In this study, we investigated the emergence of drug resistance among HIV-infected breastfeeding infants and found that two-thirds of infants who became infected before 6 mo developed resistance to one or more antiretroviral drugs, most likely because of exposure to the drugs through the mother's breast milk. Previous studies have shown that taking antiretroviral therapy while breastfeeding significantly reduces the chances that HIV-infected mothers will transmit the virus to their infants 
. However, the infants who get infected (and not put on antiretroviral therapy soon enough or left untreated) may be at risk of developing resistance to some of the antiretroviral drugs the mother is taking. The patterns of HIV viral load in infants and the timing of detection of HIV drug resistance mutations suggests that drug-resistant HIV variants were likely not transmitted. As previously reported by Mirochnick and colleagues 
, 3TC and NVP given to mothers are transmitted to their infants via breastfeeding in quantities sufficient to have biologic, but suboptimal, effect on HIV with potential risk of emergence of HIV drug resistance 
. Thus, the HIV drug resistance mutations reported here likely emerged as a result of exposure to antiretroviral drugs ingested by the infant either through single-dose NVP exposure at delivery or indirectly through ingestion of antiretroviral drugs through breast milk. This possibility has also been suggested by the findings of the 6-wk extended NVP study (SWEN) in Uganda, which found the presence of NRTI resistance in infants provided with NVP prophylaxis while the mothers were on a triple-antiretroviral regimen 
Fourteen infants were first HIV-PCR positive by week 2. Only eight of the 14, however, had detectable viral load and none of these had HIV drug resistance mutations detected at the time of diagnosis of HIV infection. The frequency of HIV drug resistance mutations increased over time; 30% of HIV-positive infants developed drug resistance mutations by week 6, 63% by week 14, and 67% by 6 mo. With only one mother–infant pair having similar HIV drug resistance patterns and no cases of HIV drug resistance mutations among the infants first HIV-PCR positive after the mothers discontinued antiretrovirals, there is little evidence to suggest that mothers transmitted resistant HIV to their infants. With single-dose NVP, Eshleman and colleagues 
reported a 46% rate of NVP-resistant mutations at 6 wk, and 55% by 12–14 wk, with subsequent waning by 12 mo using standard ViroSeq genotyping techniques. In the present study, where mothers received triple-antiretroviral regimen through 6 mo of breastfeeding and infants chronically ingested suboptimal quantities of antiretroviral drugs via breast milk, there were increased rates of HIV drug resistance mutations in infants at 14 and 24 wk compared to 6 wk, with no evidence of fading of HIV drug resistant mutations. It is most likely that the resistance was maintained over time in the HIV-infected infants because of ongoing exposure to low quantities of antiretroviral drugs in the mother's breast milk.
Our data illustrate a possible relationship between higher viral load in the HIV-infected infants and emergence of drug resistance mutations. The viral load at birth was likely partially suppressed owing to the maternal antiretroviral therapy transferred through the placenta 
. The decrease in viral load from delivery to 2 wk is presumably due to the single-dose NVP given to all infants at birth and the sharp increase in viral load from 2 wk to 6 wk coincides with the emergence of HIV drug resistance mutations from 6 wk to 6 mo ().
Kenyan National guidelines and WHO guidelines for early infant diagnosis recommend testing for HIV at 6 wk 
. With wide-scale implementation of early infant HIV diagnosis, breastfeeding HIV-infected infants could be started on antiretrovirals different from the maternal antiretrovirals to reduce risk of development of HIV drug resistance and, therefore, lower morbidity and mortality among the HIV-infected infants 
. It is possible that infection in the first few weeks (not detected by DNA PCR) could still be affected by drug pressure from the infant NVP dose, which could contribute to the observed NVP resistance at 6 wk.
One major limitation in this study was that HIV drug resistance genotyping was performed on maternal plasma samples and not on breast milk, which is a relatively secluded biologic compartment with differences in drug concentrations and may possibly have had drug resistance HIV variants that could be passed to the infants; however, genotypic results from the majority of the infants at the first time of diagnosis revealed the absence of resistant strains. We previously showed that drug concentrations in breast milk and bloodstream differ, and that biologically significant, but suboptimal, amounts of antiretrovirals for treatment of HIV infection could be passed to the infant via breastfeeding 
. Breast milk concentrations of NVP were ~70% of maternal plasma levels and in the infants were below the accepted target trough concentration for treatment of HIV. 3TC concentrations were ~2.5-fold higher in the breast milk than maternal plasma, but below optimal plasma concentrations in the infant. The high proportion of infants developing 3TC resistance mutations in this report is consistent with the sub therapeutic concentrations of 3TC. The relatively low frequency of 3TC resistance among HIV-infected infants exposed to maternal NVP/ZDV/3TC suggests that NVP may be preventing or delaying emergence of 3TC resistance. In contrast, the fact that all HIV-infected infants exposed to maternal NFV/ZDV/3TC developed a 3TC mutation may be explained by possible low levels of NFV in breast milk and consequently low quantities transmitted to the infant 
. The appearance of K65R in one-quarter of the infants with resistance () is of concern, and this might be due to a high level of 3TC in the breast milk 
. A follow-up genotypic analysis on the detection of K65R mutation in breast milk is necessary. ZDV concentrations were very low in breast milk, and, in the infants, were below the limit of quantification most of the time. None of the infants had mutations associated with ZDV resistance, which appears consistent with the low infant plasma ZDV concentrations detected 
All infants in the KiBS trial received single-dose NVP prophylaxis at birth; however, no infants exposed to maternal NFV/ZDV/3TC developed NVP drug resistance mutations. This finding suggests that this maternal regimen provided an effective “tail” with 3TC via breast milk to reduce emergence of NVP drug resistance mutations in the infants during the first few weeks when infant NVP drug levels following single-dose NVP were still present. In contrast, six of 15 infants exposed to both single-dose NVP as well as maternal NVP through breast milk developed at least one NNRTI mutation after 2 wk of age. This finding corroborates other reports that showed that extended infant NVP prophylaxis for PMTCT significantly reduces transmission of HIV to infants 
, but with a high risk of developing resistance to NVP 
. Notably, no infants exposed to maternal NFV developed drug resistance mutations which is consistent with unpublished findings in a subset of women from the KIBS study that NFV and its active metabolite (M8) transfer into breast milk in low concentrations and the resulting concentrations in dried blood spots of their breast-feeding infants for NFV were less than the limit of quantification (LOQ)−30ng/ml, and for M8 <LOQ −32ng/ml [Weidle PJ, personal communication]. In another study, NFV was present in extremely low concentrations in breast milk and M8 was not detected . Others have found no detectable NFV in the plasma of breastfeeding infants whose mothers were taking NFV as part of triple-combination antiretroviral therapy 
. The subtype distribution observed in this study follow a typical pattern of viruses circulating in this region 
. Furthermore, we did not observe preferential selection of resistance in these infants based on subtypes.
The WHO recommends exclusive breastfeeding for HIV-exposed infants in resource-limited settings where acceptable, feasible, affordable, sustainable, and where safe replacement feedings cannot be achieved 
. Owing to this recommendation, there is an urgent need to assess risks and benefits of antiretroviral drugs that are less likely to be transmitted through breast milk, thus reducing risk of exposure of infants to subtherapeutic levels of antiretroviral drugs and hence reducing the risk of development of HIV drug resistance among infants who become HIV infected 
. The differential development of drug resistance mutations in the infant depending on the maternal antiretroviral regimen may have implications for subsequent treatment options. This tradeoff for low maternal-to-child transmission rates with the use of triple-combination maternal antiretroviral therapy, which includes NVP during breastfeeding, may require more complicated treatment regimens (e.g., including a PI) to treat the small number of infants who do become HIV infected in order to reduce development of drug resistance. However, the options for antiretroviral regimens available in most resource-limited settings are quite limited and so may forestall introduction of ideal combinations that address our findings. Because HIV drug resistance mutations in most infants emerge over time, improvements in early infant HIV diagnosis and treatment programs could mitigate the problem 
before drug resistance mutations develop from exposure to maternal antiretrovirals through breastfeeding.
In conclusion, the low mother-to-child HIV transmission rates observed in the KiBS trial support the role of triple-combination maternal antiretroviral therapy as a successful PMTCT intervention among breastfeeding HIV-infected mothers 
. However, the data from this secondary analysis suggest that ingestion of antiretroviral drugs through breast milk may have contributed to the emergence of HIV drug resistance mutations in the infants, as we observed an increasing frequency of infants with HIV drug resistance mutations over the first 6 mo of life when maternal antiretroviral therapy was given during breastfeeding. Infant drug resistance mutation patterns, depending on the maternal regimen, may have implications for subsequent HIV treatment of the small number of infants, exposed to these maternal regimens, who become HIV infected. Maternal antiretroviral regimens used for PMTCT among HIV-infected breastfeeding women should be evaluated to determine evolution of HIV drug resistance mutations among infants who become HIV infected while exposed to these regimens. PMTCT programs providing maternal antiretroviral therapy during breastfeeding and those caring for infants exposed to antiretroviral through breast milk will need to be cognizant of this issue and consider monitoring these infants more closely and tailoring their treatment accordingly.