Several other studies have evaluated antiretroviral drug resistance in HIV-infected African children on HAART.15–18
Those studies include children on a variety of antiretroviral regimens (e.g
., two NRTIs with either one PI or one NNRTI). Because of the differences in design among the studies, it is difficult to compare the risk of resistance in the different cohorts. In general, NRTI and NNRTI resistance were commonly detected among children with suboptimal viral suppression.
By using the ViroSeq system, we detected NVP resistance before treatment in only one of 74 children, 35 of whom had a history of prior sdNVP exposure. That child did not achieve viral suppression on the NVP-containing treatment regimen. With the LigAmp assay, we detected Y181C in five children, one of whom did not have a history of prior sdNVP exposure; we do not know if detection of Y181C in that child reflects an inaccurate NVP exposure history, or natural occurrence of Y181C as a minority variant in the viral population. Our validation studies of the LigAmp assay included comparison of LigAmp results with other methods for minority variants analysis, including phenotypic selection in a yeast system,19
allele-specific PCR (ASPCR),20
and characterization of cloned variants. We have not detected false-positive results with LigAmp. In contrast to LigAmp, false-positive results are not uncommon with ASPCR. False-positive results in ASPCR usually reflect mismatches at binding sites in the control (nonmutant) reaction; this leads to an underestimation of wild-type HIV, with a consequent overestimation of mutant HIV. A major difference between LigAmp and ASPCR is that in LigAmp, mutant DNA is directly quantified in a fixed amount of amplified DNA. Mutant data are not normalized to wild-type data, as in ASPCR. In LigAmp, mismatches at oligonucleotide-binding sites can lead to underestimation of mutant DNA, but do not lead to false-positive results.20
For these reasons, although we cannot rule out the possibility of a false-positive result, we believe that this is unlikely to be the explanation for mutation detection in these children. A trend was noted toward detection of Y181C before treatment in children who did not achieve viral suppression on HAART, but the association was not statistically significant.
In our cohort, all 12 of the children who had a viral load >1,000 copies/ml at 48 weeks and had a genotyping result had both NNRTI and NRTI resistance. Continuing treatment for an additional 48 weeks (to week 96) led to enhanced resistance to NRTIs, reflecting selection of HIV with thymidine analogue mutations. With this combination of NNRTI and NRTI resistance, children remain susceptible to only the protease inhibitors in the currently recommended second-line regimen for children (ABC+ddI+lopinavir/ritonavir). In this cohort, immunologic and virologic treatment responses were similar in sdNVP-exposed versus
In this study, children who were not virally suppressed developed NNRTI resistance, regardless of prior sdNVP exposure.
These findings demonstrate that resistance emerges frequently in children receiving HAART who are not virally suppressed, even if they are clinically and immunologically stable. Use of viral load monitoring should be considered if feasible to reduce the emergence of multidrug resistance in children undergoing antiretroviral treatment. This is particularly important in resource-limited settings where the pediatric formulations of antiretroviral drugs are limited.