We present evidence that in NVP-exposed children, the detection of pre-treatment NNRTI resistance in plasma HIV-1 by highly sensitive methods, but not by standard bulk sequencing, may correlate with virologic failure on subsequent NVP-containing ART. This finding is similar to a recent study among adults by Coovadia et al
, which showed that low levels of NVP resistance predicted virologic failure on subsequent NVP-containing ART 11
. In that study, however, the presence of minor resistant variants was not associated with prior sdNVP exposure, and sdNVP exposure was not in and of itself associated with subsequent virologic failure (and no infants/children were followed). This is in contrast to our findings among children: we found that the presence of NNRTI resistance mutations in plasma using ASPCR was associated with prior infant NVP exposure as 12 of the 13 infants (92%) with resistance had been exposed to nevirapine; and that NNRTI resistance trended toward association with virologic failure (RR 2.4, CI 0.96-9.17, p=0.08), although this association failed to reach statistical significance in this small sample set.
The failure to detect resistance by ViroSeq in plasma from 23 infants of the 26 with known exposure to NVP demonstrates the lack of utility of this type of testing pre-ART in the majority of HIV-infected children who were exposed to NVP in the past. The delay in treatment initiation (average time between NVP exposure and ART initiation was 6.5 months in this cohort) allows resistance to fade as wild-type virus will outgrow the less fit resistant virus. Therefore, the most sensitive methods are more likely to detect resistance at the pre-ART time point.
The analysis of DNA from buffy coat samples (archived reservoir) demonstrated a high degree of resistant virus. However, the presence of resistance in the DNA was distributed fairly evenly between infants who did and did not experience virologic failure, which suggests that DNA analysis of infant samples after sdNVP is not predictive of ART success.
This study also supports previously published results among adults suggesting that individuals whose initiation of NVP-containing ART is delayed post-sdNVP exposure may have a greater chance of virologic success (although among patients with indication for treatment soon after sdNVP exposure, non-NVP-based ART is preferable) 17, 18
. Failures occurred more frequently in the NVP-exposed infants when treatment was initiated prior to their 7-month visit. The development of resistance in infants was associated with high viral load and low CD4 counts in the mother, but not associated with either viral load or CD4 count in the infant.
This analysis has several limitations. First, the sample size was small, with only 7 infants with no known NVP exposure; we have limited power to explore the effect of minor variants (and of NVP exposure itself) on subsequent ART response. Furthermore, infants were exposed to NVP by several possible mechanisms (maternal/infant sdNVP; infant sdNVP only; and NVP via breast milk alone). Each NVP exposure group is small in number, further limiting our ability to determine if route or degree of NVP exposure had an impact upon virologic treatment outcome in these infants, or if in fact failure with ART initiation before 7 months is multifactorial and related to difficulties administering medications to these infants as well as resistance. Additionally, the ability to check for NVP resistance at a set time point after NVP exposure was not possible, as earlier samples (several months pre-ART) were not available. It would be interesting to perform longitudinal analysis of minor variant resistance as it fades in the plasma after NVP exposure to determine if there is an obvious threshold of percent mutant virus below which clinical success would be predicted. In addition, the children in this study started ART at a relatively young age (6.5 months); these results may not be applicable to children starting ART at an older age (by which time NVP resistance may have declined further), nor to children exposed to NVP alone (without maternal AZT, which may have decreased the rate and amount of infant NVP resistance).
This study demonstrates that resistance after sdNVP exposure is extensive when highly sensitive assays are used to evaluate clinical samples, and likely to be missed if commercial assays are applied pre-ART. The data using highly sensitive methods are also suggestive that minor plasma resistance mutations after sdNVP may have a negative impact infant virologic treatment outcome on NVP-containing ART. These findings accentuate the importance of implementing MTCT preventions that minimize the development of antiretroviral drug resistance whenever possible; and of making more effective treatment regimens (e.g. protease inhibitor-containing regimens19
) available to NVP-exposed, HIV-1-infected infants globally.