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HIV-1 Infection: Laboratory Tests for Selecting Antiretroviral Therapy

HIV-1 Infection: Laboratory Tests for Selecting Antiretroviral Therapy

Test Guide

HIV-1 Infection

Laboratory Tests for Selecting Antiretroviral Therapy

  

This Test Guide provides an overview of laboratory tests that, in the context of the patient’s clinical and laboratory history, may be useful in selecting components of an antiretroviral (ARV)  treatment regimen. Flowcharts illustrate how these tests might be used for evaluating treatment choices at entry into HIV care (Figure 1) and when changing treatment regimens (Figure 2). A list of relevant tests is provided in Appendix 1. A summary of recommendations from the Department of Health and Human Services for laboratory testing in selecting antiretroviral therapy is provided in Appendix 2.

Figure 1. Tests for Antiretroviral (ARV) Drug Selection Prior to Treatment Initiation
Figure 2. Tests for Drug Selection When Considering a Change in Treatment Regimen

Resistance Tests

HIV drug resistance can be evaluated with genotypic or phenotypic testing. Regardless of the approach, resistance testing should be performed on samples obtained while the patient is still receiving the failing regimen or soon (<4 weeks) after discontinuation; otherwise, resistant variants may not be detected but may re-emerge if the drug is reinstated. Therapy decisions thus should also take into account the results of previous resistance testing.1

Genotypic Testing

Genotypic HIV resistance assays identify drug resistance-associated mutations in virus isolated from HIV-infected individuals. Relative to phenotypic testing, genotypic testing is generally considered to have the advantages of rapid turnaround time (typically about 1-2 weeks), lower cost, and enhanced detection of resistance-associated mutations in mixed virus populations.1

Quest Diagnostics offers a genotype assay (HIV-1 Genotype; test code 34949) that establishes the HIV-1 subtype and tests for mutations associated with resistance to currently available protease inhibitors (PIs), nucleos(t)ide reverse transcriptase inhibitors (NRTIs), and nonnucleoside reverse transcriptase inhibitors (NNRTIs). A separate genotype assay is offered to test for mutations associated with resistance to the integrase inhibitors raltegravir, elvitegravir, and dolutegravir. The sequencing results are interpreted with a rules-based algorithm developed by Quest Diagnostics scientific directors and academic associates. The patient report form provides the actual mutations detected as well as the drug resistance patterns predicted from these mutations.

Phenotypic Testing

Phenotypic resistance tests assess the ability of HIV to replicate in the presence of selected antiretroviral drugs, without regard to specific mutations present in the viral population. These assays use a recombinant HIV-1 virus consisting of a reference strain with a cloned insert from the patient’s virus spanning the protease and the reverse transcriptase genes. The resulting recombinant virus is then transfected into cultured cells, which are exposed to increasing concentrations of each drug to find the concentration that inhibits viral replication by 50% (IC50). This value is compared to the IC50 of a wild-type reference strain to calculate a “fold change“ in phenotypic drug susceptibility. The fold change is evaluated against a clinical cutoff value to determine whether the virus remains susceptible to each drug. Because phenotypic testing reflects the net effect of HIV mutations, it may be particularly useful in highly treatment-experienced patients.

Table 1 summarizes some of the key differentiating features of genotypic and phenotypic testing.

Table 1. Differentiating Features of Genotypic and Phenotypic HIV Resistance Testing

Genotype

Phenotype

Relatively rapid turnaround time (1-2 weeks)

Lower cost

Greater sensitivity than phenotype for the
   detection of resistance-associated mutations
   in mixed virus populations

Qualitative assessment of resistance
   (resistance likely or not likely)

Useful in most clinical settings that call for
   resistance testing

• Direct in vitro measurement of drug
   susceptibility of patient virus population

• Presents sum effect of mutations on
   susceptibility to each drug (fold-change
   value)

• Useful in patients with complex mutation
   patterns

 

What the Guidelines Say

Genotypic assays are currently recommended at all stages of disease management that call for ARV resistance testing (Table 2),1,2 mainly because of their generally lower cost, faster turnaround time, and more sensitive detection of resistance mutations in mixed viral populations. The addition of phenotypic analysis is recommended for treatment-experienced patients with complex HIV drug resistance mutation patterns, particularly those related to protease inhibitors. Phenotypic analysis may also be relevant for patients initially infected with multidrug-resistant virus. Given the complexity involved in managing treatment-experienced patients, expert advice is recommended when assessing options for those developing virologic failure.1

Table 2. Use of HIV Drug Resistance Testing at Different Stages of Disease Management1,2
Indication Resistance Test
Acute HIV infection Genotype
Newly diagnosed chronic HIV infection Genotype
Treatment initiation Genotype
First or second treatment failurea Genotype
Treatment failure in patients with complex treatment
history or mutation pattern
Genotype, preferably with addition of
phenotype

a

Patients initially infected with multidrug-resistant virus may have complex mutation patterns and benefit from addition of phenotypic resistance testing.

Other Tests Used in Selecting HIV ARVs

HIV-1 Coreceptor Tropism Assay

Analysis of HIV-1 coreceptor tropism helps determine eligibility for treatment with CCR5 antagonists. HIV-1 utilizes the CD4 cell surface receptor and either the CCR5 or CXCR4 chemokine receptor to infect cells. The only FDA-cleared CCR5 antagonist currently available, maraviroc, inhibits HIV-1 entry by binding to the CCR5 coreceptor and is effective only against R5-tropic viruses that exclusively utilize the CCR5 coreceptor. CCR5 antagonists do not effectively inhibit X4-tropic viruses, which exclusively utilize the CXCR4 coreceptor, or dual/mixed (D/M)-tropic viruses, which utilize both coreceptors. Thus, testing for coreceptor tropism is recommended for patients being considered for treatment with a CCR5 inhibitor and may also be helpful for evaluating virologic failure during treatment with an CCR5 antagonist.1

Although most early studies of CCR5 antagonists assessed viral coreceptor tropism with a phenotypic assay, a more rapid genotypic assay is now available that analyzes the third variable loop (V3) of the HIV-1 envelope gene, the primary determinant of tropism.3 To enhance sensitivity for detection of minority X4 virus in D/M viral populations, next-generation DNA sequencing (ultradeep sequencing [UDS]) is used. UDS performance is comparable to phenotypic tropism tests for predicting clinical response to maraviroc therapy.3,4 The Quest Diagnostics test is also comparable to a high-sensitivity phenotypic test in distinguishing between virologic responders and nonresponders.5 Guidelines consider phenotypic testing the preferred method because of its high sensitivity.1 However, genotypic testing is a guideline-approved alternative because of its lower cost and faster analytical times.1

HIV-1 coreceptor tropism testing requires sufficient levels of HIV-1 RNA (typically ≥1,000 copies/mL). Because successful antiretroviral therapy can suppress HIV-1 RNA to well below this level, patients receiving suppressive therapy may not have enough RNA for tropism to be determined. One option in such cases is to test for HIV-1 proviral DNA (HIV-1 DNA that has integrated into the host genome). Because proviral DNA persists despite suppressive therapy, it can be used to determine tropism status when HIV-1 RNA is undetectable.6 Although guidelines indicate that proviral HIV-1 DNA can be used to determine HIV-1 tropism status in patients with undetectable viral loads (eg, before switching to a maraviroc-containing regimen), they note that the clinical utility of this approach has not yet been determined.1

HLA-B*5701 Typing

This assay helps predict hypersensitivity to the NRTI abacavir, which occurs in roughly 2% to 9% of patients.7 Susceptibility to this hypersensitivity reaction has been linked to a specific human genetic variation known as HLA-B*5701. Pharmacogenetic screening for the HLA-B*5701 allele is recommended for abacavir-naïve patients before initiation of abacavir treatment.7,8 Selection of a non-abacavir regimen would then be recommended for patients with the HLA-B*5701 allele. Testing is also recommended for patients who are reinitiating abacavir treatment, if their HLA-B*5701 status has not been previously determined and they have no history of adverse reaction to abacavir. This test uses PCR followed by hybridization with sequence-specific oligonucleotide probes to detect the HLA-B*5701 allele.
Appendix 1. Laboratory Tests Used in Guiding Selection of Antiretroviral Drugsa
Test Code Test Name Primary Clinical Use or Differentiating Factors
34949 HIV-1 Genotypeb Detect mutations associated with resistance to RTIs and PIs
91692 HIV-1 Genotype (RTI, PI, Integrase Inhibitors)b Detect mutations associated with resistance to RTIs, PIs, and INSTIs
94015 HIV-1 Genotype and Coreceptor Tropism, Ultradeep Sequencingb,c Detect mutations associated with resistance to RTIs and PIs and determine eligibility for therapy with CCR5 antagonist (genotypic assay)
16868 HIV-1 Integrase Genotypeb Assess mutations associated with resistance to the integrase inhibitors raltegravir, elvitegravir, and dolutegravir
94014 HIV-1 Coreceptor Tropism, Ultradeep Sequencingb,c Determine eligibility for therapy with CCR5 antagonist (genotypic assay)
91299 HIV-1 Coreceptor Tropism, Proviral DNAb Determine eligibility for therapy with CCR5 antagonist in patients with viral load <1000 HIV-1 RNA copies/mL
19774 HLA-B*5701 Typingd Assess risk of abacavir hypersensitivity reaction

Viral Load with Reflex to Resistance Testing

34471 HIV-1 RNA, Quantitative PCR with Reflex to Genotypec,e Evaluate need for treatment modification; detect mutations associated with resistance to antiretroviral drugs
90926 HIV-1 RNA, Quantitative Real-Time PCR with Reflex to Integrase Genotypec,e
91691 HIV-1 RNA, Quantitative Real-Time PCR with Reflex to Genotype (RTI, PI, Integrase) c,e

INSTI, integrase strand transfer inhibitor; PI, protease inhibitor; RTI, reverse transcriptase inhibitor.

a

For additional testing options, consult the Quest Diagnostics online Test Center (QuestDiagnostics.com/TestCenter).

b

This test was developed and its performance characteristics have been determined by Quest Diagnostics. It has not been cleared or approved by the U.S. Food and Drug Administration. This assay has been validated pursuant to the CLIA regulations and is used for clinical purposes.

c

Reflex tests are performed at an additional charge and are associated with an additional CPT code(s).

d

Typing performed by using AS-PCR with reflex to the FDA-cleared LABType® SSO Kit. The AS-PCR portion of the test was developed and its performance characteristics have been determined by Quest Diagnostics.

e

Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche Molecular Systems, Inc.

Appendix 2. Summary of DHHS Testing Recommendations for Selecting Antiretroviral Treatment1

Clinical Setting

Testing Recommendation

General Recommendations

 

ART-naïve patients

  • Genotypic resistance testing, regardless of whether ART is initiated immediately or deferred
  • If ART deferred, consider repeat resistance testing when therapy is initiated
  • INSTI genotypic resistance if considering an INSTI and transmitted INSTI resistance is a concern
  • Screen for infection with HBV and HCVa
 

Patients with virologic failure

  • Drug resistance testing while patient is taking prescribed ART, or, if not possible, within 4 weeks of discontinuing ARTb:
    • –  If HIV RNA levels ˃1,000 copies/mL

      –  If HIV RNA levels ˃500 copies/mL but <1,000 copies/mL, testing may not be successful but should still be considered

      –  If plasma viral load <500 copies/mL, testing usually not recommended

  • Genotypic assay preferred for first or second treatment failure

Specific Recommendations

 

Patients with known or suspected complex drug-resistance mutation patterns

  • Genotypic and phenotypic testing
 

Patients failing INSTI-based regimens

  • INSTI genotypic resistance assay
 

Patients with virologic failure on a CCR5 antagonist, or patients being considered for CCR5 antagonist treatment

  • Coreceptor tropism assay (phenotypic or genotypic)
  • Sequencing assay with proviral DNA obtained from peripheral blood mononuclear cells if plasma HIV-1 RNA below the level of detectionc
 

Patients being considered for an abacavir-containing regimen

  • HLA-B*5701 typing assay

ART, antiretroviral treatment; DHHS, Department of Health and Human Services; INSTI, integrase strand transfer inhibitor.

a

Coinfection of HIV and either hepatitis B or hepatitis C virus may affect the choice of ART. Reference 1 provides guidance on ART selection in coinfected individuals.

b

Resistance testing may still provide useful information more than 4 weeks after discontinuation of ART, but previously selected resistance mutations can be missed.

c

Clinical utility remains to be determined.

References

  1. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Department of Health and Human Services. https://aidsinfo.nih.gov/
    contentfiles/lvguidelines/adultandadolescentgl.pdf
    . Updated July 14, 2016. Accessed August 22, 2016.

  2. Günthard HF, Saag MS, Benson CA, et al. Antiretroviral Drugs for Treatment and Prevention of HIV Infection in Adults: 2016 Recommendations of the International Antiviral Society-USA Panel. JAMA. 2016;316:191-210.

  3. Swenson LC, Mo T, Dong WW, et al. Deep V3 sequencing for HIV type 1 tropism in treatment-naive patients: a reanalysis of the MERIT trial of maraviroc. Clin Infect Dis. 2011:53:732-742.

  4. Swenson LC, Mo T, Dong WW, et al. Deep sequencing to infer HIV-1 co-receptor usage: application to three clinical trials of maraviroc in treatment-experienced patients. J Infect Dis. 2011;203:237-245.

  5. Kagan RM, Johnson EP, Siaw M, et al. A genotypic test for HIV-1 tropism combining Sanger sequencing with ultradeep sequencing predicts virologic response in treatment-experienced patients. PloS One. 2012;7:e46334.

  6. Vandekerckhove LP, Wensing AM, Kaiser R, et al. European guidelines on the clinical management of HIV-1 tropism testing. Lancet Infect Dis. 2011;11:394-407.

  7. Ziagen® [prescribing information]. Research Triangle Park, NC: GlaxoSmithKline; 2015.

  8. Information for Healthcare Professionals: Abacavir (marketed as Ziagen) and abacavir-containing medications. http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/ucm123927.htm. Updated August 15, 2013. Accessed August 22, 2016.
     

 
 

Content reviewed 10/2016

 

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* The tests listed by specialist are a select group of tests offered. For a complete list of Quest Diagnostics tests, please refer to our Directory of Services.