Overview of HIV-1 drug resistance testing assays

INTRODUCTION — 

The use of drug resistance testing has become an integral part of human immunodeficiency virus (HIV) clinical care. The first clinical description of HIV resistance to an antiretroviral agent was published in 1989, when patients taking zidovudine monotherapy accumulated mutations within the reverse transcriptase gene, resulting in a marked increase in drug resistance [1]. Subsequently, HIV variants developed resistance to every available antiretroviral agent that has been identified. The evolution of drug resistance has significant clinical implications for choosing effective antiretroviral regimens.

This topic will provide an overview of HIV drug resistance testing. The interpretation of these tests and the approach to selecting an antiretroviral therapy (ART) regimen for patients with drug resistance mutations are discussed elsewhere. (See "Interpretation of HIV-1 drug resistance testing" and "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy" and "Evaluation of the treatment-experienced patient failing HIV therapy" and "Selecting antiretroviral regimens for treatment-naïve persons with HIV-1: General approach".)

RATIONALE FOR HIV DRUG RESISTANCE TESTING — 

The use of HIV drug resistance testing to guide antiretroviral therapy (ART) in treatment-naïve and treatment-experienced individuals leads to better viral suppression and has been associated with improved survival [2-5]. As an example, in an observational study of more than 2699 patients with HIV who were eligible for genotypic and phenotypic testing between 1999 and 2005, drug resistance testing was associated with improved survival (adjusted hazard ratio, 0.69 [95% CI 0.51-0.94]) after controlling for demographics, CD4 cell count, HIV RNA level, and intensity of clinical follow-up [3].

GENOTYPIC VERSUS PHENOTYPIC ASSAYS

Overview of the assays — The most common way to test for HIV drug resistance is through the use of genotypic assays [6,7]. In patients with a more extensive treatment history, and possibly more complex resistance mutational patterns, phenotypic resistance testing may supplement genotype testing.

Genotypic and phenotypic assays detect drug resistance in fundamentally different ways, although the results generally correlate with each other.

●Genotypic assays – Genotypic drug resistance assays detect the presence of specific drug resistance mutations in the regions of the HIV genome, typically those encoding protease, reverse transcriptase, and integrase. These assays predict the impact of the different mutations on each associated antiretroviral medication. (See 'Genotypic resistance assays' below.)

●Phenotypic assays – Phenotypic drug resistance assays measure (rather than predict) the extent to which an antiretroviral drug inhibits virus replication in vitro. (See 'Available phenotypic assays' below.)

Results are reported as a fold-change measured resistance to each drug in the patient isolate as compared with a laboratory reference strain without resistance. (See 'Interpreting phenotypic assays' below.)

Both genotypic and phenotypic assays have been limited by insensitivity to minority resistance variants comprising 1 to 20 percent of the virus population [8,9]. This limitation is important because once antiretroviral therapy (ART) is discontinued, a wild-type virus may re-emerge to replace the drug resistant virus as soon as four to six weeks after therapy is stopped. Thus, absence of a detectable resistance mutation must be interpreted with caution in a patient who has recently discontinued ART, as it may exist at levels below the assay's limit of detection. Other techniques have been developed to improve the detection of these minority species compared with standard drug resistance assays, and some genotypic assays are becoming more sensitive in detecting these minority resistance variants. (See 'Deep sequencing' below.)

Which assay to use — The choice of assay depends in part on the patient's treatment history and the expected complexity of their drug resistance profile.

Approach for most patients — In most patients, genotypic assays are preferred, since they are more readily available, have a shorter turnaround time, and a lower cost compared with phenotypic assays. These assays may also be more sensitive for detecting "mixtures" of resistant and wild-type viruses.

This approach is consistent with HIV treatment guidelines, which recommend a genotypic assay as the preferred testing method in patients whose resistance mutation patterns are known or not expected to be complex [4,10-12]. This includes treatment-naïve patients and patients with virologic failure or suboptimal viral load reductions while on a first or second antiretroviral regimen. In these situations, mutation patterns can be interpreted by available algorithms that adequately predict their impact on subsequent regimens and virologic response. (See 'Genotypic resistance assays' below.)

Extensive treatment history and possible complex drug resistance profile — In patients with more extensive treatment history and possibly more complex resistance mutational patterns, phenotypic resistance testing may supplement genotypic testing. In such patients, interpreting genotypes can be challenging since it can be difficult to interpret the clinical significance of a specific mutation, or interactions between them, when many mutations are present. As an example, some mutations cause resistance to certain drugs but increase susceptibility to others, while other mutations may impact viral fitness. (See "Interpretation of HIV-1 drug resistance testing", section on 'Viral characteristics'.)

Phenotypic tests measure resistance more directly and can assess relative susceptibility and interactions among mutations. This includes mutations that may not have been recognized to date as causing resistance or combinations of mutations that may have a different effect on susceptibility compared with each individual mutation alone or currently considered co-occurrences.

Low or suppressed viral loads — Proviral DNA genotypic drug resistance testing may be useful when switching a patient's antiretroviral regimen when the HIV viral load is nondetectable and there are no prior drug resistance testing results available or if there is low-level viremia and a plasma HIV RNA genotypic assay is unlikely to be or is not successful [12]. (See 'Genotypic resistance assays' below.)

The results of proviral DNA genotypic testing should be combined with all prior genotypic and phenotypic test results, if available, to construct a cumulative genotype that incorporates all current and previously detected drug resistance mutations. The clinical utility of proviral DNA genotypic resistance testing alone has yet to be fully determined [12], and the results must be interpreted with caution. Although these assays aim to detect drug resistance mutations archived in the proviral DNA, not all mutations are archived or captured by the assay, and some of the previously existing drug resistance mutations might not be detected [13,14].

GENOTYPIC RESISTANCE ASSAYS — 

Genotypic assay reports typically list a series of drug resistance mutations detected in the viral sequence generated from the patient's isolate, which is then compared with a reference wild-type sequence.

Available genotypic assays — Commercially available genotypic assays to detect HIV-1 resistance include US Food and Drug Administration (FDA)-approved kits as well as a variety of in-house assays performed by reference laboratories. HIV-2 resistance assays are only available in research or public health laboratories. (See 'HIV-2 drug resistance testing' below.)

Genotypic assays sequence regions of the HIV genome (commonly protease, reverse transcriptase, and integrase genes) that have been polymerase chain reaction (PCR)-amplified from the viral quasispecies circulating in a patient's plasma. When genotype tests were first introduced, standard tests only included testing for nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), and protease inhibitor (PI) resistance. In some settings, integrase strand transfer inhibitor (INSTI) resistance testing may need to be requested separately.

Some genotypic drug resistance assays are designed to sequence the proviral HIV DNA archived within host peripheral blood mononuclear cells and detect drug resistance mutations (see 'Low or suppressed viral loads' above) or determine viral tropism. (See 'Tropism assays' below.)

Interpreting genotypic assays — Genotypic resistance assay reports provide the individual mutations (eg, M184V, the signature mutation for lamivudine or emtricitabine resistance, where Methionine at position 184 of the viral reverse transcriptase is substituted by a Valine) and the predicted impact associated with these mutations (eg, "susceptible," "low-level resistant," or "high-level resistant"). (See "Interpretation of HIV-1 drug resistance testing".)

These interpretations are made using two general approaches, which are based on accumulated knowledge bases:

●Rule- or opinion-based algorithms defined by experts in the field of HIV drug resistance testing. These algorithms are based upon a synthesis of the genotypic definitions of resistance:

•The mutation confers phenotypic resistance when introduced into a drug-sensitive laboratory strain of HIV.

•The mutation is selected for during serial in vitro passage of virus in the presence of drug.

•The mutation is selected for during clinical therapy with that drug.

•The presence of the mutation in clinical isolates is associated with phenotypic resistance and virologic failure.

Over 20 rules-based genotypic interpretation systems have been proposed [15,16], with good correlation among them [17].

●Databases and machine learning systems, in which genotype is correlated with phenotype or virological response to a new treatment regimen [4,17].

No direct prospective comparisons of the genotypic interpretations using these two approaches have been made. Collaborative groups have been formed to ensure that interpretation of genotypic resistance mutational patterns remains up to date as new data emerge and the information reflects agents used in the clinic [17].

A few examples of commonly used genotypic resistance mutation lists and interpretation systems that can be helpful to clinicians and investigators and are transparent, updated regularly, and publicly available include:

●The International Antiviral Society-USA (IAS-USA) provides an updated list of mutations associated with HIV drug resistance to all FDA-approved drugs, based on evaluations by a panel of experts (www.iasusa.org) [18].

●The Stanford HIV Drug Resistance Database (hivdb.stanford.edu) provides access to a rules-based genotypic resistance algorithm, which assigns a penalty score for each drug resistance mutation, based on published studies of in vitro, clinical outcome, and expert opinion [19]. Some information on expected fold-change in IC50 of drug resistance mutations patterns can be obtained by searching the database, which is drawn from published studies and some unpublished clinical trials data.

●Agence Nationale de Recherches sur le SIDA (https://hivfrenchresistance.org) provides access to rules-based genotypic resistance tables that list mutations conferring resistance to antiretroviral drugs, based on correlation between mutations and virological outcome from patients failing antiretroviral therapy.

There are insufficient data to recommend one type of interpretation system over another. Evidence suggests that many of these systems have a similar ability to predict virologic outcome [20,21]. Caution should be used when interpreting resistance testing to investigational or recently approved antiretroviral agents. In addition, if a genotype was interpreted in the past using older algorithms, it should be reinterpreted using updated algorithms with new information as drug resistance interpretations can change over time [22,23].

PHENOTYPIC RESISTANCE ASSAYS

Available phenotypic assays — Current phenotypic assays measure the ability of a laboratory virus that contains a specific viral gene from the tested patient sample to grow in the presence of different concentrations of an antiretroviral drug. Phenotype testing is performed using recombinant virus assays (RVAs) [24,25]. These replaced the slow, labor-intensive cell culture method initially used [26] and have allowed phenotyping to be done on a larger scale.

Available RVAs use recombinant (chimeric) viruses commonly composed of protease, reverse transcriptase, and integrase gene sequences from viruses circulating in the tested patient's plasma, which are inserted into the genetic backbone of a laboratory reference strain of HIV. The resulting chimeric DNA clone is transfected into mammalian cells to produce an infectious virus that expresses the protease, reverse transcriptase, and integrase enzymes from the tested patient's HIV strain. The drug susceptibility of this chimeric virus can then be measured in a phenotypic resistance assay by exposing that virus to different drug concentrations and measuring their impact on the viral capacity to grow. Modifications of the RVA are also available for fusion and CCR5 inhibitors [27-30].

When validating an assay, studies are done to define the inter-assay and inter-laboratory assessment of assay repeatability and robustness. Earlier definitions of drug resistance for phenotypic assays were based on the technical reproducibility of the assay and did not necessarily reflect clinically relevant definitions of resistance [31].

HIV phenotypic resistance testing in the United States is only offered through one reference laboratory, and only the PhenoSense assay is available (https://monogrambio.labcorp.com/resources/phenotyping/phenosense). An older assay, entitled Antivirogram (Janssen Diagnostics, Belgium), was withdrawn from the market due to a decline in demand. However, a clinician may still encounter an old Antivirogram test result when reviewing a patient's prior drug resistance history.

Interpreting phenotypic assays — Phenotypic drug resistance reports also summarize which drugs the patient's isolate is estimated to be susceptible, possibly resistant, or resistant to. Unlike genotyping, phenotyping incorporates actual measurement of resistance rather than predicted. These estimations are derived from the provided drug-specific fold-increase in resistance based on clinical and biological cutoffs. This information can guide providers in antiretroviral regimen design.

Results are usually presented as fold-increase in resistance, defined as the drug concentration required to inhibit viral replication in the patient's isolate, compared with wild-type virus. Replication capacity, which may be reported, measures the patient's viral replication with no drug presence versus a wild-type reference virus. This provides an estimation of viral fitness, which can be informative in some complex cases. (See "Interpretation of HIV-1 drug resistance testing", section on 'Basic terminology'.)

There are several limitations to phenotype testing. As an example, clinical cutoffs, which can be derived by correlating changes in drug susceptibility in treatment-experienced patients, from the time of virologic failure to treatment response, are best verified by clinical trials, yet this has not been performed for many agents. There is also no consensus on the appropriate magnitude or timing of the virologic response that should be used to define drug-specific thresholds, which can also be influenced by the activity of the background antiretroviral regimen. Thus, clinically significant phenotypic resistance must often be inferred from 'biological cutoffs', which are determined by comparing the patient's drug susceptibility to that obtained from multiple treatment-naïve patients, which provide a reference for comparison. However, this does not offer insight into how the patient's isolate will respond to a specific drug and assumes that all treatment-naïve patients will respond equally well to that drug [32].

OTHER DRUG RESISTANCE ASSAYS

Tropism assays — HIV requires both the CD4 receptor and a coreceptor in order to enter CD4+ T cells. This coreceptor can be CCR5 or CXCR4. Viruses that use CCR5 as their coreceptor are referred to as R5 viruses; those that use CXCR4 are X4 viruses. An individual may have R5 viruses, X4 viruses, or a combination of both [12]. Most transmitted viruses are R5 viruses; X4 viruses usually emerge later in infection.

The frequency of exclusive R5 virus varies depending on the patient's treatment history; in treatment-naïve patients, exclusive R5 virus is found in approximately 80 to 85 percent [33,34]; in treatment-experienced patients with late-stage disease, it is found in only 50 to 56 percent [35-37].

Indications — Testing for viral tropism is recommended when considering the use of a CCR5 antagonist (maraviroc), which only inhibits R5 virus [12]. Coreceptor tropism testing may also be helpful for patients who demonstrate virologic failure on a CCR5 inhibitor.

Types of tropism assays — Both genotypic- and phenotypic-based assays have been developed to determine viral tropism, although there are much more clinical trial data regarding the use of phenotypic tropism assays [38,39]. The United States Department of Health and Human Services (DHHS) HIV treatment guidelines recommend phenotypic assays as the preferred method for tropism testing; a genotypic assay can be considered as an alternative [12].

●Phenotypic tropism assays – There are two types of phenotypic assays (Trofile assay, LabCorp, Burlington, NC; and Phenoscript assay, VIRAlliance, Paris, France) [38,40-45]. Similar to the previously mentioned phenotypic drug resistance methods, these assays use laboratory viruses that express patient-derived viral envelope proteins [38]. The laboratory-generated pseudovirus is used to infect cell lines that express either CCR5 or CXCR4; results of these infectivity assays determine the tropism of the patient's isolate.

Although there were problems with the sensitivity of earlier assays, subsequent tropism assays are able to detect CXCR4-utilizing viruses at very low levels (ie, 0.3 percent of the viral population) [44,46]. These new phenotypic tropism assays compare well to older established methods that used cell lines (eg, MT-2 cells) that do not express CCR5 to determine tropism [38,43].

The main limitations of these assays are the turnaround time needed for results (approximately three to four weeks from specimen collection) and the requirement for a plasma HIV RNA level ≥1000 copies/mL. Even with adequate viremia, tropism cannot be determined on a small number of patient samples. (See "Overview of antiretroviral agents used to treat HIV", section on 'CCR5 antagonists (Maraviroc)'.)

●Genotypic tropism assays – Genotypic assays sequence specific regions in the env gene [39,47]. Envelope, a major protein of HIV, determines HIV tropism. As with other genotypic methods, sequence algorithms are used to predict tropism.

When compared to early-generation phenotypic assays, population-based (rather than deep sequencing-based) genotypic assays are specific but have lower sensitivity for the detection of CXCR4-using viruses [48]. However, when used retrospectively to determine tropism in patients enrolled in clinical trials of maraviroc, newer genotypic assays performed as well as or better than the original Trofile assay in predicting virologic response and clinical outcomes [47-49].

The use of deep sequencing of the envelope protein can detect and quantify low prevalence subpopulations of CXCR4-using HIV within a large set of clinical isolates [50,51]. This method surpassed the ability of the original Trofile assay to predict viral suppression and the likelihood of switching tropism after maraviroc exposure.

European guidelines favor genotypic testing for determining coreceptor usage. In the United States, DHHS HIV treatment guidelines still recommend that a phenotype be used to determine coreceptor tropism. Although there are more data correlating phenotypic tropism results to outcomes than with genotypes, the latter are less expensive and results return much more rapidly. As a result, genotype tropism testing is now an alternative option for clinical practice [12].

In patients with an undetectable viral load or low-level viremia (eg, HIV RNA <1,000 copies/mL), HIV proviral DNA tropism testing can be used to help determine co-receptor usage [12,29]. As an example, this assay may be helpful when changing a patient with undetectable HIV RNA levels to a new regimen that contains maraviroc [52,53]. If only R5 virus is detected, maraviroc could be considered for use in the new regimen. However, caution should be used when interpreting the results from these assays, as the clinical utility of this approach is still under investigation [12].

ASSAYS USED IN THE RESEARCH SETTING

Deep sequencing — Genotypic (eg, Sanger sequencing) and phenotypic assays are typically unable to detect low-abundance drug resistant variants that are present at less than approximately 20 percent of the viral quasispecies in a given sample. Deep sequencing, also referred to as next-generation sequencing (NGS), can detect minority variants present at ≥1 percent of the viral quasispecies using techniques whereby hundreds of thousands (virtually all) of the individual viral molecules in a sample are sequenced in a single assay run [9,54-56]. The derived multiple sequence reads are aligned and analyzed to evaluate the prevalence of low-abundance drug resistant HIV strains. Data suggest that the presence of low frequency-resistant variants detected by deep sequencing can be associated with inferior treatment responses for certain antiretroviral classes [9,57-61] which may be missed by standard genotypic testing [9,56]. However, the significance of finding low-level drug esistant variants depends upon the specific antiretroviral agent and its genetic barrier to resistance.

Deep sequencing techniques were retrospectively applied to a subset of 264 baseline samples from treatment-naïve patients within a large clinical trial [9]. A significantly higher proportion of mutations were detected by deep sequencing than by standard genotypic sequencing (28 versus 14 percent, respectively). Among patients who initiated treatment with an antiretroviral regimen that combined nucleoside and non-nucleoside reverse-transcriptase inhibitors (NRTIs and NNRTIs), all those who had an NNRTI-associated resistance mutation identified by deep sequencing experienced virologic failure over a prolonged follow-up period.

Subsequent data have shown that low-level variants with isolated protease inhibitor (PI)-associated mutations were not predictive of virologic failure; this is likely due to the higher genetic barrier to resistance for ritonavir-boosted PI-based regimens [62]. In contrast, subjects with low-level resistant variants with extensive NRTI-associated mutations had a high rate of virologic failure, as would be expected.

One deep sequencing study found that the detection of low-level drug resistant variants was more closely correlated with the patient's prior treatment history than with results of deep sequencing. These results confirm the importance of obtaining a thorough medical history [63]. (See "Evaluation of the treatment-experienced patient failing HIV therapy".)

Whether and how to incorporate drug resistance data derived from deep sequencing is an active area of research and some commercial companies conducting genotypic drug resistance testing have already started to incorporate these more sensitive assays.

Point mutation assays — Point mutation assays detect single mutations instead of conventional genomic sequencing, for example by the differential hybridization of oligonucleotide probes to the wild-type virus and mutant variants [64]. These assays must be individually tailored for each mutation they are designed to detect. False-positive or false-negative test results can occur due to binding site variability [56]. These assays are not yet incorporated into clinical care; however, their low cost makes them good candidates for drug resistance epidemiology studies, where information may be needed only on the most common drug resistance mutations [65,66].

Clonal sequencing assays — Clonal sequencing assays can detect minority viral species but are labor intensive; approximately 100 to 300 clones must be sequenced in order to identify a viral variant present in only 1 or 2 percent of the viral quasispecies [67]. These assays are used only for research.

HIV-2 drug resistance testing — There are no US Food and Drug Administration (FDA)-approved HIV-2 genotypic or phenotypic testing for drug resistance or tropism validated for clinical use [12]. Resistance testing is only available through research facilities. (See "Treatment of HIV-2 infection", section on 'Assays for drug resistance testing'.)

SUMMARY AND RECOMMENDATIONS

●Rationale for drug resistance testing – The guidelines-recommended use of HIV drug resistance testing to guide antiretroviral therapy (ART) in treatment-naïve and treatment-experienced individuals leads to better viral suppression and has been associated with improved survival.

●Commonly used resistance assays – Commonly used drug resistance assays can be categorized as either genotypic or phenotypic. Genotypic assays detect the presence of specific drug resistance mutations, eg, in the regions of the HIV genome encoding protease, reverse transcriptase, and integrase, and predict their impact. By contrast, phenotypic assays measure the extent to which an antiretroviral drug inhibits virus replication in vitro. (See 'Overview of the assays' above.)

●Which assay to use

•Approach for most patients – In most settings, traditional genotypic assays are preferred due to faster turnaround time, greater sensitivity for detecting mixtures of resistant and wild-type virus, and lower cost. (See 'Approach for most patients' above.)

•Persons with extensive treatment history and possible complex drug resistance – Phenotyping may be used to supplement genotyping in treatment-experienced patients with multiple drug resistance mutations. Phenotype testing is better able to determine the clinical significance of complex mutation patterns. (See 'Extensive treatment history and possible complex drug resistance profile' above.)

•Persons with low or suppressed viral loads – Some genotypic drug resistance assays are designed to analyze HIV proviral DNA located in host peripheral blood mononuclear cells and detect archived drug resistance mutations. Proviral genotyping may be useful when switching a patient's antiretroviral regimen when the HIV viral load is nondetectable and there are no prior drug resistance results available. (See 'Low or suppressed viral loads' above.)

●Other resistance assays

•Tropism assays – Testing for viral tropism should be performed when considering the use of the CCR5 antagonist maraviroc since this agent only inhibits virus that uses the CCR5 coreceptor to enter the CD4 cells. Both genotypic- and phenotypic-based assays have been developed to determine viral tropism, although there are more clinical trial data regarding the use of phenotypic tropism assays. (See 'Tropism assays' above.)

•Deep sequencing – Deep sequencing, also referred to as next-generation sequencing (NGS), can detect minority variants present at ≥1 percent of the viral quasispecies using a technique whereby hundreds of thousands (virtually all) of the individual viral molecules in a sample are sequenced in a single assay run. (See 'Deep sequencing' above.)

Other assays that detect low-abundance mutations are also being developed. (See 'Assays used in the research setting' above.)

ACKNOWLEDGMENT — 

The UpToDate editorial staff acknowledges Michael Kozal, MD, who contributed to an earlier version of this topic review.