While sustained suppression of plasma HIV-1 RNA below the limits of detection with standard assays is possible in many infected individuals, it is clear that virus still persists in cellular reservoirs for prolonged periods of time [1,2]. Nevertheless, there is little evidence that viral evolution is occurring in this reservoir or that it will be a source of drug resistant virus in the future . In addition to the cellular reservoir, other pharmacologically privileged areas such as the central nervous system and the genital tract may act as an additional sources of residual virus in those with ‘undetectable’ levels of plasma HIV-1 RNA. Several groups have reported data using investigational assays that measure plasma HIV-1 RNA levels below 50 copies/ml, showing that many patients on potent antiretroviral (ARV) therapy have detectable levels present below the limits of detection of standard assays [3,4]. It is not yet known what the clinical implications are of having quantifiable viral levels in this range or what is the source of this low level viremia. Defining these issues may have implications in the clinic as well as towards our understanding of HIV-1 pathogenesis. In fact, recent investigation of novel ARV treatment strategies has provided insight into these areas of interest.
McKinnon and colleagues report in this issue of AIDS data from stored specimens collected during the course of a clinical trial of maintenance therapy with a single pharmacologically boosted protease inhibitor (PI). The results of this study were reported by Arribas et al.  and showed that 20 of 21 who remained on a combination drug regimen maintained undetectable levels of plasma HIV-1, with the lone ‘failure’ having left the study because of toxicity. In contrast, 17 of 21 who simplified their combination therapy to lopinavir/ritonavir (LPV/r) alone remained suppressed, with one of the four not suppressed being lost to follow-up and the other three experiencing viral rebound. The investigators in the current analyses used a modified RT–PCR assay with a lower limit of quantitation of 3 copies/ml to further explore the impact removing two drugs from a three-drug regimen have on low level plasma HIV-1 RNA. They found that in the controls as well as those that remained suppressed on LPV/r alone there was no change in plasma HIV-1 RNA over time. In contrast, those experiencing viral rebound had an increase in plasma HIV-1 RNA detected with the highly sensitive assay prior to experiencing viral rebound identified by standard methods.
From a clinical perspective these data show that the majority of subjects who were suppressed on a three-drug regimen maintained suppression with LPV/r alone, consistent with a recently reported open-label study simplifying combination therapy to atazanavir (ATV) with ritonavir (RTV) . The results also suggest that the more sensitive assay may have some utility in identifying early failure; however, the numbers are small and the clinical relevance of identifying an increase in HIV-1 RNA at levels below versus above 50 copies/ml is not known.
The strategy of using RTV-boosted PI monotherapy has been further studied in a series of relatively small, controlled studies presented at the recent XVI International AIDS Conference. Each study had a unique design that provides further insight into the potential role of this treatment strategy. One study randomized treatment naive subjects to dual nucleoside reverse transcriptase inhibitors (NRTI) with LPV/r versus LPV/r alone, the latter group including 83 subjects. They found that viral suppression was common in both groups but detection of viral levels in the 50–400 copies/ml range was more common in the monotherapy arm than in controls . Cameron and colleagues studied treatment naive subjects that were randomized to receive dual NRTI with LPV/r versus efavirenz (EFV). Ninety-two of the subjects in the LPV/r group that achieved plasma HIV-1 RNA levels below 50 copies/ml during the first year of therapy were then simplified to LPV/r alone . Once again, those on monotherapy with LPV/r alone were more likely than controls to have plasma HIV-1 RNA levels between 50 and 400 copies/ml. In contrast, a third study randomized those on combination therapy with sustained plasma HIV-1 RNA levels below 50 copies/ml for a median of 19 months to continue the same or simplify to LPV/r alone. They found that the majority of subjects in both groups maintained plasma HIV-1 RNA levels below 50 copies/ml . The design and results of the latter study approximates that of the original pilot clinical trial used in the current analysis of McKinnon et al., and the study of Swindells and colleagues , where simplification to monotherapy with a RTV-boosted PI occurred after a prolonged period of viral suppression. Together the results of these studies suggest that what is going on just above the current bar of the clinically used assay (50–400 copies/ml) and just below the bar (3–50 copies/ml) may be relevant and ultimately defined by the type of treatment and duration of suppression at the time of simplification to monotherapy. It may be that getting to very low levels of virus – those only appreciated with a 3-copy assay – requires the use of more potent combination therapy for longer periods of time, and only then can therapeutic simplification consistently maintain virologic control. This is in keeping with the observations of Di Mascio and colleagues that it can take many months to get plasma HIV-1 RNA levels from 50 to less than 3 copies/ml, a threshold unappreciated with standard assays used in the clinic today . Further analyses of subjects from all of these studies, along with potentially applying the more sensitive assay used by McKinnon et al. may shed further light on these issues and define the optimal and most rationale approach to moving ARV maintenance strategies forward.
The use of the more sensitive assay also has the potential to provide insight into the source of HIV-1 detected in those with persistent viremia below the level of detection of current assays. As proposed by the authors the leading hypotheses are ongoing rounds of low level replication associated with insufficient drug potency or the release of virus from reservoirs. Reservoirs may include long-lived cells that are either persistently or latently infected or pharmacologically privileged sites where viral replication is ongoing despite suppression in plasma. Based upon the results of the current study insufficient potency in plasma seems unlikely as reducing the number of active drugs in the regimen was not associated with an increase in plasma HIV-1 RNA. The possibility that low level viremia is a result of the release from a cellular reservoir cannot be excluded based upon the results of this study, nor the possibility that virus is being released from compartments in which NRTI and PI achieve insufficient levels. Future maintenance ARV strategies will hopefully address these issues by defining drug penetration in compartments of potential clinical relevance such as the central nervous system and genital tract, by assessing both changes in drug levels and HIV-1 RNA .
Although this study demonstrated that increases in viremia detected by assays measuring between 3 and 50 copies/ml predicted virologic failure, only further analyses will determine whether this assay provides relevant clinical information above and beyond those currently available in the clinic. In the meantime this highly sensitive tool does appear to be useful for exploring HIV-1 pathogenesis and defining the activity of novel ARV agents and treatment strategies.
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