Resistance Testing

Background

Genotype and phenotype testing for resistance currently is commercially available for all nucleoside reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase inhibitors that have been approved by the U.S. Food and Drug Administration (FDA). In addition, standardized genotype testing is commercially available for enfuvirtide (a fusion inhibitor).

Neither genotype nor phenotype predicts which antiretroviral (ARV) drugs will be active in a particular patient, only ARVs that are not likely to be active. Nevertheless, studies comparing the use of resistance testing with expert opinion alone have shown that resistance testing can improve virologic control of HIV. Resistance testing is used to guide subsequent treatment for patients whose antiretroviral therapy (ART) is failing and for those whose viral load is not completely suppressed after starting therapy. It also is used to select an initial regimen that is likely to be effective for patients who have never been treated, and it is recommended for all patients with HIV infection (both acute and chronic) upon entry into care, whether or not ART is to be initiated. In addition, resistance testing is recommended for pregnant women who are not on ART and for those who are on ART but have a detectable HIV viral load.

Genotype Tests

Genotype testing works by amplifying and sequencing HIV taken from a patient to look for mutations in the HIV reverse transcriptase, protease, integrase, or envelope genes that are known to correlate with clinical resistance to ARV drugs. Genotype tests generally can detect mutations in plasma samples with HIV RNA levels of >1,000 copies/mL, but sometimes are successful with viral loads of 500-1,000 copies/mL. Species representing 20% or more of the amplified product usually can be detected by current techniques, but minor species may not be detected. Resistance mutations that developed in the past during treatment with certain ARV medications may be archived as minor species and become invisible to genotype testing (as early as 4-6 weeks) after the drug is discontinued. These resistance mutations may reemerge and cause drug failure, however, if the previous drug is used again. By contrast, mutations acquired at the time of infection (from a transmitted virus that was already resistant) appear to persist for years, although the duration is not known precisely and may vary by mutation.

A genotype test takes 1-2 weeks to complete. The results are reported as a list of the mutations detected; most reports also include an interpretation that indicates the drug resistance likely to be conferred by those mutations (see "Limits of Resistance Testing," below).

Note that the standard genotype tests detect only mutations that may affect reverse transcriptase inhibitors and PIs; a specific genotype for the integrase inhibitor (or fusion inhibitor) class must be ordered if there is concern for resistance to this class. Resistance to CCR5 antagonists is uncommon; a change in coreceptor tropism is a more likely cause of virologic failure for patients taking maraviroc. For such patients, a coreceptor tropism assay should be obtained. Afterwards, if resistance to CCR5 antagonists is still suspected, a resistance test can be obtained.

Genotype results must be interpreted carefully. Because mutations can become invisible to the genotype testing process when the selective pressure of a drug is removed, a thorough ARV history, a review of any past resistance tests, and expert clinical assessment are necessary to put the results of a genotype test in proper perspective and to identify options for further treatment (see "Limits of Resistance Testing," below). A compilation of the most common HIV mutations selected by the three classes of antiretroviral agents is available. Other resources useful in understanding resistance testing and interpreting test results include the information complied by the International Antiviral Society-USA and the Stanford University HIV Drug Resistance Database.

A "virtual phenotype" is a genotype that is compared with a databank of patient samples that have been analyzed by paired genotype and phenotype testing. The patient's genotype is matched to a banked genotype, and the patient's phenotype is then predicted on the basis of the phenotypes paired to the banked genotype. A virtual phenotype can be completed in the same amount of time as a genotype. Results are reported as a genotype (listing the mutations detected) as well as a predicted fold change in the 50% inhibitory concentration (IC50) of each drug to the patient's virus (see "Phenotype Tests," below). The predicted susceptibility of the patient's virus to each drug is then reported, based on biologic and clinical cutoffs.

Phenotype Tests

Phenotype testing works by splicing the HIV reverse transcriptase and HIV protease genes from a patient's virus into a standardized laboratory strain, which is then grown in the presence of escalating concentrations of ARV drugs. The test measures the IC50 of each drug against the virus in vitro. Results are reported as fold change in IC50, as compared with a drug-susceptible control strain or with a previous test of the same patient's blood. The predicted susceptibility of the patient's virus to each drug is then reported, based on what is known about the correlation between fold change in IC50 of that drug and clinical resistance. As with genotype testing, the phenotype may not be able to detect resistance if the HIV RNA level is low (<1,000 copies/mL) and may not detect minor species. Therefore, a thorough history of ARV use and resistance tests, as well as expert interpretation, are essential for determining the significance of the results (see "Limits of Resistance Testing," below). A phenotype takes 2-3 weeks to complete.

Choosing Between Genotype and Phenotype

Genotype testing is faster and cheaper than phenotype testing, and it can detect emerging resistance, that is, virus with a mixture of strains of which some may be sensitive and some may be resistant to a given drug, as long as they are present in sufficient quantity. It is therefore generally recommended (including by the U.S. Department of Health and Human Services [HHS]) for ART-naive patients and for patients whose first or second ART regimens are failing. Additionally, in some instances it is very important to know the exact viral mutation pattern in order to predict the virus's response to subsequent therapy, particularly in patients with virologic failure on a regimen containing an NNRTI or integrase inhibitor. HHS recommends phenotype testing when patients are suspected of having complicated or multidrug resistance patterns (for example, in the setting of extensive prior ART), or when patients are found to have such patterns on genotype testing (especially resistance to PIs).

Using Genotype and Phenotype Tests at the Same Time

Genotype and phenotype tests have a few complementary properties that may, in some circumstances, make it desirable to use both tests at the same time. This strategy is especially advantageous when trying to devise a regimen for patients who have been exposed to many ARV agents and have few remaining treatment options, and for whom the development of additional resistance could be particularly dangerous. For example, early mutations may appear on a genotype before increases in inhibitory concentrations are detectable on a phenotype. Phenotype testing can detect loss or gain of drug efficacy caused by complex interactions of mutations that, by themselves, would not be predictive. In some cases, results of the genotype and the phenotype may be discordant; in these cases, consultation with an expert is recommended.

Recommendations for Resistance Testing

An overview of when genotype and phenotype testing is and is not recommended is presented in Table 1.

Table 1. Resistance Testing Recommendations
Clinical SettingRecommendationTestRationale

Adapted from 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. Accessed December 1, 2013.

Acute HIV infectionRecommended
  • Genotype
  • Determine whether drug-resistant virus was transmitted, to help design an initial regimen or to change a regimen accordingly.
  • If ART is deferred, consider repeating genotype at the time of ART initiation.
Chronic HIV infection before starting ARTRecommended
  • Genotype
  • Determine whether drug-resistant virus was transmitted to help design an initial regimen.
  • Perform upon entry to care (as close to the time of infection as possible), because transmitted drug-resistant virus is more likely to be detected earlier in the course of HIV infection.
  • If ART is deferred, consider repeating genotype at the time of ART initiation.
  • Consider integrase genotypic resistance assay if integrase inhibitor resistance is a concern.
  • Coreceptor tropism assay
  • Perform coreceptor tropism assay if use of a CCR5 antagonist is being considered.
Virologic failure during ARTRecommended
  • Genotype if failure of 1st or 2nd regimen
  • Both phenotype and genotype if multiple prior regimens, suspicion of mutations to multiple ARVs (especially PIs), or evidence of such mutations on genotype
  • Determine the role of resistance in drug failure and optimize the selection of active drugs for the new regimen, if indicated.
  • Perform while patient is taking ARVs, or ≤4 weeks after discontinuing ARVs.
  • If virologic failure on integrase inhibitor or fusion inhibitor, obtain specific genotypic testing for resistance to these to determine whether to continue them.
  • Coreceptor tropism assay
  • Perform coreceptor tropism assay if use of a CCR5 antagonist is being considered.
Suboptimal suppression or viral loadRecommended
  • Genotype or phenotype, depending on clinical situation (as above)
  • Determine role of resistance: identify active drugs for new regimen.
Pregnant womenRecommended
  • Genotype or phenotype, depending on treatment history (as above)
  • Perform before initiation of ART or prophylaxis.
  • Perform for all on ART with detectable HIV RNA levels.
  • Optimize the selection of active drugs for ARV regimen.
After discontinuation (>4 weeks) of ARVsNot Usually Recommendedn/a
  • Assist in selecting optimal regimen to achieve maximal viral suppression.
  • Drug-resistance mutations may decrease in number and become undetectable on assays.
Plasma viral load <500 HIV RNA copies/mLNot Usually Recommendedn/a
  • Resistance assays are likely to be unsuccessful in patients with HIV RNA <500 copies/mL, because of the low number of RNA copies. In those with levels >500 but <1,000 copies/mL, testing may fail but can be considered.

Antiretroviral-Naive Patients

With treatment-naive patients, resistance testing may reveal resistance mutations that were acquired at the time of infection, through infection with a strain of HIV that had already developed ARV resistance. Current guidelines recommend genotype testing for recently infected patients and for ARV-naive, chronically infected patients before initiation of therapy. It is important to test as early as possible in the course of HIV infection, to increase the likelihood of detecting transmitted mutations. The rationale for resistance testing in ARV-naive patients is twofold: 1) The prevalence of primary resistance is substantial, particularly in locations with a high prevalence of persons taking ART; and 2) Unknowingly starting a patient on ARV medications to which his or her virus is already resistant may risk failure of the initial regimen, rapid acquisition of additional resistance mutations, and curtailment of future treatment options.

Limits of Resistance Testing

As discussed above, drug-resistant HIV evolves in response to selective pressure applied by the ARV drugs in the patient's system. Specific resistance mutations develop in response to the pressure exerted by specific drugs (M184V, for example, evolves in response to lamivudine or emtricitabine). The presence of viral resistance suggests that a particular drug (and drugs with similar resistance patterns, or cross-resistance) is unlikely to be successful in suppressing viral replication.

In contrast, the absence of resistance to a drug on a genotype or phenotype test does not necessarily indicate that the drug will be effective, particularly if that drug (or drugs sharing cross-resistance) has been used previously. If a particular drug is discontinued, the viral strains harboring the mutations that confer resistance to that drug may decrease below the threshold of detection by the resistance assay, so the resistance test may not reveal certain resistance mutations. In such situations, minority populations of resistant viruses may exist in reservoirs and may emerge rapidly under selective pressure if that drug is restarted, or if drugs with similar or overlapping resistance patterns are used. The implications of archived mutations are twofold: 1) Resistance tests are most reliable when the patient is still taking the failing regimen; and 2) Resistance testing should be interpreted in the context of both the drugs that the patient was taking at the time of the test and the drugs that the patient had been exposed to previously (i.e., the patient's ARV history). In addition, it is important to review any previous resistance tests, which may show resistance mutations that were not revealed on subsequent testing.

Resistance Testing in Patients with Virologic Failure

As discussed in the chapter Antiretroviral Therapy, factors other than resistance may cause failure of ART; these include nonadherence, drug-drug interactions, and malabsorption. Therefore, before assuming drug failure, it is important to assess the causes of ARV regimen failure. If resistance is suspected, resistance testing should be done while the patient is taking the failing regimen, for the reasons noted above.

Patient Education

Advise patients of the following:

  • Resistance testing can improve the likelihood of virologic control of HIV.
  • Most treatment guidelines recommend resistance testing in certain circumstances.
  • Both genotype and phenotype testing can detect resistance only if it exists in at least 20% of the viral species present in a patient (known as the dominant species). Minor species may harbor resistance that remains undetected by either test.
  • In general, a patient's viral load must be at least 1,000 copies/mL for either test to be reliable, although samples with >500 copies/mL sometimes can be analyzed.
  • Resistance tests are most reliable when performed while a patient is still taking a failing regimen, or within 4 weeks after stopping.
  • Neither test predicts which drugs will be active in a particular patient, only drugs that are not likely to be active.

References

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Abbreviations for Dosing Terminology

BID
twice daily
BIW
twice weekly
IM
intramuscular (injection), intramuscularly
IV
intravenous (injection), intravenously
PO
oral, orally
Q2H, Q4H, etc.
every 2 hours, every 4 hours, etc.
QAM
every morning
QD
once daily
QH
every hour
QHS
every night at bedtime
QID
four times daily
QOD
every other day
QPM
every evening
TID
three times daily
TIW
three times weekly