Although a large number of studies have addressed first-line antiretroviral therapy in HIV-infected patients, less has been done to investigate patient management following treatment failure, particularly in countries with limited resources . Genotypic resistance testing after failure is recommended to build the new regimen, possibly combining at least two active drugs, to maximize the likelihood of regaining viral suppression [2–4].
The MOBIDIP study , addressing optimization of second-line regimens in Africa, has shed some light on this topic. This multicenter, randomized, open label, superiority trial, included 265 patients (73% women) with CD4+ cell counts above 100 cells/μl and previous treatment failure, currently receiving a boosted protease inhibitor (bPI) with two nucleoside reverse transcriptase inhibitors (NRTIs) for at least 48 weeks with virological control (at least two consecutive HIV RNA <200 copies/ml in the previous 6 months). The study included two arms: one with darunavir/ritonavir 800/100 mg once daily or lopinavir/ritonavir 200/50 mg twice daily (monotherapy arm), the other with the same bPIs in combination with lamivudine 300 mg once daily (dual therapy arm). Cumulative past genotype indicated that 29% of patients harbored virus with at least three thymidine analogues mutations (TAMS) and 96% with M184V, which confers high-level resistance to lamivudine. The authors hypothesized that the addition of lamivudine could increase efficacy because of residual antiviral activity despite resistance and/or the M184I/V fitness cost . The patients were to be followed up at weeks 4, 12, and every 3 months thereafter up to week 96. Virological failure was defined as confirmed HIV RNA above 500 copies/ml, which led to the NRTIs reintroduction (lamivudine with tenofovir), as per protocol. Treatment failure was defined as follows: HIV RNA above 500 copies/ml, discontinuation of bPI or reintroduction of NRTIs. At week 48, the treatment failure rate with bPI monotherapy and bPI with lamivudine was 24.8 and 3%, respectively [odds ratio (OR) 10.6, 95% confidence interval (CI) 3.6 - 42.1]. Consequently, the monotherapy arm was discontinued.
Indeed, the study is unusual in the design and somewhat unexpected in the results. As outlined by the authors themselves, a standard of care (SOC) arm (i.e. a triple drug regimen) was missing, which makes the study difficult to replicate, at least in Europe. In addition, the study did not exclude patients with a low nadir CD4+ cell count (53% of patients had nadir CD4+ cell counts below 100 cells/ml), whereas previous monotherapy trials [6,7] clearly indicated the underperformance of bPI monotherapy compared with the SOC, in that setting. Despite these caveats, the study conveys relevant information on antiretroviral switch strategies. The novelty in MODIBIP is the dual therapy with a bPI with lamivudine in patients with previous evidence of M184V virus. In fact, the efficacy of such a dual combination has been shown in four switch trials (with up to 96-week results), confirming similar rates of virological suppression compared with the reference bPI triple arm, but all in the absence of any preexisting primary resistance mutations [8–11]. Thus, such trials tested a dual regimen with two fully active drugs, as opposed to the MOBIDIP study wherever virtually all patients had a history of lamivudine-resistant virus.
Although the role of the archived mutations with regard to the risk of failure is still controversial , including a drug, which is expected to work marginally (if at all) in a dual therapy maintenance strategy is not recommended [3,4]. Nonetheless, the rate of virological suppression at week 48 for the dual arm in the MOBIDIP trial was surprising (98%), advising to rethink the interpretation of past genotypic resistance data. In this regard, the MOBIDIP study provides a unique contribution to our understanding of the role of lamivudine, in spite of past resistance, whenever combined with a high genetic barrier drug such as a bPI. On the other hand, several studies have provided somehow complementary information by removing genotypically inactive NRTIs to spare both toxicity and costs. For instance, removing one or two predictably inactive NRTIs has been reported in a proof-of-concept study of 31 virosuppressed patients being treated with a four-drug or five-drug regimen, without affecting the virological success at week 48 . Similarly, the OPTIONS trial  randomized omitting or adding inactive NRTIs in 360 treatment failing patients, confirming that virological success at week 48 can be achieved without including predicted inactive NRTIs, provided that regimens had at least two fully active agents. However, these data, rather than supporting the absence of NRTI antiviral activity in the presence of resistance mutations, do confirm that virological suppression can be achieved or maintained with dual, triple or quadruple therapy devoid of NRTIs, but including at least two fully active agents [15,16]. In the setting of viremic patients with multidrug-resistant HIV, old data suggest that only the individuals who discontinued NRTIs (differently from protease inhibitors or NNRTIs) from their current regimen had immediate increases in HIV RNA levels, with disappearance of the M184V mutation, suggesting some kind of residual antiviral activity of this class of drugs, despite predicted resistance .
Whether or not including NRTIs to rescue NNRTI-based first-line failures has been a focus of three randomized trials (18–21) comparing the efficacy of bPI with two to three NRTIs versus bPI with raltegravir second-line therapy (Table 1).
The SECOND-LINE study  randomized 541 patients failing their first-line therapy to receive either a bPI (lopinavir/r) with two to three NRTIs (271 patients) or a bPI with raltegravir (270 patients). The NRTIs were selected by genotypic testing (73%) or protocol-specified algorithm. Notably, as many as 60% of cases had M184V and around 30% cases had multiple TAMs or multi-NRTI resistance. At week 48, 81 versus 83% had HIV RNA below 200 copies/ml in the NRTI and raltegravir group, respectively, without any difference according to the NRTIs selection system. Results were confirmed for HIV RNA below 50 copies/ml. At week 96, the findings were similar as 80.4 versus 76% had HIV RNA less than 200 copies/ml.
The EARNEST study [19,20] randomized 1277 patients failing their first-line regimen in sub-Saharan Africa, to three options: bPI monotherapy (lopinavir/r), bPI combined with raltegravir or bPI combined with two to three NRTIs without resistance testing. Unexpectedly, the 96-week virological response was 86% for both the raltegravir and the NRTI-based arm. By contrast, the success rate was inferior in the protease inhibitor monotherapy arm (61%), which was then discontinued. Subsequent resistance testing on stored baseline samples, again showing extensive NRTI resistance, indicated that the standard genotypic resistance algorithms did not predict in-vivo antiviral activity of NRTIs . In fact, at week 144, 81% of patients in the raltegravir-based arm had HIV RNA less than 400 copies/ml compared with 89% of patients on NRTIs predicted to be inactive (P = 0.02). Although previous NRTI resistance may have been a proxy of adherence in this setting, it is noteworthy that the success rate was much lower with bPI monotherapy compared with bPI with inactive NRTIs (61 and 89%, respectively, at week 96), indicating that predicted inactive NRTIs still helped in virological control, resembling MOBIDIP results.
Finally, the SELECT study (A5273)  randomized 515 patients failing their first-line regimen in resource-limited settings to bPI (lopinavir/r) combined with either raltegravir or two to three NRTIs (selected from an algorithm: zidovudine after failure of tenofovir, and vice versa). At week 48, the proportion of patients with HIV RNA less than 400 copies/ml was 92 versus 91% in the raltegravir and NRTI group, respectively. Again, NRTI resistance at baseline was extensive, with 90% M184V and 50% multi-NRTI resistance.
Thus, the above mentioned three studies failed to show superiority of raltegravir-based regimens and supported the significant antiviral activity of NRTIs in second-line treatment, despite genotypic resistance. Accordingly, WHO treatment guidelines do not recommend resistance testing before switching to second-line regimens in low-income countries . However, the studies cannot indicate which or how many NRTIs are needed to achieve virological re-suppression, as all patients were on two or three NRTIs combined with lopinavir/ritonavir. Although in the context of suppressed viremia, the MODIBIP study answered this question, clearly showing the benefit of recycling lamivudine only.
Persistent antiviral activity of lamivudine, in spite of archived M184I/V resistance mutation, has been suggested also in another single arm dual regimen DOLULAM study . In this observational cohort, 27 highly pretreated and virologically suppressed patients (similarly to the MOBIDIP trial) were switched to dolutegravir with lamivudine, with 24 of 27 (89%) maintaining virological suppression up to 2 years, and three patients discontinuing treatment for reasons other than virological failure. As in the MOBIDIP study, past lamivudine resistance did not appear to impact the virological response to dual therapy as the M184I/V mutation was previously detected in HIV DNA or RNA in 17 of 27 (63%) patients . Again, these patients had a long duration of virological suppression before switching (median 6.4 years)  and lamivudine was added to a high genetic barrier agent (i.e. dolutegravir).
Although dual therapy simplification studies [5,23] support the efficacy of lamivudine despite previously documented resistance, a mechanistic explanation for this unexpected finding is at present lacking. The M184V virus has been extensively characterized as a low-fitness variant in vitro  and shown to antagonize dolutegravir resistance . Accordingly, in an in-vivo pilot study, even lamivudine monotherapy was superior to complete treatment interruption in patients on treatment failure harboring an M184V virus . An ultradeep sequencing analysis of baseline samples from the DOLULAM study detected the lower fit M184I variant mostly contained in nonfunctional HIV DNA, a finding consistent with preferential loss of such variant and/or preferential targeting of the 184 reverse transcriptase codon by the cellular innate defense APOBEC system . Additional information on the kinetics of M184I/V mutation in patients enrolled in key studies such as MOBIDIP would be relevant to challenge the paradigm that any HIV variant ever occurring remains ready to resume replication forever.
Nowadays, treatment de-intensification following prolonged suppression of virus replication is possible [8–11,28]. The role of time spent on virological suppression before switching in blunting the effect of historical resistance mutations is also suggested by past raltegravir simplification studies. Indeed, the SWITCHMRK  and the SPIRAL  studies both replaced a bPI with raltegravir in virosuppressed patients, while maintaining the NRTI backbone, despite past treatment failure and/or NRTI resistance in approximately 30% of cases. At week 48, SPIRAL, but not SWITCHMRK, showed similar rates of virological success compared with the control arm. In fact, as addressed by the same authors , such a difference may be because of the longer duration of virosuppression in the SPIRAL (median time >6 years) compared with the SWITCHMRK study (<3 years, as inferred from treatment duration data). Findings from the large EUROSIDA cohort  confirm such an observation. In this perspective, a long-lasting replication control may favor exhaustion of specific variants from the HIV reservoir or maintenance below a biologically significant threshold, particularly for those with impaired fitness. Unfortunately, clinical studies do not generally provide information on how much time elapsed since the last detection of a specific mutant, how long the patient remained with that mutant and to what viremia levels did it replicate. All this information would be helpful in understanding the kinetics and impact of M184V-harbouring viruses.
Taken together, these observations challenge the common assumptions underlying resistance-testing interpretation, support a residual activity of NRTIs despite present or past primary resistance mutations and open the opportunity for rethinking the potential contribution of recycling NRTIs. Certainly, before translating this strategy into clinical practice, more data are needed. However, should this approach be confirmed, two of the oldest agents in the HIV armamentarium (bPI and lamivudine) could be promoted to play a role in maintenance strategy with an effective, simplified and less expensive option. On the basis of these premises, a recent and thoughtful commentary  argued for the potential of a universal second-line fixed-dose combination therapy. The option is relevant as M184V mutation is the most frequent mutation in treatment failing patients [5,14,19] and lamivudine is well tolerated. Although more attractive for limited resources countries, a dual regimen including lamivudine may be an opportunity also for middle-income or high-income countries, as long as the universal treatment is expected to increase the healthcare costs.
Conflicts of interest
There are no conflicts of interest.
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* Diego Ripamonti and Maurizio Zazzi contributed equally to the writing of the article.