Detection of pre-existing minority Y181C mutants encoding NNRTI resistance was associated with a more than 3-fold increased risk of virological failure to initial ART with efavirenz-based regimens in ART-naive HIV-1-infected subjects in the presence of perfect adherence. The increased risk persisted across subjects with diverse baseline characteristics, including those with plasma HIV-1 RNA levels greater than or equal to, or less than 100,000 copies/mL; the risk magnitude was considerable and clinically relevant. Importantly, the impact of the presence of low-abundance Y181C mutants on the risk of virologic failure was diminished among non-adherent subjects. These findings confirm the importance of pre-existing resistant viruses present as minority members of the viral quasispecies in determining the virologic outcome of ART, particularly in the case of drugs with a low genetic barrier to resistance. They also underscore the clinical need for improving the sensitivity of genotypic drug resistance assays.
Mutations Y181C and K103N were chosen for the ASPCR analysis because they are the most frequent NNRTI resistance mutations found after virological failure to nevirapine and efavirenz. Minority Y181C and K103N mutants were detected by ASPCR in nearly 40% of subjects with wildtype virus by standard genotypic testing. This prevalence represented an almost 9-fold increase in the detection of primary NNRTI resistance when the results of ASPCR plus population sequencing (44%) were compared to population sequencing alone (5%).
In the current study, we did not detect an association with the presence of low-abundance K103N mutants and increased risk of virologic failure. This observation contrasts with previous studies, including our own finding in the same study population of a significantly increased risk of virologic failure when K103N was detected by population sequencing. However, this discrepancy may be attributable to the relatively small number of subjects with low-abundance K103N mutants identified by ASPCR.
It is noteworthy that all of the mutants identified by ASPCR in our study were present at levels below 1%. Although these low levels could represent underestimation due to polymorphisms at the primer binding sites in the target sequences, it would be surprising if this were the case in every subject tested. A more likely explanation is that Y181C and K103N mutants present at higher levels had already been identified by population sequencing, since samples from those subjects were not retested by ASPCR. This interpretation is consistent with data generated by ultradeep pyrosequencing [22
], which found that NNRTI-resistant mutants were either present at relatively high levels (>20%, and thus detectable by population sequencing) or at low levels (generally below 1%–5%). These findings suggest that ultrasensitive resistance assays should have sufficient sensitivity to detect variants present at less than 1%–5% of the plasma virus population.
Our study extends the findings of two earlier studies. A retrospective case-control analysis from the U.S. Centers for Diseases Control and Prevention (CDC) applied a modified ASPCR technique to baseline samples drawn from two clinical trials of efavirenz-containing first-line regimens [33
]. Presence of minority mutations at RT codons 103, 181 or 184 was associated with an 11-fold increased odds of virological failure, but these mutations were detected in only a small number of subjects (7/95 with virologic failure and 2/221 with virologic suppression). The contribution of each individual mutation to the risk of virologic failure could not be assessed.
Analysis of baseline resistance by ultradeep pyrosequencing in the Flexible Initial Retrovirus Suppressive Therapies (FIRST) study, which compared initial ART strategies including an NNRTI, PI or both [34
] found that pre-existing minority NNRTI-resistant variants more than tripled the hazard of virological failure in ART-naive subjects starting NNRTI-based therapy [22
]. Similarly, all 4 subjects in the PI arm in whom PI-resistant minority variants were detected experienced virologic failure, but the numbers were too small to show a statistically significant increase in the risk of virologic failure.
The clinical application of ASPCR or any other resistance assay requires a precise refinement of thresholds that identify subjects at greatest risk of virologic failure. We were unable to define a threshold level of mutants that distinguished between subjects with virologic failure and subjects without virologic failure with high sensitivity and specificity. Although subjects with minority Y181C variants were at greater risk of virologic failure, 70% of these subjects nevertheless achieved long-term viral suppression on their initial efavirenz-based regimen. In post-hoc exploratory analyses, we were unable to identify factors that explained this difference. The high sensitivity of ASPCR may capture natural fluctuations within the quasispecies over time that are not necessarily clinically significant. Conversely, the other two studies addressing the clinical relevance of minority variants used higher thresholds for detecting minor variants. Because the modified ASPCR method used in the CDC study [33
] was designed to detect mutant viruses above the natural quasispecies frequency of each mutation, the actual threshold for detecting the K103N and Y181C mutants was 0.9% and 1.0%, respectively, which is at least two orders of magnitude higher than with our approach. Indeed, the clonal frequencies of the resistant variants in the CDC study ranged between 0.6% and 12.5%, suggesting a lower sensitivity of the ASPCR method used by the CDC, relative to ours. Similarly, due to the error rate of pyrosequencing, the cut-off for detecting minority variants in the FIRST study [22
] was established at 1%. Whereas the CDC and the FIRST studies could have missed clinically relevant minority mutants, a number of minority mutants detected in our study did not contribute to virologic failure during the study period. Determining the optimum threshold to maximize sensitivity and specificity requires analysis of a larger number of samples than available in studies performed to date.
Although the presence of pre-existing low-abundance Y181C mutants was associated with a greater risk of virologic failure, other EFV resistance mutations were more commonly found at the time of virologic failure. Similar results were obtained in the FIRST study [22
]. It is possible that presence of the Y181C mutants was a marker for presence of other, undetected NNRTI mutants that emerged under efavirenz selection. Alternatively, the low-level EFV resistance conferred by Y181C could have allowed ongoing virus replication that led, in turn, to the later accumulation of other NNRTI resistance mutations such as K103N or G190S. Persistence of Y181C might have been selected against by the coadministration of zidovudine, since Y181C increases HIV-1 susceptibility to that drug [32
]. Because we did not attempt to detect minority NRTI-resistant variants at baseline, we cannot fully rule out that preexistence of some of such variants (e.g. M184V) could have influenced the risk of virological failure.
In conclusion, low-abundance NNRTI-resistant variants significantly increased the risk of virologic failure to initial antiretroviral therapy with efavirenz among adherent subjects; these minority mutants did not add to the risk of failure with non-adherence. More sensitive resistance assays could improve the clinical management of HIV-infected subjects. The clinical application of such assays, however, will require further technical developments, a better understanding of the role of low-abundance resistant variants in different clinical scenarios and, refinement of assay thresholds that identify patients at greatest risk of virologic failure.