In this pooled analysis, we found that the presence of minority HIV-1 drug resistant variants was associated with more than twice the risk of virologic failure in patients receiving an initial NNRTI-based ART regimen in an analysis that controlled for medication adherence, ethnicity, baseline CD4 cell count, and HIV-1 viral load. The presence of minority variants was associated with 2.5–3 times the risk of virologic failure at either high or low levels of medication adherence. The association of minority variants with virologic failure was dose-dependent and most prominent in those with NNRTI-resistance mutations.
Multiple factors contribute to the risk of ART failure. Adherence to antiretroviral therapy is a major predictor of viral suppression and disease progression28–30
. In this analysis, we found that the risk of virologic failure associated with the presence of minority drug-resistant variants was similar to that conferred by suboptimal medication adherence. Patients with both minority drug-resistant variants and suboptimal medication adherence had a 10-fold risk of virologic failure when compared to those with wild-type virus and excellent adherence. However, optimal medication adherence did not completely compensate for the higher risk of virologic failure in the presence of minority drug-resistant HIV-1 variants.
Interestingly, ethnicity was found to be a significant predictor of virologic failure and in particular, white participants had a lower risk of virologic failure compared to black and Hispanic participants. This risk differential was not due to differing rates of minority variant detection. While some studies have shown no association of race or HIV-1 subtype with initial treatment response31
, a secondary analysis of the A5095 trial uncovered an interaction between ethnicity and adherence, and found a greater effect of non-adherence on virologic failure in black participants32
. It is interesting to note that the effect of ethnicity on virologic failure seen in our analysis was present even after adjusting for the level of medication adherence. The relationship between ethnicity and virologic failure most likely is mediated by factors such as socioeconomic status, drug and alcohol use, or other factors not accounted for here that may correlate with adherence and could contribute to residual confounding. Another potential explanation for these findings could be related to the recent report that cytochrome P450 polymorphisms affect NNRTI pharmacokinetics and treatment outcome in a race-specific manner33
Minority drug-resistant variants detected by ultrasensitive assays could arise from a few sources. Those found at higher proportions may represent transmitted drug resistance that have been replaced by wild-type revertants over time34
or resulted from multivariant transmission35, 36
, whereas mutations present at extremely low frequencies (much less than 1% of the viral population) could be due to de novo
mutations resulting from errors introduced during viral replication37
or laboratory artifacts from reverse transcription and PCR amplification. The presence of spontaneously appearing minority drug-resistance mutations has been described in HIV samples collected in the pre-ART drug era7
. It has been proposed that minority drug-resistant variants present at extremely low levels may not have a significant clinical impact. While we found a dose-dependent effect of minority drug-resistant variants on risk of virologic failure, this increased risk was significant even at very low minority variant frequencies (<0.5% and 10–99 copies/mL). A recent study reported a strong correlation between virologic failure and the presence of ≥2,000 copies/ml of K103N-containing HIV-1 whereas patients with <2,000 copies/ml of K103N did not show an increased risk of virologic failure19
. One explanation for the difference between these results and those of the current analysis is that the earlier study used an assay with a limit of detection for minority drug-resistant variants of 0.5% of the virus population and therefore identified only a limited number of participants with resistant variants present at low copy numbers. Other possible explanations include the lack of Y181C measurement in that study and differences between studies of the NRTI component of the regimen. Nevertheless, it is clear that not all patients in whom minority drug-resistant variants are identified will experience virologic failure and a frequency-dependent effect of the minority drug-resistant population is clearly evident from the current pooled analysis. Further research is needed to identify additional factors that contribute to the risk of virologic failure.
This analysis has several limitations. In order to combine patient-level data from studies with different study designs, statistical adjustments were required such as limiting the inclusion of patients from case-control studies to only those patients with virologic failure and using a stratified Cox proportional hazard model in which virologic failure patients outside of the cohort studies were only counted at the time of failure. Although this approach has been validated in previous studies24, 25
, we confirmed the robustness of our findings in sensitivity analyses limited to data obtained only from the cohort studies. In addition, studies that contributed data to this analysis had differences with regard to assay methodology, sensitivity, and resistance mutations detected. The assay with the highest limit of detection was the HIV-SNaPshot assay (2%)17
, whereas allele-specific PCR assays had lower limits of detection (down to 0.003%). The study that contributed the second largest number of participants and the largest proportion of virologic failures utilized the most sensitive assay20
. As expected, patients from that study made up the greatest proportion of those with minority drug-resistant variants (72%). Nevertheless, the increased risk of virologic failure associated with presence of minority drug-resistant variants persisted even when this study was removed from the analysis. Visual inspection of the Kaplan-Meier curves () suggests that the increased risk of virologic failure associated with minority variants may be most prominent early in the course of treatment. Such a result would not be unexpected and would mean that the hazard ratios presented (which represent the average hazard ratio over the entire study period) may underestimate the effect of minority drug-resistant variants during the early treatment period. Another limitation involves the types of drug-resistance mutations studied. All studies measured the levels of K103N, but only 6 studies evaluated the presence of Y181C (44% of total patients) and only a minority of the total study population was tested for the presence of M184V (23%) or K65R (17%). Consequently, our ability to detect a significant association of NRTI-resistance mutations and risk of virologic failure or a difference in effect between K103N and Y181C minority variants was limited. Because only a subset of participants were tested for the presence of other NNRTI-resistance mutations, our results most likely underestimate the effect of minority NNRTI-resistant variants on virologic failure as a significant proportion of those categorized as having no detectable minority variants may have had unmeasured Y181C or other NNRTI-resistance mutations.
The findings of this pooled analysis demonstrate that minority HIV-1 drug resistance mutations, and NNRTI resistance mutations in particular, confer a greater than 2-fold risk of virologic failure for individuals on a first-line NNRTI-containing ART regimen. Using the most sensitive test for NNRTI-resistance mutations, approximately 11 patients would need to be screened prior to initiating an NNRTI-based ART regimen to avoid one case of virologic failure. These data provide a rationale for developing standardized clinical assays for the detection of minority NNRTI-resistant variants. As NNRTI-based regimens are the most commonly prescribed first-line antiretroviral therapy, the clinical use of ultra-sensitive HIV drug resistance screening could help identify individuals at greatest risk of virologic failure and allow ART to be tailored appropriately.