Interpretation of HIV-1 drug resistance testing

INTRODUCTION — 

Managing patients with human immunodeficiency virus (HIV) drug resistance has become less common. Advances in drug development have allowed for more potent antiretroviral therapy (ART) regimens that are easier to take, resulting in fewer persons who develop drug-resistant strains of HIV [1]. However, there remains a subset of patients who were infected with or develop drug-resistant variants, which can challenge an HIV clinician.

While drug resistance mutation patterns can be complex, there are basic principles that underlie effective utilization of drug resistance data to optimize the design of treatment regimens. This topic will review the drug resistance patterns found in association with different antiretroviral agents and describe how that knowledge can impact the selection of an appropriate treatment regimen in patients with transmitted drug resistance or virologic failure [2].

The details in this topic refer mainly to drug resistance patterns seen with HIV-1 subtype B, which is the most prevalent clade in the United States, Europe, Japan, and Australia, and on which the majority of the HIV drug resistance knowledge base is built. Available data generally support applying this knowledge to all HIV-1 subtypes, though this is an active area of research [3-7].

Topic reviews that discuss the different types of drug resistance tests and provide detailed guidance on selecting an ART regimen are found elsewhere. (See "Overview of HIV-1 drug resistance testing assays" and "Evaluation of the treatment-experienced patient failing HIV therapy" and "Selecting antiretroviral regimens for treatment-naïve persons with HIV-1: General approach" and "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy" and "Treatment of HIV-2 infection".)

FACTORS CONTRIBUTING TO DRUG RESISTANCE — 

Multiple factors influence the development of HIV drug resistance, including the biology of HIV, genetic barriers to resistance, regimen potency, pharmacokinetics of antiretroviral drugs, and medication adherence. Drug-resistant mutations can predate the initiation of a specific drug regimen (transmitted drug resistance), or can develop when viral replication continues in the face of ongoing drug pressure (acquired drug resistance).

Viral factors — HIV infection is characterized by high rates of replication, with more than 10⁹ virions produced daily [8]. In addition, HIV reverse transcriptase, which is responsible for replicating the viral genome, is error-prone. The combination of the high rate of replication and the frequent introduction of mutations during each round of replication leads to the occurrence of randomly generated mutations, some of which confer drug resistance [9]. The resulting population of genetically related, but distinct, HIV variants in a patient is referred to as a "quasispecies."

Viruses with spontaneous mutations are often defective and incapable of replication. However, some mutations can persist in archived proviral HIV DNA in resting memory host cells if the mutant virus has a selective growth advantage over wild-type virus [10]. If viral replication is not fully suppressed by an antiretroviral regimen, these mutants can re-emerge. Depending on their level of drug resistance, these mutants can either cause overt virologic failure or lead to a more drug-resistant virus by the gradual accumulation of additional resistance mutations.

Regimen-related factors — There are several regimen-related factors that contribute to the development of resistance.

●Genetic barriers to resistance – HIV can develop high-level resistance to some drugs with only a single mutation, while other drugs require multiple mutations for high-level resistance. Drugs in the former situation are referred to as having a "low genetic barrier" to resistance. (See "Overview of antiretroviral agents used to treat HIV".)

Agents that have a low genetic barrier to resistance include the nucleoside reverse transcriptase inhibitor (NRTI) lamivudine (and the closely related agent emtricitabine), the non-nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and rilpivirine, and the integrase strand transfer inhibitors (INSTIs) raltegravir and elvitegravir.

By contrast, a high resistance barrier is seen in the protease inhibitor (PI) class and with the INSTIs dolutegravir and bictegravir. Resistance to these agents usually requires the accumulation of multiple mutations.

●Regimen potency – The potency of an antiretroviral regimen is a crucial determinant of viral load suppression. If the viral load is not fully suppressed, continued virus replication in the presence of the drug can lead to the accumulation of mutations. This can lead to the development of viral resistance, even to drugs with a high genetic barrier to resistance. As an example, unboosted PIs, which are no longer recommended, had a high genetic barrier to resistance but achieved low serum trough concentrations; thus, virus could continue to replicate and accumulate mutations that led to high levels of resistance. This problem was offset by the use of pharmacokinetic "boosting" with ritonavir or cobicistat. (See "Overview of antiretroviral agents used to treat HIV", section on 'Protease inhibitors (PIs)'.)

●Pharmacokinetic factors – There are several ways that pharmacokinetic factors can lead to drug resistance.

•Low serum concentrations – Low serum concentrations of an antiretroviral agent can be seen as a result of drug or food interactions, poor absorption, or secondary to the pharmacokinetics of a specific agent (eg, unboosted PIs as described above). Continued virus replication in the presence of the drug can lead to the accumulation of resistance mutations.

•Varying half-lives – Resistance can result when individual components of an HIV antiretroviral regimen have varying plasma half-lives. In this case, discontinuation of the regimen can lead to functional monotherapy. As an example, when the combination of efavirenz-emtricitabine-tenofovir disoproxil fumarate is discontinued, the half-life of efavirenz is longer than the half-lives of emtricitabine or tenofovir disoproxil fumarate (TDF), and the patient may be at risk of developing efavirenz resistance.

•Genetic factors – Pharmacokinetic profiles can vary significantly in different patient populations. This pharmacokinetic variability was well illustrated in a study of 152 patients who were receiving efavirenz, which is metabolized primarily by cytochrome p450 2B6 isoenzymes [11]. A G-to-T polymorphism at position 516 of CYP2B6 was associated with higher plasma efavirenz concentrations and slower clearance [12].

Medication adherence — Nonadherence to antiretroviral therapy (ART) is closely associated with incomplete viral suppression and the development of drug-resistant HIV variants [13]. The relationship between adherence and drug resistance is complex and influenced by drug potency, pill burden, and pharmacokinetics [14,15]. (See "Evaluation of the treatment-experienced patient failing HIV therapy", section on 'Nonadherence'.)

Therapeutic drug monitoring (TDM) is of theoretical utility to ensure that there is adequate drug exposure [16]. However, the role of TDM in clinical practice remains controversial because of the lack of prospective studies demonstrating that TDM improves clinical outcomes in patients with drug-resistant HIV. In addition, plasma NRTI concentrations are not thought to be as important clinically due to the intracellular location of their active forms. This is an active area of research [17].

BASIC TERMINOLOGY — 

This section defines terms that are used when discussing drug resistance.

Viral characteristics — Several terms can be used to describe HIV. These include:

●Wild-type virus – "Wild-type" virus refers to the original parent virus that was predominant prior to the use of antiretroviral medications. A patient may become infected by either a wild-type or a resistant strain. As there are many HIV viral variants that are considered "wild type," all are compared to a reference strain in laboratories that perform drug resistance testing.

●Viral fitness – Viral fitness generally refers to the ability of one virus to out-compete a second virus in a defined environment [10,18]. In general, wild-type virus replicates more efficiently (ie, is more "fit") in cell culture than resistant virus and overgrows mutant variants in the absence of drug selection pressure.

In the setting of ART, drug-resistant virus is selected, despite that it is less fit in the absence of the drug [10]. Viral variants with reduced fitness may be less pathogenic, although studies examining the correlation of HIV replication fitness with clinical outcomes have not given consistent results [19].

●Replication capacity – Replication capacity refers to the ability of the virus to replicate in comparison to another reference virus. Replication capacity can either decrease or increase in the presence of certain mutations.

Some patients demonstrated transient immunologic benefit despite ongoing viremia [20,21]. This may have been related to the selection of a "less fit" drug-resistant virus population. Viral fitness is inferred through measurement of replication capacity, which can be obtained from one phenotypic assay, PhenoSense HIV (Labcorp/Monogram Biosciences, Clinical Reference Laboratory). (See "Overview of HIV-1 drug resistance testing assays".)

However, the primary goal is always to complete viral suppression, which is associated with durable immunologic benefits. Viral suppression is usually achievable with currently available antiretroviral medications, even among patients with multi-class drug resistance [16,22]. (See "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy".)

Mutations and polymorphisms — A mutation in the context of HIV drug resistance is an amino acid change, defined as a difference between a patient's virus and a reference HIV strain. (See 'Viral characteristics' above.)

By convention, mutations are indicated by a codon number representing a position in the protein, preceded by a letter indicating the consensus amino acid in wild-type (drug-susceptible) virus and followed by a letter indicating the amino acid substitution in the mutant virus. As an example, "M184V" indicates that methionine has been substituted for valine at amino acid codon 184, in this case in the reverse transcriptase enzyme. The notation "M184M/V" indicates the presence of a mixture of wild-type (M) and mutant (V) virus.

Signature and accessory mutations exist.

●Signature mutation – A "signature mutation" is a mutation that is typically associated with resistance to a particular drug. As an example, the K103N mutation is the signature mutation for resistance to efavirenz, a non-nucleoside reverse transcriptase inhibitor (NNRTI). Other signature mutations are described below. (See 'Specific drug resistance mutations' below.)

●Accessory mutations – When HIV continues to replicate under drug selection pressure, additional, more minor mutations may accumulate over time. These accessory or secondary mutations can further increase drug resistance or can compensate for the loss of viral fitness induced by the primary, more major mutations. Thus, it is important to switch antiretroviral therapy (ART) rather than expose a patient with ongoing viremia to the same drugs in order to avoid the accumulation of additional resistance mutations [16]. (See "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy", section on 'Management of patients with incomplete viral suppression'.)

In a given sample, some detected amino acid changes from the wild-type virus that are less significant for drug resistance may occur. These amino acid substitutions are often termed "polymorphisms" and do not on their own confer phenotypic or in vitro resistance to a particular antiretroviral agent [10]. Polymorphisms do not require drug pressures to occur. Resources that help interpret drug resistance testing should indicate drug susceptibility when these polymorphisms are identified. (See 'Resources to interpret resistance testing' below.)

Cross-resistance — The term "cross-resistance" refers to resistance to drugs other than the drug that selected the mutation(s). As examples, the M184V mutation associated with lamivudine confers cross-resistance to the related drug emtricitabine, and the K103N mutation selected by the NNRTIs efavirenz and nevirapine confers cross-resistance to each other. However, a specific mutation may not cause cross-resistance to all agents within a class. Although virus expressing the K103N is resistant to nevirapine and efavirenz, other NNRTIs, such as etravirine and rilpivirine, maintain activity if there are no other additional NNRTI mutations. Similarly, a mutation at integrase position Y143 can confer resistance to raltegravir but has little effect on dolutegravir susceptibility. (See 'Non-nucleoside reverse transcriptase inhibitors' below and 'Integrase strand transfer inhibitors' below.)

Hypersusceptibility — On occasion, resistance to one drug can lead to increased susceptibility, or hypersusceptibility, to another drug. For example, the M184V mutation that confers lamivudine resistance causes hypersusceptibility to zidovudine and tenofovir compared with wild-type virus [23]. Another example is the thymidine analog mutation (TAM) T215Y, which is associated with efavirenz hypersusceptibility when present [24].

SPECIFIC DRUG RESISTANCE MUTATIONS — 

Our understanding of HIV drug resistance continues to evolve, as new mutations, drugs, and drug classes develop and as more information is obtained regarding associations between them and response to therapy. The spectrum of drug resistance mutation patterns can range from simple to complex, and a large knowledge base has been accumulated since the first HIV drug resistance mutations have been reported [25].

This section will review drug resistance mutations that impact regimen selection. Discussions of HIV drug resistance assays and regimen selection for persons with resistance are presented in separate topic reviews. (See "Overview of HIV-1 drug resistance testing assays" and "Evaluation of the treatment-experienced patient failing HIV therapy" and "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy".)

Nucleoside reverse transcriptase inhibitors — Nucleoside reverse transcriptase inhibitors (NRTIs) can select for resistance mutations when used in a nonsuppressive regimen or when taken inadequately. The specific mutation pattern may be associated with varying degrees of NRTI cross-resistance. In general, medications for which a single mutation is sufficient to lead to resistance include lamivudine, emtricitabine, tenofovir, or abacavir, whereas resistance to zidovudine (and the less used stavudine) usually requires multiple mutations. A summary of NRTI resistance mutations can be found on the International Antiviral Society – USA and the Stanford HIV Drug Resistance Database websites and in related publications, which are updated regularly [16,26-29].

Emtricitabine and lamivudine — Lamivudine and emtricitabine are NRTIs with a low genetic barrier to resistance. Lamivudine and emtricitabine both select for the M184V mutation. This is the most prevalent NRTI-associated mutation seen in treated patients, in part because of the widespread use of these medications. It is often the first mutation to appear [30].

M184V (as well as the less common M184I mutation) causes high-level in vitro resistance to both lamivudine and emtricitabine. It also causes a modest decrease in susceptibility to abacavir [31]. However, M184V confers hypersusceptibility to tenofovir and zidovudine and, therefore, partially reverses thymidine analog mutation (TAM)-mediated resistance to these drugs [32]. (See 'Hypersusceptibility' above and 'Zidovudine (and the less used stavudine)' below.)

The M184V mutation also leads to decreased replication capacity of HIV [33]. Thus, a viral isolate with this mutation is "less fit" than wild-type virus [34]. Continuation of either lamivudine or emtricitabine, with maintenance of the M184V mutation, leads to a persistent mean 0.5 log decrease in viral load from baseline. (See 'Viral characteristics' above.)

Some clinicians maintain lamivudine or emtricitabine in a regimen despite the presence of the M184V mutation because of the safety, tolerability, and residual partial virologic activity of these drugs as well as the beneficial effect on susceptibility to zidovudine, stavudine, and tenofovir [35]. This is probably most important in regimens containing NRTIs, especially tenofovir. (See "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy".)

Tenofovir — Tenofovir is available as one of two tenofovir prodrugs: tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF). Resistance considerations are generally the same for both agents, although the higher intracellular tenofovir concentrations achieved with TAF may give it greater activity against some NRTI-resistant viruses. A more detailed review of these agents is found elsewhere. (See "Overview of antiretroviral agents used to treat HIV", section on 'Tenofovir'.)

K65R is the signature mutation associated with tenofovir resistance [36]. When it occurs, it confers significant cross-resistance to abacavir and lamivudine [37]. By contrast, the K65R mutation induces hypersusceptibility to zidovudine [38], similar to that seen with the M184V mutation. (See 'Emtricitabine and lamivudine' above.)

First-line tenofovir failure has also been associated with other, less common mutations at positions K70E and Y115F [39,40]. In addition, tenofovir resistance can be mediated by the presence of multiple TAMs, selected by the two thymidine analogs zidovudine and stavudine as discussed above. (See 'Zidovudine (and the less used stavudine)' below.)

These mutations are most often found in persons failing an initial regimen with a low barrier to resistance (eg, a non-nucleoside reverse transcriptase inhibitor (NNRTI)-containing regimen). However, available data suggest virologic suppression can still be achieved in some patients when tenofovir is continued and used with a second-generation integrase strand transfer inhibitor (INSTI) or a boosted protease inhibitor (PI). The ultimate choice of regimen depends on prior treatment regimens and viral failure episodes and durations as well as specific mutation profiles determined by drug resistance testing. A discussion of regimen selection in patients with drug-resistant virus is presented in a separate topic review. (See "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy", section on 'Patients with drug-resistant virus'.)

Abacavir — The common mutation associated with abacavir is L74V [41,42]. Less commonly, abacavir selects for other mutations, including K65R, Y115F, and M184V. When L74V is present in combination with M184V, there is further loss of susceptibility to abacavir. However, the presence of both mutations increases susceptibility to zidovudine and tenofovir [38]. In addition, L74V mutant isolates are less fit than wild-type virus [43]. (See 'Viral characteristics' above.)

Zidovudine (and the less used stavudine) — Zidovudine (and stavudine) are thymidine analogs, which share common resistance patterns. Although these agents are no longer recommended as first-line ART because of their toxicity [16,44,45], it is important to understand their effect on drug resistance in patients who were previously exposed to these agents.

Common resistance mutations include M41L, L210W, T215Y, D67N, K70R, and K219Q/E/N [46]. These substitutions are referred to as TAMs because they are selected by the two thymidine analogs (zidovudine and stavudine). Regimens containing a thymidine analog can select for TAMs and additional mutations, such as E40F, E44D/A, and V118I as well as K43Q/N, E203K, H208Y, D218E, K223Q/E, and L228H/R.

TAMs typically emerge via one of two "pathways":

●Pattern 1 includes the M41L, L210W, and T215Y mutations.

●Pattern 2 involves the D67N, K70R, and K219Q/E/N mutations [47,48].

However, there can be mixtures of both patterns present in some patients [47,48]. TAMs continue to accumulate when patients continue failing ART regimens.

The first TAM pathway listed above confers cross-resistance to almost all NRTIs and the nucleotide analog, tenofovir [27,49,50]. The second pathway confers resistance primarily to zidovudine (and stavudine) but causes less cross-resistance to abacavir, tenofovir, and the less-used didanosine. Since zidovudine and stavudine are both thymidine analogs, resistance to zidovudine results in cross-resistance to stavudine and vice versa.

TAMs are uncommon with early failure of a thymidine analog-containing regimen. At least three TAMs are usually required before virologic activity of zidovudine (or stavudine) is completely lost. However, continuation of a thymidine analog-containing regimen in the presence of ongoing viremia will result in the accumulation of additional TAMs, leading to greater resistance and NRTI cross-resistance [32,51]. (See 'Mutations and polymorphisms' above and 'Multinucleoside resistance mutations' below.)

In the case of multiple TAMs, phenotypic drug resistance testing may be helpful for determining whether any NRTIs are likely to have partial activity [30]. However, the use of fully active agents is preferred whenever feasible; extensive NRTI resistance may require the use of an ART regimen that includes a combination of agents from other drug classes. (See "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy".)

Multinucleoside resistance mutations — While the most common cause of broad NRTI cross-resistance is the combination of multiple TAMs with M184V [32], two other resistance patterns can also lead to significant NRTI cross-resistance:

●The Q151M complex is associated with resistance to all NRTIs, with the least effect on tenofovir. It usually occurs in combination with two or more accessory mutations A62V, V75I, F77L, and F116Y.

●The T69 insertion causes resistance to all NRTIs, including tenofovir, when it is accompanied by one or more TAMs at codons 41, 210, or 215.

These mutations are usually selected by older NRTI combinations that included didanosine plus either zidovudine or stavudine. When these mutations are present, the use of an NRTI-sparing regimen may be considered. (See "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy".)

Integrase strand transfer inhibitors — There are five main INSTIs approved for use. Raltegravir, elvitegravir, dolutegravir, and bictegravir can be used in treatment-naïve and treatment-experienced patients. Cabotegravir, the newest INSTI was approved in December 2021 to be used with rilpivirine as part of a long-acting injectable complete antiretroviral regimen to replace a stable oral regimen in patients with viral suppression. (See "Use of long-acting cabotegravir-rilpivirine in people with HIV".)

HIV integrase is essential for viral replication; integrase inserts viral DNA into the cellular genome through two catalytic reactions. Loss of integrase activity disrupts the viral life cycle. Due to a different mechanism of action, there is no cross-resistance of these agents with drugs from other classes.

Raltegravir and elvitegravir — Raltegravir and elvitegravir (which is only available as part of a one-pill combination with emtricitabine (FTC) and TDF or TAF) are first generation INSTIs with potent in vitro activity against both wild-type and HIV isolates that are resistant to other drug classes. Both phenotypic and genotypic integrase resistance tests are commercially available. (See "Overview of HIV-1 drug resistance testing assays".)

If resistance develops, there is virtually complete cross-resistance between raltegravir and elvitegravir. Signature mutations in the integrase gene associated with resistance to raltegravir include Q148H/K/R, Y143C, and N155H, which develop along three key resistance pathways. The primary mutations for elvitegravir are Q148R, E92Q, and T66l. Mutations G140A/C/S can significantly contribute to both raltegravir and elvitegravir resistance. Additional mutations occur at codons 92, 118, 138, 147, and 263 [27,52].

Mutations at codon 148 are associated with the highest level of integrase resistance and become more prevalent with ongoing failure. As an example, in a longitudinal analysis of a phase II study, there was a trend for the N155H mutation to be replaced by the Q148H mutation, which became predominant over time [53]. These data suggest that viruses with the Q148H mutation, in combination with other secondary mutations, have greater replicative fitness in vitro than viruses with the N155H mutation [54,55]. (See 'Viral characteristics' above.)

Dolutegravir and bictegravir — Dolutegravir and bictegravir are second-generation INSTIs that have a higher barrier to resistance than raltegravir, elvitegravir, and likely cabotegravir.

●Dolutegravir – Dolutegravir has a higher barrier to resistance than raltegravir or elvitegravir. In addition, dolutegravir is active against raltegravir- and elvitegravir-resistant virus with Y143C- or N155H-mediated resistance. However, susceptibility can be reduced in the presence of mainly four pathways including mutations R263K, G118R, N155H, and Q148H/R/K [56,57].

Twice-daily dolutegravir dosing is recommended in patients who are INSTI-experienced if a viral isolate is found to have resistance to raltegravir and elvitegravir but remains susceptible to dolutegravir or if INSTI resistance is clinically suspected. However, dolutegravir, even when dosed twice daily, only appears to have partial activity against virus with the 148 mutation [58].

●Bictegravir – Bictegravir was approved by the US Food and Drug Administration (FDA) for use in a combination pill with TAF-FTC [59-62]. Like dolutegravir, bictegravir has a high barrier to resistance, and emergent resistance patterns have not been seen in clinical trials evaluating these patient groups [60-64].

In vitro, mutations at positions M50I and 263K confer reduced susceptibility to bictegravir [59]. In addition, in vitro cross-resistance studies have suggested that mutations at G140A/C/S and Q148H/RK will confer reduced susceptibility to bictegravir, especially when additional INSTI mutations are present [59,60]. As bictegravir is used more broadly, a better understanding of resistance patterns should emerge.

Cabotegravir — Cabotegravir, a structural dolutegravir analog, is available as part of a first-of-its-kind long-acting antiretroviral regimen, in combination with rilpivirine. The regimen was approved in January 2021 by the US FDA as an intramuscular injection option for engaged and virologically suppressed patients who have no relevant drug resistance [16,65]. It is also used for pre-exposure prophylaxis to prevent HIV infection. (See "Use of long-acting cabotegravir-rilpivirine in people with HIV" and "HIV pre-exposure prophylaxis", section on 'Injectable therapy'.)

In clinical trials, most of the few treatment failures on this regimen had phenotypic resistance to cabotegravir with INSTI resistance-associated mutations (eg, E138K, G140R, Q148R, N155H) [66-69]. Further information will be acquired as this regimen is increasingly used.

Non-nucleoside reverse transcriptase inhibitors — The NNRTIs efavirenz, nevirapine, and rilpivirine have a low genetic barrier to resistance compared with PIs and the INSTIs dolutegravir, bictegravir, and cabotegravir. Doravirine is a newer NNRTI approved for use in ART-naïve persons with HIV-1, and a combination of mutations is needed to confer high-level resistance to the drug [70,71]. Etravirine, which is used in salvage regimens for patients with drug-resistant virus, has a higher resistance barrier and may be active against some efavirenz- and nevirapine-resistant viruses [72]. (See "Overview of antiretroviral agents used to treat HIV", section on 'Non-nucleoside reverse transcriptase inhibitors (NNRTIs)' and "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy", section on 'Those failing a first-line regimen containing an NNRTI'.)

Efavirenz and nevirapine — Initial NNRTI resistance mutations tend to emerge quickly in patients with detectable viremia during NNRTI-based ART. The most common mutations seen in association with efavirenz or nevirapine are K103N and Y181C [3]. There is significant cross-resistance between those two agents [49]. As a result, selection of one of these single-point mutations (ie, Y181C or K103N) confers loss of activity to both drugs [73].

K103N is selected by efavirenz more frequently than by nevirapine; Y181C is selected more commonly by nevirapine when zidovudine is not coadministered. Although efavirenz may appear phenotypically active in the presence of Y181C [73], the use of efavirenz after the emergence of the Y181C mutation on nevirapine leads to the rapid development of drug resistance and virologic failure [74].

Efavirenz and nevirapine may also select Y188L, which confers high-level resistance to some NNRTIs when present as a single mutation. Other common mutations selected by these NNRTIs include: L100I, V106A, G190S/A, and M230L. Accumulation of two or more of these common mutations is associated with broad NNRTI class resistance. Thus, first-generation NNRTIs (eg, efavirenz, nevirapine) should be discontinued in patients experiencing virologic failure and documented resistance. Maintaining NNRTI therapy in viremic patients on either efavirenz or nevirapine risks accumulation of further NNRTI resistance mutations, which may reduce the utility of second-generation NNRTIs such as etravirine. (See "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy", section on 'Management of patients with incomplete viral suppression'.)

Rilpivirine and doravirine — Rilpivirine and doravirine are second generation NNRTIs that are used primarily for treatment-naïve patients and virologically suppressed patients switching regimens.

●Rilpivirine – In clinical trials that have evaluated the efficacy of rilpivirine in treatment-naïve patients, virologic failure was associated with the emergence of the following resistance-associated mutations: K101E, V189I, Y181C, V90I, H221Y, and E138K [75,76]. E138K, the most frequently observed rilpivirine mutation, is a mutation that by itself causes high-level rilpivirine resistance as well as potential low-level resistance to other NNRTIs [27,77,78].

In studies in which rilpivirine was given with emtricitabine or lamivudine, the E138K mutation was most often seen in association with the NRTI mutation M184I or, less commonly, with M184V [75,76,79]. Although M184I/V mutations are associated with a decline in replication capacity, the coexistence of the E138K mutation appears to restore viral fitness [79], particularly when found in combination with M184I [80]. (See 'Viral characteristics' above.)

●Doravirine – Doravirine was approved by the US FDA in 2018 [71]. In clinical trials, the most frequent patterns of mutations conferring high-level doravirine resistance involved changes at codon V106 in combination with other known NNRTI resistance substitutions (eg, V106A/M with F227C, Y188L, and Y318F) [27,70,71]. In addition, other NNRTI mutations (eg, G190E/Q and M230L) can confer high-level resistance to doravirine [27,71,81]. In an analysis of NNRTI cross-resistance, clinical isolates demonstrated resistance to doravirine when Y188L was identified, alone or in combination with K103N or V106I. Also, the NNRTI mutation combination of G190A and F227L, or E138K in combination with Y181C and M230L, showed greater than 100-fold reduced susceptibility to doravirine [27,71,81]. A better understanding of the most common clinically emergent doravirine resistance profiles will likely be forthcoming as this drug is increasingly used.

Etravirine — Etravirine is a second-generation NNRTI with activity against a large proportion of NNRTI-resistant viruses isolated from patients with virologic failure on efavirenz or nevirapine [82]. The K103N mutation, which is the most common mutation associated with efavirenz failure, does not cause cross-resistance to etravirine. However, the presence of other NNRTI-associated mutations may affect etravirine potency, as noted above [83]. There are no clinical data on the use of etravirine after virologic failure on rilpivirine.

A retrospective analysis from the DUET trials identified a number of mutations that may diminish response to etravirine, including V90I, A98G, L100I, K101E/P, V106I, V179D/F, Y181C/I/V/Y, and G190S/A [83]. An increased number of mutations was correlated with a declining treatment response. In addition, although no single mutation led to high-level resistance to etravirine, some mutations (eg, those at codon 181) led to "intermediate" level resistance and appeared to have a significant negative impact on etravirine susceptibility and activity.

Phenotypic drug resistance testing is helpful in determining the residual activity of etravirine when multiple mutations are found on genotypic testing. (See "Overview of HIV-1 drug resistance testing assays", section on 'Phenotypic resistance assays'.)

Protease inhibitors — PIs are almost always given in combination with cobicistat or low-dose ritonavir. These boosting agents are used to increase serum trough concentrations of the parent drug. (See "Overview of antiretroviral agents used to treat HIV", section on 'Protease inhibitors (PIs)'.)

Pharmacologically boosted PIs achieve high serum concentrations and have a high genetic barrier to resistance; therefore, decreased drug susceptibility generally requires the accumulation of multiple mutations. When boosted PIs are used by patients without pre-existing PI resistance, treatment failure is less commonly associated with PI resistance. (See "Evaluation of the treatment-experienced patient failing HIV therapy".)

When PI-associated mutations do occur, they are classified as either primary (major) or secondary (minor) mutations. The mutations that emerge depend upon which medication is being administered. However, it should be emphasized that some data on mutations associated with failure of PI-based regimens come from studies of unboosted PIs, which are not recommended and rarely used today.

Interpreting complex genotypes can be challenging in patients with multiple PI resistance mutations. Thus, in some cases, phenotypic resistance testing may offer an advantage over genotypic testing, as it measures resistance more directly and can assess relative susceptibility and interactions among mutations.

Atazanavir — The signature mutation associated with use of unboosted atazanavir is I50L, which does not decrease susceptibility to other PIs and, in some cases, may increase susceptibility to them. Other potential mutations include I84V and N88S.

Darunavir — Pharmacologically boosted darunavir is an important agent for use in treatment-experienced patients since darunavir maintains virologic activity despite multiple PI mutations [84]. Mutations that alter the virologic response to darunavir include: 11I, L10F, V32I, L33F, I47V/A, I50V, I54L/M, G73S, T74P, L76V, V82F, I84A/C/V, L89V [27,85]. The dosing of darunavir (once versus twice daily) and the choice of boosting agent depend upon the number of PI mutations. When using phenotypic assays, an optimal response to darunavir is associated with a phenotypic fold change ≤10 [86]. (See "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy", section on 'Protease inhibitors' and "Overview of antiretroviral agents used to treat HIV", section on 'Protease inhibitors (PIs)'.)

Lopinavir — Lopinavir is only available in a coformulation with ritonavir. Several PI mutations are required before its efficacy is significantly compromised. Key mutations include L76V, V32I, I47V/A, I54L/M, V82A/F/T/S, and I84A/C/V.

Older protease inhibitors — Almost all of the older PIs are infrequently used or have been discontinued. However, patients who have taken these in the past may have drug resistance mutations related to their use.

●Tipranavir – Tipranavir is rarely used in clinical practice due to its higher rate of adverse events and higher dose of ritonavir required for boosting than other PIs [16]. It has only been used in heavily treatment-experienced patients and has a unique resistance profile [87,88]. As an example, the mutations I50V, I54L, and L76V, which confer significant resistance to darunavir, are associated with hypersusceptibility to tipranavir in vitro [2,27,89]. The use of either phenotypic resistance testing or a tipranavir mutation score has been helpful in determining the use of this agent [90].

●Fosamprenavir – Common mutations associated with fosamprenavir, an amprenavir prodrug, include I50V and I84V. These mutations are also associated with cross-resistance to other PIs, most notably darunavir. There are less clinical trial data for fosamprenavir than for other boosted PIs.

●Nelfinavir – The signature mutation associated with nelfinavir resistance is D30N, which does not affect susceptibility to other PIs. Another less common mutation associated with nelfinavir use is L90M, which causes cross-resistance to other PIs. Nelfinavir is rarely used because of its gastrointestinal side effects (eg, diarrhea), lower efficacy, and inability to be pharmacologically boosted. (See 'Cross-resistance' above and "Antiretroviral selection and management in pregnant individuals with HIV in resource-abundant settings".)

●Saquinavir – Common mutations associated with the emergence of saquinavir resistance include the unique G48V mutation as well as the L90M mutation, which is associated with PI cross-resistance. Saquinavir is no longer available in the United States and Canada.

●Indinavir – Common mutations associated with indinavir resistance include M46/I/L, V82A/F/T, and I84V. The latter two mutations are also selected by other PIs and have a significant impact on most other PIs. This drug is no longer available.

Other agents

Maraviroc — Maraviroc is a CCR5 antagonist that can interfere with the CCR5-viral interaction and inhibit viral entry. To see if maraviroc will be active, a pretreatment screening test must be utilized to assess viral tropism. (See "Overview of HIV-1 drug resistance testing assays", section on 'Tropism assays'.)

To enter the CD4 cell, HIV requires binding to coreceptor molecules, CCR5 (by R5 viruses) or CXCR4 (by X4 viruses), in addition to attaching to the CD4 receptor. However, CCR5 antagonists are not active against viruses that enter the cell by binding to the CXCR4 coreceptor or viruses that enter by using both the CCR5 and CXCR4 coreceptors (dual or mixed tropic virus). (See "Overview of antiretroviral agents used to treat HIV", section on 'CCR5 antagonists (Maraviroc)'.)

In clinical trials, virologic failure of maraviroc has been attributed to the emergence of X4 viruses. The primary mechanism for this phenomenon was the selection of pre-existing minority populations that were not detected at baseline due to the extreme diversity of viral subpopulations in treatment-experienced patients [91]. This may be a less important problem given the improved sensitivity of available tropism assays for detecting X4 or dual/mixed tropic virus. However, even the contemporary assays can miss low-frequency variants that can emerge under nonsuppressive treatment [92].

Treatment failure can also occur as a result of the emergence of maraviroc resistance mutations in R5 tropic virus, but this process has not been well characterized, and drug resistance testing is not yet commercially available. The resistance profile for maraviroc has not been fully characterized, and there are no specific associated signature mutations [16].

Enfuvirtide (T-20) — Enfuvirtide is a fusion inhibitor that binds to the envelope glycoprotein 41 (gp41) of HIV to prevent viral membrane fusion to the CD4 T cell membrane. The only approved fusion inhibitor is enfuvirtide (sometimes referred to as T-20). (See "Overview of antiretroviral agents used to treat HIV", section on 'Fusion inhibitors'.)

Enfuvirtide has a low barrier to resistance. Resistance to enfuvirtide is a result of mutations occurring in a critical sequence of 10 amino acids (positions 36 to 45) within the helical region (HR-1) of the gp41 envelope (env) gene and, to a lesser extent, in other areas of the env gene [93]. The substitutions most frequently associated with resistance to enfuvirtide include: G36D/S, I37A, V38A/E/M, Q39R, Q40H, N42T, and N43D [16].

Genotypic assays for enfuvirtide resistance are commercially available. However, there are few data to guide practitioners as to whether enfuvirtide has residual activity based on these resistance mutation patterns [18,94]. It is expected to have maximal activity when it is used for the first time; by contrast, there is likely to be minimal antiviral activity if this drug was previously utilized in a nonsuppressive regimen.

Ibalizumab — Ibalizumab is a humanized IgG4 monoclonal antibody that blocks the entry of HIV-1 into cells by noncompetitive binding to CD4 [95,96]. Ibalizumab was approved by the US FDA in 2018 for use in combination with other antiretrovirals in heavily treatment-experienced adults with multidrug-resistant HIV-1 infection who are failing their current antiretroviral regimen [95]. Decreased susceptibility to ibalizumab has been described in some patients experiencing virologic failure and may be associated with genotypic changes in the HIV-1 env protein. The clinical significance of decreased susceptibility to ibalizumab has not been fully elucidated. There is no commercial assay available to evaluate for virologic resistance to this agent in the event of virologic breakthrough on treatment.

Fostemsavir — Fostemsavir is an attachment inhibitor that blocks the gp-120 viral receptor and prevents initial viral attachment and entry to the host CD4+ cell [97-99]. Fostemsavir was approved by the US FDA in 2020 for use in combination with other antiretrovirals in heavily treatment-experienced adults with multidrug-resistant HIV-1 infection who are failing their current antiretroviral regimen [99]. Treatment for emergent mutations in patients with virologic failure in the fostemsavir clinical trials was seen at four key gp120 positions: S375, M426, M434, and M475. There is no commercial assay available to evaluate for resistance to this agent in the event of virologic breakthrough on treatment.

Lenacapavir — Lenacapavir is a first-in-class long-acting HIV-1 capsid inhibitor that binds to the interface between the viral capsid protein (P24) subunits and interferes with nuclear transport, virus assembly and release, and capsid assembly [100,101]. (See "Selecting an antiretroviral regimen for treatment-experienced patients with HIV who are failing therapy", section on 'Lenacapavir'.)

In clinical trials, lenacapavir-associated capsid resistance mutations were found in 41 percent of participants with confirmed virologic failure, with M66I being the most common mutation [102,103]. Selecting a robust optimized background regimen to support lenacapavir is a key component of its use. Lenacapavir is a moderate CYP3A4 inhibitor and may increase concentrations of some co-administered drugs, whereas its concentration may be significantly decreased by strong CYP3A4 inducers [104]. There is no commercial assay available to evaluate for resistance to this agent in the event of virologic breakthrough on treatment.

RESOURCES TO INTERPRET RESISTANCE TESTING — 

The extensive knowledge that has been acquired on HIV drug resistance testing, only briefly outlined above, serves as the basis for interpreting HIV drug resistance testing results.

The immediate resources for the interpretation of HIV drug resistance testing are the drug resistance reports that are provided by the laboratory that conducted the testing. These reports typically contain the predicted (in genotypic testing) or measured (in phenotypic testing) resistance level to each antiretroviral drug. These levels can range from fully susceptible to highly resistant, with laboratory-specific information in between. Clinicians can use this information, combined with their knowledge of the patient, ART availability, and the other factors contributing to drug resistance, to design the best next ART regimen.

Following are two commonly used genotypic resistance mutations lists and interpretation systems. These resources contain comprehensive summaries of HIV drug resistance mutations that are helpful to clinicians and investigators and are transparent, updated regularly, and publicly available:

●The International Antiviral Society-USA (IAS-USA) provides an updated list of mutations associated with HIV drug resistance to all US FDA-approved drugs, based on evaluations by a panel group of experts (www.iasusa.org/resources/hiv-drug-resistance-mutations/) [16].

●The Stanford HIV Drug Resistance Database is a rich multi-level resource for HIV drug resistance (https://hivdb.stanford.edu/). The Drug Resistance Summaries section of the website contains diagrammatic summaries of mutations by drug class with detailed mutation-specific comments. The HIVdb Program on the website provides an interpretation of specific genotypes or mutations that are input by the user.

SUMMARY AND RECOMMENDATIONS

●Importance of resistance testing – With improvements in the efficacy, safety, tolerability, and convenience of antiretroviral therapy (ART), there are fewer patients with detectable viremia and drug resistance. Nevertheless, clinicians caring for patients living with HIV need to understand the resistance patterns associated with specific antiretroviral medications so that they may appropriately select treatment regimens that will maximize the likelihood of viral suppression. (See 'Introduction' above.)

●Factors that influence drug resistance – Drug-resistant mutations can predate the initiation of a specific drug regimen or can develop when viral replication continues in the face of ongoing drug pressure. Multiple factors influence the development of HIV drug resistance, including the biology of HIV, genetic barriers to resistance, regimen potency, pharmacokinetics of antiretroviral drugs, and medication adherence. (See 'Factors contributing to drug resistance' above.)

●Terminology – When discussing HIV drug resistance, several terms can be used to describe the viral characteristics and different mutations. As an example, "wild-type" virus refers to the original parent virus that was predominant prior to the use of antiretroviral medications. In general, wild-type virus replicates more efficiently (ie, is more "fit") in cell culture than resistant virus and may overgrow mutant variants in the absence of drug selection pressure. (See 'Basic terminology' above.)

●Interpretation of specific mutations – The extensive knowledge of HIV drug resistance mutations should serve as the basis for interpreting results, together with lab-provided reports and additional available resources to optimize regimen design, assisted by consultation with an HIV specialist, as needed. (See 'Resources to interpret resistance testing' above.)

•Nucleoside reverse transcriptase inhibitors (NRTIs) – For NRTIs, a single mutation can lead to resistance to lamivudine, emtricitabine, tenofovir, or abacavir, whereas resistance to zidovudine usually requires multiple thymidine analog mutations. (See 'Nucleoside reverse transcriptase inhibitors' above.)

•Integrase strand transfer inhibitors (INSTIs) – There are several INSTIs available, raltegravir, elvitegravir, dolutegravir, bictegravir, and cabotegravir. Dolutegravir and bictegravir have higher barriers to resistance than raltegravir or elvitegravir and likely cabotegravir. Dolutegravir (administered twice daily) can be used in treatment-experienced patients with raltegravir- and elvitegravir-resistant virus with Y143C- or N155H-mediated resistance. However, susceptibility is reduced in the presence of codon 148 mutations when other mutations are present. (See 'Dolutegravir and bictegravir' above.)

•Non-nucleoside reverse transcriptase inhibitors (NNRTIs) – The NNRTIs efavirenz, nevirapine, and rilpivirine have a low genetic barrier to resistance, and single mutations can eliminate antiviral activity of these drugs. Doravirine is a newer NNRTI, and in clinical studies, a combination of mutations conferred high-level resistance to the drug. Doravirine and etravirine have a somewhat higher barrier to resistance and may be active against some efavirenz- and nevirapine-resistant isolates. (See 'Non-nucleoside reverse transcriptase inhibitors' above.)

•Pharmacologically boosted protease inhibitors (PIs) – PIs achieve high serum concentrations and have a high genetic barrier to resistance; therefore, decreased drug susceptibility generally requires the accumulation of multiple mutations. When boosted PIs are used by patients without pre-existing PI resistance, treatment failure is less commonly associated with PI resistance. (See 'Protease inhibitors' above.)

•Novel agents – No commercially available resistance assays are available for novel agents such as ibalizumab (a humanized monoclonal antibody that blocks the cell entry of HIV-1), fostemsavir (an attachment inhibitor), or lenacapavir (a capsid inhibitor).

ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Calvin J Cohen, MD, MSc, Joel Gallant, MD, and Michael Kozal, MD, who contributed to an earlier version of this topic review.