sdNVP is frequently used in MTCT prevention programs as highly effective combination ART and even short-course antenatal zidovudine for PMTCT are not yet accessible to the majority of HIV-infected pregnant women in the developing world. Unfortunately, many studies have shown that at least transient drug resistance develops in a large fraction of women after sdNVP, and drug-resistant HIV variants persist for considerable periods in patient samples at frequencies below the limit of detection of the most commonly used commercial genotyping methods.15–18,26,27
The clinical importance of such drug-resistant “minor variants” following sdNVP remains unclear. Recent reports in settings other than sdNVP for PMTCT have suggested that minor variants may contribute to virologic failure.20–22,28
These studies were in both treatment-naive patients, in whom the minor variants may represent transmitted drug resistance, and in heavily treatment-experienced patients, in whom these minor variants likely represent archived, resistant virus that reemerges with the reintroduction of ART. Additionally, a new report demonstrated that minor variant resistance at the 103 position of reverse transcriptase after sdNVP was associated with early virologic failure with NNRTI-containing ART.23
The study described here was designed to determine if the presence of low-frequency drug variants, detected by highly sensitive methods, could predict virologic failure when NVP-based ART is initiated following sdNVP exposure. The ASPCR assay was performed at positions 103 and 181 of reverse transcriptase as these mutations were present most often after sdNVP in Botswana.29
The results of this study showed that patients with minor drug-resistant variants present in the plasma at a frequency greater than 0.19%, prior to ART initiation, had a higher odds of clinical failure (odds ratio
13, 95% CI 1.27–133). Those that failed NVP-containing ART started medications closer to the time of nevirapine exposure compared to those who were successful with ART (5.5 months vs. 9.5 months, p
Minor drug-resistant variants have been proposed as a possible cause of the higher than anticipated rates of ART failure in the Thailand PHPT-2 study,11
and our results support the hypothesis of a relationship between pre-ART initiation minor resistant variants and ART outcome. Our results also provide initial data for reexamining the clinically relevant threshold of drug-resistant viruses. Although resistance detected by standard genotyping in treatment-naive individuals is well documented to decrease the likelihood of successful ART and/or to delay the time to virologic suppression,30,31
these assays are limited to detecting drug resistance mutations representing approximately 20% or more of the virus population in a patient, and there is no logical reason to presuppose that the assay sensitivity threshold is also the threshold frequency of clinical relevance. As more sensitive methods such as ASPCR have been developed that allow detection and quantification of drug resistance mutations to frequencies as low as 0.10%, it has become possible to begin to reexamine the question of minimum drug resistance frequency that may impact clinical outcome.
A clinically relevant threshold of minor resistant variants after sdNVP that helps predict successful ART likely reflects the degree of viral decay of resistant virions that has occurred (with subsequent outgrowth of wild-type virus) to allow for clinical success with ART. This theory would explain the finding in Botswana that waiting at least until 6 months after NVP exposure significantly improved virologic success with ART, at levels matching those women who received zidovudine and placebo,12
but low levels of resistance can be overcome as seen in six patients in this study who were successful with ART despite the presence of minor variants above the level of 0.19%.
Viroseq testing at the time of failure in the six individuals who had baseline minor drug-resistant variants above 0.19% demonstrated NNRTI mutations in five of the six samples. Additionally, in three of the samples, the M184V mutation was detected, which confers resistance to lamivudine. It is likely that the baseline NNRTI resistance precluded full virologic suppression, allowing lamivudine resistance to emerge. This supports the importance of the baseline NNRTI resistance, even at these low levels, in some individuals.
This study analyzed patient samples immediately prior to starting ART and not at a specified time point (e.g., 3 or 6 months) after exposure to NVP. An examination of nevirapine resistance at a standardized time point in all patients who received sdNVP, determining resistance in both the plasma and proviral DNA reservoir, would allow correlation of both reservoirs with clinical outcome. Ideally, such a sample set would show clearly the viral kinetics of the resistant variants and determine if resistant virus decay is important for treatment success. It is unlikely to explain some of the variability in the results as some resistant variants are still detectable long after NVP exposure and not associated with treatment failure. This may be related to host factors or individual viral characteristics and cannot be resolved in this study.
Additionally, the threshold of clinical relevance that was selected was not differentiated between the two different mutations and there may be different thresholds of clinical relevance given the reported greater impact that the Y181C has on viral fitness compared to the K103N mutation.32
This possibly could be examined in a large sample set where stratification based on minor variant detection thresholds could be employed, but this may not be clinically significant given the recent report by Coovadia et al.23
In their study, when looking only at the K103N mutation, a threshold of 0.20% was found to be significant in predicting success of ART.
It is notable that the CD4 counts in the cases and controls were significantly different in this study (67.5 cells/μl vs. 188.8 cells/μl, p
0.001). Persons with lower CD4 counts were more likely to fail therapy; this was also demonstrated in the Mashi study overall. However, there is no obvious biological explanation for why lower CD4 counts might contribute to minor variant drug resistance since the viral loads were not statistically different between the two groups. Indeed, in five of the six individuals who failed therapy with detectable low level drug resistance at treatment initiation, the genotype at the time of failure demonstrated NNRTI resistance. This certainly suggests that minor drug-resistant variants play a role in treatment failure, but these variants cannot be conclusively stated as the sole cause of failure, particularly as this study was unable to match patients by CD4 count. Future studies with a larger sample size should be stratified by CD4 count to clarify that minor variants contribute to clinical failure independent of CD4 count. It is unlikely that there were differences in nevirapine exposure to account for the different outcomes, as a subset of individuals examined in the Mashi study had detectable nevirapine levels in 95 of 96 (99%) women, suggesting very high adherence overall to the sdNVP intervention in the Mashi study.29
The ASPCR assay designed for this study, with its extensive primers and control plasmids, would be difficult to employ in many settings due to cost and the need for genotype information prior to primer selection. However, the data from this analysis do support the observation that waiting for a period of time after sdNVP exposure is likely to be beneficial, as these findings may represent the decay of resistant virions after sdNVP. The reality in resource-limited settings may be that waiting as long as possible after sdNVP exposure before starting ART, without jeopardizing patients' health, may better the odds of successful nevirapine-containing ART.
The observation in this study that pre-ART minor drug-resistant variants in plasma samples after sdNVP were predictive of subsequent clinical failure suggests that detection of drug-resistant viruses with greater sensitivity than current standards has clinical value. More research should be performed to see that the clinical threshold with this ASPCR assay is maintained in larger data sets, to determine the relationship between CD4 count and minor variants, and also to explore the possibility that other viral reservoirs containing resistant viruses impact clinical success. The goal of PMTCT is for better, safer interventions designed to protect mother and infant. To do this, it is necessary to continue to try to understand the impact of sdNVP and how best to treat patients who develop nevirapine resistance as a consequence.