals (routine prenatal screening recommended) |
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USPSTFb |
All individuals 15-65 yrs of age Individuals <15 yrs or >65 yrs at increased risk of infection Pregnant individual, including untested individuals who present in labor with unknown HIV status |
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aRecommendations issued initially in 2006 and current in 2018. bRecommendations issued in 2019. AAFP, American Academy of Family Physicians; ACOG, American College of Obstetricians and Gynecologists; ACP, American College of Physicians; IDSA, Infectious Diseases Society of America; USPSTF, U.S. Preventive Services Task Force |
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Laboratory testing to diagnose HIV infection is appropriate in individuals with a positive screening test result, symptoms of HIV infection, or recent exposure to an HIV-infected person, and in infants born to HIV-positive mothers.
Testing to monitor treatment is appropriate for patients who are currently receiving ART for HIV.
Laboratory Testing
Screening and Diagnostic Testing
Many types of tests are used to screen for and diagnose HIV. Rapid screening tests can be performed at the point of care (POC) and provide results within minutes; however, these tests are not as reliable as combined antigen/antibody tests (fourth generation tests), which can be used for screening or diagnosis. Confirmatory tests are supplemental tests used to confirm the diagnosis in patients with repeatedly reactive fourth generation antigen/antibody tests. Finally, follow-up tests are used if discrepancies exist between the screening and confirmatory tests.
The types of tests used in screening and diagnosis are detailed below; refer to ARUP's Human Immunodeficiency Virus in Adults and Adolescents Testing algorithm and to the ARUP HIV Screening and Diagnostic Test Offerings for an overview of testing strategy.
| Type of Test | Uses | Advantages | Limitations | Comments |
|---|---|---|---|---|
| Screening Tests | ||||
| Rapid antibody test | Screen adult for HIV |
Can be performed at POC Provides results within minutes |
Not reliable until 23-90 days after exposure |
If rapid result is negative in a patient with a possible exposure, repeat testing after window period to confirm negative result Repeat testing is recommended in high-risk patients if clinically indicated |
| Rapid antibody plus antigen test | Screen adult for HIV |
Can be performed at POC Provides results within minutes |
Not reliable until 18-90 days after exposure |
If rapid result is negative in a patient with a possible exposure, repeat testing after window period to confirm negative result Repeat testing is recommended in high-risk patients if clinically indicated |
| Fourth generation test (ie, laboratory-performed antigen/antibody test) | Screen adult for HIV |
Diagnostic window is shorter than for antibody-only tests Cheaper than NAAT Enables detection of HIV when patient is highly infectious |
Not reliable until 18-45 days after exposure Does not distinguish between the presence of HIV-1 p24 antigen, HIV-1 antibodies, and HIV-2 antibodies |
Consider repeat testing for negative results if clinically indicated after the window period following the most recent exposure |
| NAAT |
Screen infant for HIV Screen adult for HIV only in certain high-risk situations |
In infants, avoids the false-positive results that may occur with antibody tests, which may detect maternal antibodies in HIV-negative infants | Generally, not a screening test for adults due to high cost |
Infants should only be screened if born to HIV-positive mothers; testing should be performed at 14-21 days, 1-2 mos, and at 4-6 mos Adults should only be screened with NAAT in cases of recent high-risk exposure or recent possible exposure plus early symptoms of HIV infection |
| Follow-Up Tests | ||||
| Antibody differentiation test |
Confirm positive HIV screening test result Differentiate between HIV-1 and HIV-2 antibodies |
Preferred confirmatory test | May require follow-up NAAT if results are negative or indeterminate | If antibodies for both HIV-1 and HIV-2 are detected, NAAT is recommended to differentiate and rule out dual infection |
| Western blot test | Confirm positive HIV screening test result | n/a |
No longer recommended May yield false-negative or indeterminate results early in disease course |
n/a |
| NAAT | Confirm HIV-1 diagnosis |
Resolves discrepant results between fourth generation tests and confirmatory tests Resolves indeterminate results of antibody differentiation testing |
More expensive than other tests | May also provide viral load |
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n/a, not applicable Sources: CDC, 2020 ; AIDSInfo, 2020 ; Cohen, 2011 ; CDC, 2018 ; CDC, 2014 |
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ARUP offers a variety of tests for HIV screening and diagnosis. Different tests may be appropriate, depending on the needs of the patient.
| Testing Need | ARUP Laboratory Test | Comments |
|---|---|---|
| Screen infant for HIV | Human Immunodeficiency Virus 1 (HIV-1) by Qualitative NAAT (3003760) | Preferred test for diagnosis in infants; use to detect HIV-1 virus RNA; does not detect proviral DNA, which is less likely to be affected by maternal use of ART |
| Human Immunodeficiency Virus 1 (HIV-1) by Quantitative NAAT, Plasma (3000867) | Use to detect and quantify HIV-1 virus RNA, but may not detect virus in infants younger than 1 mo | |
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Diagnose or confirm HIV in adult (Components may include antigen/antibody detection [fourth generation test], HIV-1/HIV-2 antibody discrimination, and/or confirmation test) |
Human Immunodeficiency Virus (HIV) Combo Antigen/Antibody (HIV-1/O/2) by CIA, Reflexive Panel (2012674) |
Fourth generation antigen/antibody screening test Repeatedly reactive antigen/antibody screening results are confirmed with an HIV-1/HIV-2 antibody differentiation test If differentiation test results are indeterminate, HIV-1 by quantitative NAAT is added |
| Human Immunodeficiency Virus (HIV) Combo Antigen/Antibody (HIV-1/0/2) by CIA, with Reflex to HIV-1/HIV-2 Antibody Differentiation, Supplemental (2013333) |
Fourth generation antigen/antibody screening test Repeatedly reactive antigen/antibody screening results are confirmed with an HIV-1/HIV-2 antibody differentiation test |
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| Human Immunodeficiency Virus Types 1 and 2 (HIV-1/2) Antibody Differentiation, Supplemental, with Reflex to HIV-1 Quantitative NAAT, Plasma (2012669) |
HIV-1/HIV-2 antibody differentiation test used to confirm repeatedly reactive antigen/antibody screening results If differentiation test results are indeterminate, HIV-1 by quantitative NAAT is added Intended only as a follow-up test for a positive fourth-generation screening test |
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| Human Immunodeficiency Virus Types 1 and 2 (HIV-1/2) Antibody Differentiation, Supplemental (2013107) |
HIV-1/HIV-2 antibody differentiation test used to confirm repeatedly reactive antigen/antibody screening results Intended only as a follow-up test for a positive fourth-generation screening test |
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| Human Immunodeficiency Virus 1 (HIV-1) by Qualitative NAAT(3003760) | Confirms diagnosis of HIV-1 after positive antibody/antigen test and antibody discrimination | |
| Human Immunodeficiency Virus (HIV) Combo Antigen/Antibody (HIV-1/O/2) by CIA, with Reflex to HIV-1 Antibody Confirmation by Western Blot (2006526) |
Fourth generation antigen/antibody screening test Repeatedly reactive antigen/antibody screening results are confirmed by HIV-1 antibody by western blot No longer recommended |
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| Human Immunodeficiency Virus Type 1 (HIV-1) Antibody Confirmation by Western Blot (0020284) |
Confirms diagnosis of HIV-1 after positive antibody/antigen test No longer recommended |
|
| Source: AIDSInfo, 2020 | ||
Treatment-Related Testing
Refer to the ARUP Treatment-Related HIV Test Offerings table for a brief overview of the ARUP tests covered in this section.
Initial Pretreatment Tests
After an established diagnosis of HIV and before beginning treatment with ART, initial recommended laboratory tests include the HIV RNA level, CD4 T-lymphocyte cell count, tests for other infections (eg, hepatitis B virus [HBV], hepatitis C virus [HCV], and tuberculosis), and tests to assess general health, such as CBC, kidney and liver function, lipid level, and glucose level tests. A pregnancy test should be given to individuals who have a uterus and are of childbearing age before beginning treatment. ART initiation should not be delayed by these test results unless kidney or liver damage is discovered or transmitted drug resistance is strongly suspected.
Drug Resistance Tests
Drug resistance testing involves genotypic or phenotypic testing to evaluate viral strains and guide the selection of therapy regimens. These tests are designed to detect resistance to the various classes of ART drugs. Resistance testing should be performed in early HIV infection even if the patient has opted to postpone ART so that the results will be available when the individual begins therapy. However, repeat testing at the start of therapy should be considered because of the possibility that a drug-resistant virus (superinfection) has been acquired between initial diagnosis and ART initiation. Resistance testing should also occur at treatment failure and when a regimen switch is planned.
Genotypic Tests
Genotypic is preferred over phenotypic testing in most contexts (eg, in patients with known drug resistant-variant patterns and in those anticipated to have uncomplicated variant patterns) because of its faster turnaround time, lower cost, and greater sensitivity for combinations of wild-type and resistant virus. Additionally, test interpretation is more straightforward than with phenotypic tests.
Genotypic assays generally use either Sanger sequencing or next generation sequencing (NGS) to identify variants in the reverse transcriptase (RT), protease resistance (PR), and integrase genes of circulating HIV RNA, which may assist in identifying resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-NRTIs (NNRTIs), integrase strand transfer inhibitors (INSTIs), and protease inhibitors (PIs). Testing is also available to detect variants in the gp41 gene, which are associated with resistance to fusion inhibitors (ie, enfuvirtide). The International Antiviral Society–USA has a list of resistance-associated variants, and the Stanford University HIV Drug Resistance Database is a useful resource to aid in genotypic test interpretation.
Therapy-Naive Patients. Genotypic testing for resistance-associated variants is indicated in patients who have not previously been treated with ART. If transmitted resistance to INSTIs is suspected, testing for variants that cause INSTI resistance (ie, variants in integrase genes) should be considered. Testing for INSTI resistance may require a separate test when using the Sanger sequencing methodology; however, INSTI resistance can be assessed at the same time as PI, NRTI, and NNRTI resistance when using NGS.
Therapy-Experienced Patients. In patients currently being treated with ART, resistance testing is recommended in those with an unsatisfactory decrease in viral load and in those with treatment failure, as indicated by virologic failure and HIV RNA measurements >1,000 copies/mL. In patients with HIV RNA values between 500 and 1,000 copies/mL, drug resistance testing may be inconclusive but should still be considered.
In cases of virologic failure, resistance testing should be performed while the patient is still on the treatment regimen or within 4 weeks of treatment discontinuation to increase the likelihood that resistance variants will be detected. If treatment failure occurs during therapy with INSTIs, testing for INSTI resistance should be performed.
Phenotypic Tests
Phenotypic assays are used to evaluate whether the virus is able to replicate in the presence of various ART drug concentrations. Phenotypic testing should be performed in addition to genotypic testing in patients who are known or anticipated to have complex drug-resistant variant patterns.
Phenotypic coreceptor tropism assays are also preferred over genotypic assays when a CCR5 antagonist (or receptor inhibitor [RI]) is under consideration or when a patient experiences virologic failure while receiving a CCR5 antagonist (eg, maraviroc). Coreceptor tropism testing is also useful to guide treatment in ART-experienced patients with significant drug resistance or consistently high concentrations of HIV RNA.
For more information concerning variants associated with viral resistance to therapy, refer to ARUP's ViroSeq HIV-1 Antiretroviral Drug Resistance Report.
Drug Hypersensitivity Test
The HLA-B*5701 genotyping test is required by the U.S. Food and Drug Administration (FDA) before treatment with abacavir to identify patients at risk for abacavir hypersensitivity. Abacavir should not be administered if the patient is a carrier of HLA-B*5701, regardless of age. Risk of hypersensitivity is the same whether the patient carries one or two copies of the variant allele. For more information, see the ARUP Consult Germline Pharmacogenetics topic.
| Testing Need | ARUP Laboratory Test | Comments | Drug Classes Covered | ||||
|---|---|---|---|---|---|---|---|
| PIs | NRTIs | NNRTIs | INSTIs | RIs | |||
| Confirm HIV in adult and predict ART resistance via genotyping | Human Immunodeficiency Virus 1 (HIV-1) by Quantitative NAAT with Reflex to HIV-1 Drug Resistance by Next Generation Sequencing (3000870) | Includes viral load and drug resistance testing | ✓ | ✓ | ✓ | ✓ | |
| Predict ART resistance via genotyping | Human Immunodeficiency Virus 1 Drug Resistance by Next Generation Sequencing (3003853) | For patients with documented HIV-1 infection and viral load ≥500 copies/mL | ✓ | ✓ | ✓ | ✓ | |
| Human Immunodeficiency Virus Type 1 (HIV-1) GenoSure Archive (3002503) | For patients with documented HIV-1 infection and undetectable viral load | ✓ | ✓ | ✓ | ✓ | ||
| Predict ART resistance via phenotyping | Human Immunodeficiency Virus Type 1 (HIV-1) PhenoSense (3000882) | For patients with documented HIV-1 infection and viral load ≥500 copies/mL | ✓ | ✓ | ✓ | ||
| Predict ART resistance via combined genotyping plus phenotyping | HIV PhenoSense GT (0092399) | For patients with known or suspected complex drug resistance patterns and viral load ≥500 copies/mL | ✓ | ✓ | ✓ | ||
| Human Immunodeficiency Virus Type 1 (HIV-1) PhenoSense GT Plus Integrase (3001186) |
For patients with known or suspected complex drug resistance patterns and viral loads ≥500 copies/mL Use only if INSTI resistance is suspected |
✓ | ✓ | ✓ | ✓ | ||
|
Determine eligibility for CCR5 antagonist treatment (Tropism types include R5, X4, dual, and mixed) |
Human Immunodeficiency Virus Type 1 (HIV-1) Trofile Co-Receptor Tropism (3001246) | For patients with documented HIV-1 infection and viral load ≥1,000 copies/mL | ✓ | ||||
| Trofile (DNA) Co-Receptor Tropism (3002570) | For patients with undetectable viral load | ✓ | |||||
| Determine potential for hypersensitivity response to abacavir | HLA-B*57:01 for Abacavir Sensitivity (2002429) | Standard of care before the initiation of abacavir therapy, as per the FDA | ✓ | ||||
Monitoring
Quantitative viral load testing can help to determine a therapeutic plan and is used for monitoring during treatment. After the initiation of ART, patients should be evaluated within 6 weeks to assess their viral load, as well as their ability to tolerate the therapy and adherence to treatment. Appropriate monitoring tests include quantitative NAAT and quantitative CD4 lymphocyte tests. Although the absolute CD4 lymphocyte count can aid in the monitoring of HIV, CD4 testing is not required for patients once viral suppression has been established (ie, is stable). The HIV Medicine Association recommends the CD4 count be monitored every 3-6 months for the first 2 years after treatment begins; after 2 years, in patients with an undetectable viral load and CD4 counts of 300-500 cells/mm3, the CD4 count should be measured annually. CD4 monitoring can be considered optional once the CD4 count is consistently >500 cells/mm3.
| Testing Need | ARUP Laboratory Test | Comments |
|---|---|---|
| Monitor the effect of treatment | Human Immunodeficiency Virus 1 (HIV-1) by Quantitative NAAT, Plasma (3000867) | Use to detect and quantify HIV-1 to assess viral response to antiretroviral treatment |
| Lymphocyte Subset Panel 1 – CD4 Absolute Count Only (0095854) | Aids in clinical management of patients with HIV before viral suppression has been established (HIV Med Assoc) | |
| Lymphocyte Subset Panel 2 - CD4 Percent and Absolute (0095885) | Aids in clinical management of patients with HIV before viral suppression has been established (HIV Med Assoc) |
ARUP Laboratory Tests
Qualitative Chemiluminescent Immunoassay/Qualitative Immunoassay/Quantitative Transcription-Mediated Amplification
Qualitative Chemiluminescent Immunoassay/Qualitative Immunoassay
Qualitative Chemiluminescent Immunoassay /Qualitative Western Blot
Qualitative Immunoassay/Quantitative Transcription-Mediated Amplification
Qualitative Immunoassay
Qualitative Western Blot
Qualitative Transcription-Mediated Amplification
Quantitative Transcription-Mediated Amplification
Quantitative Transcription-Mediated Amplification
Quantitative Transcription-Mediated Amplification/Massively Parallel Sequencing
Refer to the ARUP Treatment-Related HIV Test Offerings table for more information on the following tests.
Quantitative Transcription-Mediated Amplification/Massively Parallel Sequencing
Massively Parallel Sequencing
Polymerase Chain Reaction (PCR)/Sequencing
Polymerase Chain Reaction (PCR)/Culture
Polymerase Chain Reaction (PCR)/Culture
Polymerase Chain Reaction (PCR)/Culture
Polymerase Chain Reaction (PCR)/Culture
Polymerase Chain Reaction (PCR)/Culture
Polymerase Chain Reaction/Fluorescence Monitoring
Refer to the ARUP HIV Monitoring Test Offerings table for more information on the following tests.
Quantitative Transcription-Mediated Amplification
Quantitative Flow Cytometry
Quantitative Flow Cytometry
References
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AIDSinfo - Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents
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AIDSinfo - Guidelines for the Use of Antiretroviral Agents in Pediatric HIV Infection
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Medical Experts
Hillyard
McMillin
Reimer
Slev
Refer to the HIV Diagnostic Tests at a Glance table for more information about the following tests.