chemokine receptor blocking agents, and agents that target CD4 binding. Other studies found that clinical resistance to ENF did not affect susceptibility to other classes of inhibi- tors (Fikkert et al 1003; Rimsky et al 1998; Greenberg and Cammack 2004; Ray et al 2007). Coreceptor usage HIV fusion is a multi-step process that involves conformational changes in both gp41 and gp120. Several in vitro studies have suggested that ENF susceptibility may be linked to coreceptor usage (Derdeyn et al 2000; Reeves et al 2002). However, data from a large number of clinical isolates from the TORO trials showed that virologic response to ENF was independent of baseline coreceptor usage (CCR5, CXCR4, and dual tropic- viruses) (Melby et al 2006). This study also showed that changes in viral load were similar across baseline coreceptor usage. Non-clade B infection Although ENF was originally developed based on the gp41 sequence of clade B, antiviral activity has been shown against non-B viral subtypes (Villahermosa et al 2003; Greenberg and Cammack 2004; D’Arrigo et al 2007; Holguin et al 2007). Although there was substantial variability in gp41 within and between subtypes, primary resistance to enfu- virtide was rare (Villahermosa et al 2003; D’Arrigo et al 2007; Holguin et al 2007) in untreated non-B individuals. Baseline IC50 values for ENF-naïve individuals were similar to other reports for ENF (Holguin et al 2007). Although ENF has shown susceptibility to non-B strains, it is not part of the current WHO approach to treatment guide- lines for resource-limited settings (Gilks et al 2006). Current limitations for ENF in resource-limited settings include cost, availability of sterile syringes and water, and availability of medical staff to instruct proper injection technique and to monitor and treat injection site reactions. However, Brazil, the ﬁrst developing country to implement policies that provide universal access to free antiretrovirals, has included enfuvirtide in its treatment guidelines (Nunnet al 2007). Although the cost of this drug has been implicated as a major component of the doubling Brazil’s total drug expenditures, it should provide more treatment options for salvage therapy for patients who have limited treatment options (Nunn et al 2007). It remains unclear if other developing countries will follow the Brazil model and adopt enfuvirtide in their treatment guidelines. Viral ﬁtness and evolution Understanding the mechanisms by which resistance muta- tions affect viral ﬁtness and evolution, particularly among Therapeutics and Clinical Risk Management 2008:4(2) Enfuvirtide therapy patients with signiﬁcant antiretroviral experience is critical in determining effective therapy regimens (Deeks et al 2005). Viral ﬁtness or replication capacity measures the association between mutations that confer resistance and changes in the ability of the virus to infect cells and reproduce. Fitness levels for a given mutation may vary depending on the pres- ence of other resistance-associated mutations or compensa- tory mutations as well as other viral characteristics such as coreceptor usage and experimental conditions. Viruses that harbor mutations at positions 36–45 were found to be less virulent than wild-type in the absence of ENF, however in the presence of ENF these viruses displayed greater replicative ﬁtness than wild-type (Lu et al 2004). Although ENF confers selective pressure against gp41, the env region must retain its resistance against neutralizing antibodies or it will be selected against in vivo (Moore and Doms 2003). Reeves et al introduced HR-1 mutations that have been shown to confer ENF resistance into both CCR5-tropic and CXCR4-tropic strains (Reeves et al 2005). They found that these mutations reduced infection and fusion efﬁcacy, and delayed fusion kinetics indicating reduction in viral ﬁtness. However, they also found that some of these mutations conferred enhanced sensitivity to neutralizing monoclonal antibodies, suggesting that these mutations may make the virus more susceptible to humoral immune responses. Labrosse et al found that in 5/6 patients who failed ENF therapy, the emergence of resistance mutations did not reduce env replicative capacity (Labrosse et al 2006). Further, they found that during ENF therapy the dominant env quasi-species differed from the dominant quasi-species circulating before ENF was initi- ated and persisted even after ENF interruption. Deeks et al examined the level of continued antiviral activity of ENF in the presence of incomplete viral suppression and ENF resis- tance mutations (Deeks et al 2007). Interruption of ENF only while remaining on a stable optimized background regimen resulted in a small but signiﬁcant increase in plasma viremia and a small but signiﬁcant decrease in CD4+ T cell counts suggesting a modest residual antiviral activity by ENF. ENF resistance mutations decreased rapidly during the interrup- tion and in most patients were undetectable by week 16 in a clonal analysis. Kitchen et al examined the viral evolution of these clones from 9 of these patients taken at three time points: before ENF, during ENF failure and after interrup- tion of ENF therapy (Kitchen et al 2006). They found strong evidence supporting ongoing viral evolution during ENF therapy and interruption, suggesting that the loss of ENF resistance mutations was due to back-mutation and not recall of presumed archive ENF-susceptible strains. This result is 437