This study showed high post-partum antiretroviral resistance rates in antiretroviral-naïve women receiving PLAT. The main determinants of resistance selection during pregnancy were the characteristics of the antiretroviral regimen chosen to prevent MTCT.
Virtually all women receiving dual therapy developed the M184V mutation. This mutation confers high-level resistance to lamivudine and emtricitabine[25
] and is associated with an increased risk of virological failure of treatment combinations including these drugs.[18
] Thymidine analogue resistance mutations (TAMs) were detected in few women but were also more frequent in those receiving dual therapy. A high proportion of women receiving triple-drug therapy also selected resistance mutations during pregnancy. Based on allele-specific PCR testing, 50% of women treated with three drugs developed the M184V mutation. Moreover, although few women received nevirapine in this study, almost 40% of these women had non-nucleoside analogue (NNRTI) resistance mutations detected at the postpartum visit. On the contrary, selection of PI resistance was rare in women treated with nelfinavir.
Use of dual therapy and duration of zidovudine exposure, which reflects the overall duration of antiretroviral therapy, were the only two variables that were independently associated with an increased risk of M184V detection after delivery. Similarly, the post-partum detection of the K103N mutation was independently associated with exposure to nevirapine and duration of exposure to zidovudine and lamivudine. These findings strongly argue against using dual therapy to prevent MTCT whenever triple therapy is available.
The fact that resistance mutations were so frequently detected among women receiving triple therapy contrasts with previous estimates from Latin America and Caribbean countries,[27
] and suggests that PLAT was less effective than expected at continuously suppressing HIV-1 RNA levels. In the univariate risk factor analysis, women who had all HIV-1 RNA levels above 400 copies/mL during the study were 2.7 times more likely to have the M184V mutation detected postpartum than those remaining aviremic through delivery. Similarly, subjects receiving nelfinavir were 4 times less likely to develop postpartum M184V than those not receiving this drug. These findings suggest that women treated with drugs with high genetic barrier to attain resistance are less likely to develop the M184V mutation. None of these variables, however, remained independently associated with risk of postpartum resistance in the multivariate analyses.
The pre-existence of primary resistance mutations could also have explained the high frequency of postpartum resistance found in this study. The prevalence of primary resistance increased during the last decade in pregnant women in the US.[28
] Using allele-specific PCR in women enrolled in WITS with similar characteristics to those included in this study, we previously reported a 9.4% prevalence of primary lamivudine and emtricitabine resistance, and a 6.3% prevalence of nelfinavir resistance between 1998 and 2004.[8
] As most women included in this analysis started antiretroviral therapy before WITS enrolment, pre-treatment resistance data was only available from one third of women. Based on this limited number of subjects, we did not observe an association between pre-existing resistance and the postpartum selection of M184V or K103N.
We did not evaluate treatment adherence; therefore we cannot rule out an association between suboptimal adherence and the observed rates of postpartum resistance. Ethnicity and hard drug or alcohol consumption have been previously associated with lower adherence to antiretroviral therapy and worse virological outcomes,[29
] however, these factors were not associated with postpartum resistance in this study.
Finally, altered drug pharmacokinetics due to physiological changes occurring in women during pregnancy could have favored the existence of suboptimal drug levels during pregnancy or prolonged drug elimination in the postpartum period. Exposure to most PIs, including nelfinavir, is reduced in HIV-1-infected women during pregnancy due to increased intestinal and/or hepatic CYP3A activity. [32
] Pregnant women, as well, have increased nevirapine clearance and lower plasma concentrations than non-pregnant women, although plasma levels are largely influenced by body weight [37
]. Chaix et al[38
] found that selection of nevirapine-resistance strongly correlated with higher median nevirapine plasma concentration. Prolonged nevirapine elimination after delivery in subjects with higher plasma levels could have allowed viral replication in the presence of suboptimal nevirapine levels after delivery.
In concordance with previous studies,[8
] allele-specific PCR increased the frequency of detection of key resistance mutations relative to population sequencing of plasma viruses. Several studies have shown that pre-treatment detection of minority NNRTI-resistant variants more than triples the risk of virological failure to subsequent NNRTI-based therapy.[17
] Therefore, the results of this study have important clinical implications for women receiving PLAT during pregnancy. It is well established that women selecting lamivudine, emtricitabine or NNRTI-resistant mutants during PLAT are at a higher risk of failing subsequent NNRTI-based antiretroviral therapy, particularly if treatment is started within 6 to 12 months after delivery.[18
Whereas ASPCR is several orders of magnitude more sensitive than viral population sequencing, it has a number of limitations that currently prevent its routine clinical application, i.e.: a) ASPCR interrogates only one codon per experiment; b) proportion measurements might be biased in the presence of polymorphisms at primer sites, and c) minority variant cut-offs that predict antiretroviral therapy outcomes with high sensitivity an specificity have not been established.
In spite of the limited numbers in some of the categories assessed and the limited pre-treatment resistance data available, our findings suggest that triple-drug therapy should be the preferred MTCT prevention approach to preserve future treatment options for mothers. When possible, antiretroviral regimens to prevent MTCT should include drugs with high genetic barrier. Nelfinavir-based therapy is no longer a preferred regimen for MTCT,[41
] but the findings of this study likely apply to other PIs.[27
] In women treated with nevirapine-based regimens the optimal timing of nevirapine interruption and length of continuation of other concomitant agents merits further inquiry to avoid active viral replication in the presence of suboptimal nevirapine levels. All efforts should be undertaken to ensure optimal adherence to antiretroviral therapy during pregnancy. Lastly, given that resistance mutations selected during pregnancy will wane after PLAT interruption, performing postpartum genotypic resistance testing within 1 to 2 months after delivery would be highly informative for designing future treatment regimens for women exposed to PLAT and may be useful in guiding the choice of antiretroviral regimen postpartum.