Human Immunodeficiency Virus 1, Antiretroviral Drug Resistance Testing - HIV Drug Resistance

Immediate treatment with antiretroviral therapy (ART) is recommended for all individuals with a diagnosis of human immunodeficiency virus (HIV). Many different drugs and multidrug regimens may be used successfully to suppress viral replication (see U.S. Department of Health and Human Services Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents). However, resistance can develop to any drug class or regimen.   Drug resistance testing is recommended before the start of therapy to guide selection of treatment regimens and should be performed for treatment guidance when treatment failures occur.  Drug resistance variants can be detected using phenotypic and genotypic methods, but genotypic methods are preferred in most circumstances.  See Drug Resistance Testing below.

Quick Answers for Clinicians

What is transmitted drug resistance and how does it relate to antiretroviral testing?

Human immunodeficiency virus (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-naïve patients before therapy initiation.

Should antiretroviral therapy be delayed until drug resistance test results are obtained?

The World Health Organization recommends that antiretroviral therapy (ART) begin within 7 days of a human immunodeficiency virus (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.  This 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. 

What is the optimal timing for resistance testing in cases of treatment failure?

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 human immunodeficiency virus (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 levels below the limit of detection of genotypic or phenotypic tests.  However, even low levels of drug-resistant virus can lead to treatment failure when ART is reinitiated. 

Which antiretroviral drug classes and regimens are commonly used in HIV?

The U.S. Food and Drug Administration (FDA) has approved seven different classes of drugs for antiretroviral therapy (ART) use in HIV.  The various ART drugs are available in both single- and multidrug formulations. Drug resistance to all classes can develop, and the use of three or more highly active antiretroviral therapy (HAART) agents is used to counter this tendency.  Treatment generally involves two nucleoside reverse transcriptase inhibitors (NRTIs) with one of the following: an integrase strand transfer inhibitor (INSTI), a non-NRTI (NNRTI), or a protease inhibitor (PI) with a pharmacokinetic enhancer.  In most patients with HIV, this multidrug approach helps to suppress virus replication.  See the U.S. Department of Health and Human Services (DHHS) Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents  for a comprehensive list of drugs and drug combinations approved for use by the FDA.

Indications for Testing

Laboratory testing in connection with ART for HIV is appropriate for:

  • Patients newly diagnosed with HIV
  • Patients previously diagnosed with HIV who delayed ART (testing should be repeated if previously performed)
  • Patients who experience treatment failure during ART

Laboratory Testing

Initial Testing

Following an established diagnosis of HIV and before beginning treatment with ART, 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], 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 women 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 Testing

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. 

Genotypic Testing

Genotypic is preferred over phenotypic testing in most contexts because of its faster turnaround time, lower cost, and greater sensitivity for combinations of wild-type and resistant virus.  Test interpretation is more straightforward than with phenotypic tests.  Genotypic testing is also preferred in patients with known drug resistance variant patterns and in those anticipated to have uncomplicated variant patterns.  Combined genotypic and phenotypic tests are also available, as are tests that quantify HIV RNA and then reflex to genotypic or phenotypic tests to investigate possible drug resistance.

Most genotypic assays use Sanger sequencing to identify variants in the reverse transcriptase (RT), protease resistance (PR), and integrase genes of circulating HIV RNA in plasma.  These assays can identify 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).  Test interpretation requires awareness of the variants that might result from various ART drugs and the connection between those variants and possible resistance to other drugs.  The International AIDS 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-Naïve Patients

Genotypic testing for variants in RT and PR genes 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. 

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 levels >1,000 copies/mL.  In patients with HIV RNA levels 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 regimen or within 4 weeks of treatment discontinuation, if possible; testing during this period increases the likelihood that resistance variants will be detected.  If treatment failure occurs during therapy with INSTIs, testing for INSTI resistance should be performed to evaluate whether the patient can be given INSTIs in future regimens. 

Phenotypic Testing

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 resistance variant patterns. 

Other Testing for Resistance to Specific Drugs

Coreceptor Tropism Testing for CCR5 Antagonist Resistance

Coreceptor tropism assays are recommended if a CCR5 antagonist is under consideration, as well as for patients who experience virologic failure while receiving a CCR5 antagonist (ie, maraviroc).  Phenotyping coreceptor tropism assays are preferred to genotyping assays.  Coreceptor tropism usage “shifts” can occur during the disease course, so testing must be repeated at treatment failure.  This testing is also useful to guide treatment in ART-experienced patients with significant drug resistance or consistently high levels of HIV RNA. 

In patients with an undetectable viral load who are being considered for treatment with a CCR5 antagonist, a proviral DNA tropism assay can be used, but caution is advised because the clinical efficacy of this test is still being explored. 

HLA-B*5701 Testing for Abacavir Hypersensitivity

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. 

Monitoring

After ART initiation, patients should be evaluated within 6 weeks to assess their ability to tolerate the therapy and adherence to treatment.  Genotypic or phenotypic resistance testing should occur at treatment failure and also when a regimen switch is planned.  In patients who experience virologic failure, resistance testing should be performed while the patient is still receiving ART or within 4 weeks of discontinuation to increase the likelihood that the resistant virus will be detected if present. 

ARUP Laboratory Tests

Genotypic Testing

HIV-1 genotyping provides antiretroviral susceptibility information for PIs, NRTIs, and NNRTIs

Intended for patients with viral load >1,000 copies/mL

May not detect HIV-1 populations <20% of the total population

HIV-1 genotyping provides antiretroviral susceptibility information for PIs, NRTIs, NNRTIs, and INSTIs

Intended for patients with viral load >1,000 copies/mL

HIV-1 genotyping provides antiretroviral susceptibility information for protease inhibitors (PI), reverse transcriptase inhibitors (NRTI, NNRTI), and integrase inhibitors (INI)

Preferred test is HIV-1 genotype and integrase inhibitor resistance by sequencing

HIV genotyping provides susceptibility information for INSTIs

Intended for patients with viral load >500 copies/mL

HIV-1 genotyping provides antiretroviral susceptibility information for PIs, NRTIs, and NNRTIs

Intended for patients with undetectable viral load or low-level viremia

Phenotypic Testing

HIV-1 phenotyping provides antiretroviral susceptibility information for PIs, NRTIs, NNRTIs

Intended for patients with documented HIV-1 infection with viral load >500 copies/mL

Combined Phenotypic/Genotypic Testing

HIV-1 combined pheno- and genotyping provides antiretroviral susceptibility information for PIs, NRTIs, NNRTIs, and INSTIs

Preferred test for patients with known or suspected complex drug resistance patterns (eg, suboptimal virologic response to treatment and viral load rebound)

Intended for patients with documented HIV-1 infection and viral loads >500 copies/mL

HIV-1 combined pheno- and genotyping provides antiretroviral susceptibility information for PIs, NRTIs, and NNRTIs

Useful in clarifying and confirming results from HIV genotyping tests

Intended for patients with viral load >500 copies/mL

HIV-1 Quantitative Tests With Reflex to Phenotypic or Genotypic Tests

Detect and quantify HIV-1

Determine HIV drug resistance by DNA sequencing (if HIV-1 by quantitative nucleic acid amplification tests (NAAT) result is ≥3.0 log copies/mL, then HIV-1 genotype by sequencing will be performed)

Detect and quantify HIV-1; includes phenotypic determination (if HIV-1 by quantitative NAAT result is ≥500 copies/mL, then HIV PhenoSense GT will be performed)

Other Testing for Resistance to Specific Drugs
Coreceptor Tropism Testing for CCR5 Antagonist Resistance

HIV-1 phenotyping determines tropism (CCR5 or CXCR4) of HIV-1 virus in patients being considered for CCR5 antagonist therapy

If patient’s viral load is <1,000 HIV-1 copies/mL, refer to tropism assay

Use to detect HIV-1 coreceptor tropism to determine an individual's eligibility for CCR5 antagonist.

HLA-B*5701 Testing for Abacavir Hypersensitivity

Standard of care before abacavir therapy per FDA

Predict risk of abacavir hypersensitivity syndrome

Screen before reinitiation of treatment in individuals who have previously tolerated abacavir but whose HLA-B*57:01 status is unknown

Medical Experts

Contributor
Contributor

McMillin

Gwendolyn A. McMillin, PhD
Professor of Clinical Pathology, University of Utah
Scientific Director, Mass Spectrometry Platform; Medical Director, Clinical Toxicology and Pharmacogenomics, ARUP Laboratories
Contributor

Reimer

Larry G. Reimer
Associate Dean for Curriculum and Graduate Medical Education, School of Medicine; Adjunct Professor, Department of Internal Medicine; Professor, Pathology, University of Utah
Chief of Clinical Microbiology, Veteran's Affairs Medical Center

References

Additional Resources
Resources from the ARUP Institute for Clinical and Experimental Pathology®