Using assays validated for drug resistance mutations in HIV-1 clinical samples, we identified, in ART-naïve persons from the US and Canada, a substantial number of minority mutant viruses at levels above the natural quasispecies frequency of each mutation. The preponderance of minority resistance mutations implies that a considerable proportion of transmitted drug resistance decayed to low levels by the time of HIV-1 diagnosis. The detection of minority resistance mutations in 17% of the wild-type virus group, part of a US cohort of newly diagnosed HIV-1–infected persons that had 20% bulk sequence-detectable primary drug resistance, suggests that bulk sequencing missed 40% of the resistance samples in this population. The frequent occurrence of drug resistance likely reflects a high prevalence of ART in these locations and suggests that transmission of HIV-1 from antiretroviral drug-experienced persons expressing virus is not uncommon.
Minority HIV-1 variants increased the observed frequency of transmitted multi-drug resistance genotypes in the cross-sectional study group by one-third, emphasizing that drug-naïve individuals may harbor hidden resistance to drugs from different classes. The several cases of low-frequency drug resistance mutations that we identified is likely an underestimation of minority resistance prevalence, because only eight of the nearly 40 codons in PR and RT that are associated with drug resistance were evaluated [25
]. Clonal sequencing and resistance mutation linkage analysis (see Figure S1
and Text S2
) have shown that multiple minority drug resistance mutations can be present and that these mutations may coexist unlinked from majority mutations in ART-naïve persons.
The presence of unlinked mutations in multidrug-resistant ART-naïve persons might reflect mutations that were once linked in oligoclonal populations and then independently reverted as a result of mutation (fitness) modulations or immune selection [15
]. Alternatively, some dissociated mutations could have been a result of infections with more than one drug-resistant variant, possibly through needle sharing or, less frequently, by sexually acquired superinfection [28
]. Of note, we found that in the wild-type virus group minority mutations were equally prevalent in recently infected persons and in those infected for longer duration (17% of both groups). This might suggest that a considerable proportion of transmitted drug resistance decays very rapidly. A better understanding of drug resistance transmission and mutation decay requires sensitive testing of longitudinal samples from a large number of acutely infected persons with dissimilar drug resistance mutation genotypes.
With the knowledge that minority drug resistance can be considerable in ART-naïve populations, we then assessed the impact of low-frequency resistance mutations on treatment responses in a case-control study of persons with no treatment-relevant mutations detected by conventional genotyping. In the baseline samples of this previously ART-naïve group we were again able to identify minority treatment-relevant mutations. The minority mutations were significantly associated with virologic failure using the Fisher exact test (p = 0.0038) and in a logistic model. These results suggest that minority transmitted resistance mutations can be clinically important.
We observed in the case-control study what appeared to be a stronger association with treatment failure for participants with minority resistance than that seen for resistance-associated mutations detected by bulk genotyping [22
]. A virologic explanation for these seemingly disparate outcomes is currently not evident, and the finding could be biased by only a few samples having any of the three relevant resistance mutations. Notably, a similar observation was recently made with low-level RT-simian–human immunodeficiency virus (SHIV) mutants in a macaque infection model. In that study, minority NNRTI mutations were associated with virologic failure, whereas viruses with resistance mutations at higher levels detectable by sequencing were successfully suppressed by the NNRTI regimen [29
]. It is possible that some minor variants have higher phenotypic resistance than the dominant mutants, presumably due to additional mutations. These minor variants could be less fit than majority mutants in the absence of drug, but more fit under drug pressure, leading to virologic failure. Additional studies are required to examine these possibilities.
While we report a significant association between poor virologic suppression and minority levels of three mutations relevant to two EFV-based regimens, the impact of a particular minority resistance mutation on other drug regimens is not entirely understood. Further investigation with sensitive tests will help define the clinical significance of individual mutations in the context of specific drug (or drug class) combinations. Data from those studies could assist with the selection of regimens that are most active for persons infected with drug-resistant viruses.
A limitation of our study is that, as with all self-reported histories, some participants with resistance mutations may not have disclosed previous exposure to antiretroviral drugs and, therefore, did not represent cases of primary drug resistance. Additionally, the small number of participants with detectable minority resistance mutations in the case-control study may have allowed for an artifactual association with virologic failure. It may be possible that the higher median viral load in the virologic failures versus the treatment successes allowed for increased opportunities to detect minority mutations in failures; however, this was not evident from the viral loads for the failures with resistance, which were comparable to the successes. A possible explanation for the low number of detectable minority variants is that NNRTI resistance mutations were not as prevalent during the period of the GSK studies as they are today, as seen in the populations sampled for our cross-sectional analysis. Additionally, the participants in the case-control study may have had infections of comparatively longer duration with substantially greater decay of resistance to undetectable levels. Nevertheless, our findings are in agreement with three other studies that reported a significant association between treatment failure and minority NNRTI resistance mutations [18
The prevalence of primary drug resistance in geographic areas where ART is common suggests that initial therapies can be significantly impacted; therefore, efforts to prevent transmission from ART-experienced persons are of great importance. The association between minority resistance and poor virologic suppression suggests a need for practical and sensitive testing to identify drug-resistant variants before treatment.
In conclusion, the data from drug-naïve persons demonstrate that sensitive testing improves detection of HIV-1 drug resistance mutations and, therefore, could be valuable not only to HIV-1 surveillance but also to ART management, particularly when treatment history is unavailable.