Despite the availability of at least 14 Food and Drug Administration (FDA)-approved antiretroviral drugs, only a minority of HIV-infected individuals achieve a durable suppression of viral replication receiving antiretroviral therapies [1–3]. Toxicity issues, poor drug compliance, lack of potency, and pharmacokinetic interactions seem to account for this limited benefit of therapy in clinical practice [4,5]. In the absence of the maximal suppression of viral replication while a patient is under antiretroviral therapy, the selection and accumulation of drug resistance mutations is inevitable. Because cross-resistance to agents within each of the three currently available classes of drugs occurs to a variable extent, the effectiveness of subsequent antiretroviral regimens is always limited [6–9].
Different mutations are selected after exposure to distinct antiretroviral agents within the same class. Primary mutations appear earliest and tend to be specific for each compound, meanwhile secondary (or compensatory) mutations tend to accumulate later and are shared by drugs within the same family [9–11]. Therefore, as soon as one failing regimen is replaced by a new drug combination, a maximally suppressive effect will probably be attained. Conversely, the longer a patient remains on a submaximally suppressive drug regimen, the greater is the likelihood of secondary mutations developing, and the greater the risk of subsequent cross-resistance and drug failure using drugs within the same class [12,13]. The importance of considering changing treatment in accordance with genotyping mainly in early virological failures and not after longer delays has been pointed out in recent Spanish guidelines for the use of drug resistance testing . After long periods of failure, resistance can be presumed for each of the compounds in the failing combination, and cross-resistance as a result of the accumulation of secondary mutations might also be assumed.
Sequencing protease inhibitors
Virological failure on nelfinavir is associated with the selection of a D30N mutation in two-thirds of patients, which reduces susceptibility to the drug without compromising the activity of other protease inhibitors (PI), which can safely be used as part of rescue regimens . Moreover, the D30N mutation reduces the replication capacity of the virus to some extent, which can be of further benefit . However, approximately a third of individuals fail on nelfinavir as the first PI develops the L90M mutation, which causes broad PI cross-resistance, precluding their use as part of salvage interventions [16,17]. In this situation, switching to regimens containing non-nucleosides or using dual PI combinations seems to be the best option. Therefore, drug resistance testing in patients experiencing an early failure under a nelfinavir-containing regimen may allow us to design the most appropriate rescue combination (Fig. 1).
Similarly, amprenavir seems to select for I50V in most instances, which is very specific and does not compromise the activity of other PI. Although this mutation does not affect viral fitness as does the nelfinavir-associated D30N, early failures on amprenavir can be successfully managed by switching to other PI-containing regimens .
Lopinavir (formerly ABT-378) is the latest PI to be approved by the FDA. It is co-dosed with ritonavir, and has an extremely high genetic barrier. Significant resistance to lopinavir is only recognized after at least 11 PI mutations have accumulated . Similarly, tipranavir is a non-peptidic molecule that retains its antiviral activity against broadly PI-resistant strains . As for lopinavir, the genetic barrier for tipranavir is much higher than for current PI, and there does not appear to be any obvious combination of mutations that clearly confer resistance to the drug. Therefore, both lopinavir and tipranavir might be good options as part of salvage regimens after failing on PI combinations. Moreover, because they exert an extremely high potency, it might be argued that these compounds should also be the preferred choice when a decision to use PI is made as part of first-line treatment.
Until recently, it was thought that the overlapping resistance profile of non-nucleoside reverse transcriptase inhibitors (NNRTI) precluded their sequential administration. For efavirenz, the K103N mutation is almost always selected when failing therapy, compromising the activity of any of the current NNRTI . In contrast, the Y181C mutation selected by nevirapine mainly annuls the susceptibility to this compound, whereas efavirenz remains active. The Y181C mutation develops within weeks in individuals failing nevirapine , much faster than is typical for mutations resulting in high-level resistance to PI. Although failures on nevirapine can be rescued by switching to PI-containing regimens, many patients might be reluctant to begin PI, being aware of their adverse effects and difficult dose schedules. Patients failing on nevirapine can be rescued with efavirenz? Several groups have shown that most persons failing on nevirapine select the Y181C mutation, although the concomitant use of zidovudine seems to favour the selection of K103N . In the absence of zidovudine, most individuals experiencing early failure on nevirapine develop the Y181C mutation, and retain at least intermediate sensitivity to efavirenz . In a clinical study , one third of subjects failing on nevirapine again achieved undetectable viraemia levels after nevirapine was replaced by efavirenz, and this favourable outcome correlated with the lack of K103N or other NNRTI-associated mutations at the time of switching. Therefore, genotyping seems to be a necessary component of any NNRTI sequencing strategy if the patient is reluctant to begin PI (Fig. 1).
A phenomenon of drug-dependent stimulation has recently been demonstrated for clinical isolates derived from some patients failing NNRTI, and carrying mutations at positions 230, 241, 245 and 270 . This observation is unique and might favour the removal of NNRTI as a class as soon as they lose their original antiviral activity. Otherwise, continuing drug pressure might provide a replicative advantage to resistant viruses. In the absence of more definitive data on the clinical impact of this phenomenon, it might be reasonable to exclude those mutations before attempting to rescue with NNRTI individuals failing other compounds of the same family.
Sequencing nucleoside analogues
The development of resistance to nucleoside reverse transcriptase inhibitors (NRTI) seems to occur mainly by two mechanisms. First, through the loss of affinity of the reverse transcriptase for the nucleotide analogue, as occurs as a consequence of the selection of M184V or L74V genotypes conferring resistance to lamivudine or didanosine, respectively. Second, resistance emerges as a result of post-replicative repair through pyrophosphorylysis of the analogue chain-terminated viral DNA. Rescue of reverse transcription by pyrophosphorylytic removal of zidovudine monophosphate from a chain-terminated transcript appears to be the primary mechanism of resistance to zidovudine. In fact, the pattern of acquisition of mutations over time (codons 70, 215, 41, 219) follows the optimal accommodation of both zidovudine triphosphate discrimination and increased pyrophosphorylysis.
During the past year it has become clear that classic zidovudine mutations may arise using other NRTI, as in 15–30% of individuals failing stavudine [27–29], and less frequently with didanosine [30,31]. Interestingly, phenotypic assays based on recombinant virus assays have not been able to demonstrate a significant increase in stavudine/didanosine resistance in the face of these ‘typical’ zidovudine substitutions [28,29], although using more sensitive methods, some authors have been able to recognize low but consistent degrees of stavudine resistance in isolates carrying classic zidovudine mutations . These low levels of resistance might be clinically relevant, as it has been suggested examining a large database .
There are at least two different pathways for the selection of HIV-1 resistant to multiple nucleosides. The first involves the Q151M mutation, together with four additional changes at positions 62, 75, 77, and 116. The second mechanism is based on rearrangements coding for different amino acid insertions following positions 67–69. The codon 151 mutant complex confers multi-nucleoside resistance through subtle effects on the conformation of the deoxyrivonucleotide triphosphate-binding pocket , meanwhile T69SXX inserts seem to reduce NRTI activity through increasing pyrophosphorylysis [35,36]. The prevalence of each of the multi-nucleoside-resistant genotypes is low in pre-treated individuals, in the order of 3–4% for codon 151 complex , and 1–2% for D67/T69 inserts . After examining 40 clinical isolates carrying inserts, a French group  concluded that more than a 10-fold loss of susceptibility primarily affects zidovudine and lamivudine, meanwhile it is noticed in approximately two-thirds of cases for stavudine and abacavir, and in only 15% of cases for didanosine.
All the information provided above on the mechanisms of resistance to NRTI stresses the complexity of sequencing these compounds. As a rule, zidovudine mutations compromise to some extent the response to almost all the remaining drugs within this class. As stavudine failures are accompanied by these ‘thymidine-associated mutations’ in only 15–30% of instances [27–29], starting with stavudine should be preferred to starting with zidovudine (Fig. 1). The results of recent clinical trials (ALBI , START-I ) support this view. On the other hand, lamivudine and didanosine failures tend to be associated with mutations that minimally affect the response to subsequent NRTI. Moreover, both M184V and L74V compromise virus fitness [42,43], which might be of benefit at least for a short period of time. Therefore, any of these drugs might be a good choice as the first NRTI.
Primary resistance, which means the loss of susceptibility to drugs noticed in individuals never exposed to treatment, is becoming a growing issue in certain regions [44–54] (Table 1). The relatively high rate of primary resistance for some drugs, e.g. zidovudine and lamivudine, should not be forgotten, and must be considered before recommending drugs for first-line treatment. Otherwise, an impaired response to first-line combinations as a result of the presence of already pre-existing mutations might further complicate rescue interventions.
Sequencing drugs of different classes
Up to now, the development of resistance to NRTI and NNRTI was considered a separate phenomenon. However, interactions between changes occurring after failing NRTI and the NNRTI binding pocket have recently been noticed [55,56]. Some individuals carrying 41L and 215Y genotypes showed a decreased susceptibility to NNRTI. They often harboured changes at positions 98 and 108, that when coexisting with classic zidovudine mutations led to a 31-fold (108I) or sixfold (98S) resistance to nevirapine. Therefore, the response to NNRTI can be reduced to some extent in individuals pre-treated with NRTI, in particular with zidovudine.
Despite the fact that the fast approval of adefovir has been declined by the FDA, the drug might play a role in multi-experienced patients failing their current therapy . Those carrying the classic M184V associated with lamivudine resistance seem to be hypersensitive to adefovir  and the new nucleotide molecule, tenofovir. Therefore, sequencing nucleotide analogues after failing on lamivudine might be a reasonable option, although it might be necessary to keep lamivudine together with nucleotides in the rescue combination for preserving this beneficial interaction.
The author would like to thank Dr Diane Havlir for the critical review of this manuscript.
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