In early phase 1 studies of TVR, it has been shown that resistant variants are rapidly selected in patients who received 14 days of TVR monotherapy. Assessment of the viral population 3-7 months after the end of TVR-dosing by clonal sequencing showed the predominance of WT virus in the majority of patients 
. However, whether the viral population in these patients eventually returned to baseline state or whether resistant variants persisted at low levels is unknown.
This study was designed to investigate whether the selection of resistant variants after short-term TVR monotherapy results in long-term persistence of these variants. To address this question, a highly sensitive UDPS analysis of resistance was carried out on plasma samples taken after a median follow-up of 4 years after participation in the clinical phase 1 trial of 14-day TVR monotherapy 
. At this follow-up time point, frequency of resistance mutations was low and in general not increased compared to baseline frequencies. In addition, no significant overall change in quasispecies diversity, expressed by genetic distance or Shannon entropy, was present comparing the follow-up time point to baseline.
To our knowledge, this is the first study that investigated the frequency of protease inhibitor-resistant variants at baseline and after treatment using UDPS. Using UDPS, clonal and population sequencing, the sporadic presence of naturally occurring resistance mutations at low frequencies have been reported by others 
. In the present study, mutations associated with resistance were detected in baseline samples of naïve patients in 6 out of 12 patients, with frequencies of less than 0.1% in 4 of these 6 patients. Of note, the observed baseline resistant variants were not predictive of the presence of resistance at the end of the 14 days dosing period as in all but one patient resistant variants were detected at a level exceeding 10% post-dosing. Furthermore, the low level presence of resistance mutations at baseline did not necessarily result in selection of that variant during treatment as shown by the baseline A156T mutation in patient 5. Interestingly, mutations R155K or R155T, which are key mutations for resistance to both linear and macrocyclic protease inhibitors in genotype 1a, were not detected in any of the baseline or follow-up samples. At the follow-up time point, frequency of resistant variants was in general not increased.
Only patient 15 had a small but statistically significant increase in the low-level resistant variants, V36M and T54S. Interestingly, the T54S mutant, that was considered enriched in patient 15 relative to baseline, was not observed by clonal sequencing of 88 clones at EOT 
. In addition, the phylogenetic analysis of clonal sequences of this patient suggests that the most recent common ancestors of the two clusters with resistant variants present at the follow-up time point are estimated to have an origin of 126 and 63 days before the long-term follow-up time point for the V36M and the T54S respectively. This suggests that in this patient the variants with the T54S and V36M mutations sequenced at follow-up are naturally occurring variants that arose after treatment.
Using conventional population or clonal sequencing others have reported a gradual decline to WT virus population after treatment discontinuation of either short term monotherapy or combination treatment with interferon for longer treatment periods 
. In other viral infections treated with DAAs, such as human immunodeficiency virus or hepatitis B virus infections, mechanisms to improve the fitness of resistant variants, such as selection of compensatory mutations, enable the resistant variants to persist. The short dosing period of 14 days was perhaps insufficient for the development of adaptive mutations that may restore fitness to WT levels. It is possible that with longer treatment durations, the fitness of resistant variants could be compensated by additional mutations that enhance the replication efficiency 
. However, the evolution of resistant variants may be limited by the implementation of stopping rules that instruct to discontinue use of TVR in patients who are likely to have virologic failure. Furthermore, the currently approved TVR combination regimen includes PEG-IFN and RBV, which synergistically suppress virus replication thereby reducing the likelihood of occurrence and persistence of mutations.
There are some limitations to this study. First, there is a theoretical possibility of oversampling but as viral loads of all samples exceeded 105
IU/ml (or 5*105
copies/ml), viral RNA input was at least 104
virus copies per test, demonstrating that redundancy or oversampling was not a problem in the UDPS test set up. Second, while at EOT resistant variants were detected in all patients 
, at follow-up the results from three patients (patient 6, 9 and 10) were missing due to unsuccessful amplification or UDPS. However it is unlikely that this has affected the conclusion of our study, as these sequence failures were random. Third, the intrinsic error rate of the UDPS technique may have caused some of the variability that was observed. A cut off of 0.1% or even 0.5% is often used for reliable detection of mutants based on plasmid controls 
. If we would have implemented such a cut off in this study, observed variation at resistant sites would have been even less than the limited variation already present, as most of the observed variation at resistance associated sites was present at a level of less than 0.5%. In stead, sequencing errors in our system set up seem to occur at a much lower level than 0.5%. This can be inferred from the fact that observed variation at the resistance associated sites was very limited with 100% WT amino acid residues and nucleotide conservation in most samples, as shown in and . The little variation that was observed resulted in amino acid changes that have been described as polymorphisms of resistance associated mutations, indicating that these mutations do not result in non-viable virus and could well be true variation.
Results from a previous study by Susser et al. 
who used clonal sequencing indicate that at long-term follow-up after initial TVR-monotherapy the majority of the viral population consisted of wild-type variants. Our study confirms and extends the results from this study as we demonstrate that in most patients, frequencies of resistant NS3 variants after 4 years of a 14 day monotherapy course measured by an extremely sensitive sequence analysis technique are comparable to baseline. Since HCV is not known to be archived, patients could potentially be retreated in the future with more expanded combination therapy regimens that still contain TVR or other protease inhibitors from the same class. Indeed, in a recent study, such quadruple combination regimens, consisting of PEG-IFN, RBV, a protease- and a polymerase inhibitor was very powerful 
. However, re-treatment clinical trials are necessary to fully understand the implications of resistance.