Although studies have shown that taking antiretroviral therapy (ART) while breastfeeding significantly reduces the chances that HIV-infected mothers will transmit the virus to their infants, the babies who are infected (and not put on ART soon enough or left untreated) may be at risk of developing resistance to some of the antiretroviral drugs the mother is taking, according to a late-breaker presentation Tuesday at the Fifteenth Conference on Retroviruses and Opportunistic Infections in Boston.
This was most likely because of exposure to the drugs through the mother’s breast milk, according to Dr Clement Zeh of the Kenyan Medical Research Institute and US Centers for Disease Control, who presented the resistance data from the Kisumu Breastfeeding Study. “When evaluating PMTCT regimens, it is important to determine the evolution of drug resistance among the infants,” he recommended.
However, these findings must also be put in the context of other evidence showing that exclusive breastfeeding offers HIV-exposed infants the best chances of survival in resource limited settings where replacement feeding options are not accessible, feasible, affordable, sustainable and safe (AFASS), and that breastfeeding mothers with HIV in need of treatment for their own health should be taking ART.
The Kisumu Breastfeeding Study
The Kisumu Breastfeeding Study is an open label study assessing the safety and efficacy of zidovudine (AZT), lamivudine (3TC), and either nevirapine (NVP) or nelfinavir (NFV) from 34 weeks' gestation through 24 weeks' postpartum for prevention of mother-to-child transmission (PMTCT) among HIV-infected breastfeeding mothers. Findings from the study describing very low rates of HIV transmission were also reported this week at the conference.
A few key points are important for the following discussion. Infants were tested for HIV via dried blood spots (DBS) collected within the first week of delivery, and again at weeks 2, 6, 14, and at months six, nine, twelve, eighteen, and twenty-four. However, the first real time test occurred at week 14, and if positive, stored DBS were retrospectively tested to determine the time of first positive sample, and viral load and resistance analyses (using the ViroSeq HIV-1 genotyping system) were also performed on the samples from both the HIV positive infants and their mothers.
Overall, out of 502 live-born babies, 24 infants were infected by six months (while mothers were on ART) and 5 infections occurred afterwards (indicating that these mothers were still breastfeeding).
Twelve of the infants were infected at the time of first sampling, and two more by week 2, indicating those infants who were most likely infect at birth or during delivery — in other words, half of the 24.
No evidence of resistance could be detected in eight of 12 tested at the time of the 0-2 week samples. Later, however, there was an increasing frequency of detectable resistance over time: in six out of 20 of the HIV positive infants at week 6, 14 out of 22 by week 14, and 16 out of 24 by time of the six-month sample. Yet, none of the infants who became infected after mothers discontinued ART showed evidence of resistance.
There were possible differences in the likelihood of positive babies becoming resistant depending upon the regimen the mother was taking.
Fourteen of the 287 infants were exposed to maternal AZT, 3TC and nevirapine were PCR positive at 6 months. Six of these 14 developed resistance.
Ten out of 200 infants whose mother had CD4 cell counts over 250 and so were exposed to maternal AZT, 3TC and nelfinavir were PCR positive by 6 months — all of these developed drug resistance.
However the patterns of resistance were different in ways that would have implications for the infant’s future treatment. All six of those exposed to nevirapine-based ART developed at least one mutation conferring resistance to the non-nucleoside analogue reverse transcriptase inhibitor class (which includes nevirapine and efavirenz), 4 developed the M184V resistance mutation to 3TC, and 1 the D67DN mutation to AZT.
Among the ten exposed to nelfinavir, all were resistant to 3TC, and two also had AZT-related mutations. However, no resistance could be detected to protease inhibitors or to NNRTIs (the babies had been given a single dose of nevirapine at birth).
How did the resistance occur?
The evidence Dr Zeh presented suggests that most of the resistance evolved in the infected infants due to exposure of sub-optimal levels of antiretrovirals via the mother’s breast milk.
First, there was very little evidence to suggest that mothers transmitted resistant virus to the infant. Among the 24 mothers whose infants were infected at six months, 12 had undetectable viral load at the end of the six months post-partum; seven with detectable viral loads had no resistance while five developed drug resistance.
Of the five mothers with drug resistance, two of the infants had no mutations, two mothers and infants had discordant resistance mutation patterns and only one had overlapping mutations perhaps suggesting possible transmitted resistance.
Of note, the resistance analysis was only performed on blood samples from the mother — analysis was not performed on breastmilk, which is a protected compartment of the body where there can be both different levels of drugs (see below) and different patterns of resistance.
The resistance was not due to direct treatment of the infants with ART because only two were on ART by month 24, and stored samples showed that they were already resistant before ART was begun.
As alluded to earlier however, it is most likely that resistance evolved independently over time in the infected children because of exposure to antiretroviral drugs in the mother’s breastmilk.
Last year, researchers from the same team presented pharmacokinetic data showing that antiretroviral levels in breastmilk can be much different than in the bloodstream, and that biologically significant but potentially sub-therapeutic levels could be transferred to the infant.
In that analysis, breastmilk concentrations of nevirapine were about 70% of normal plasma levels. In most of the breastfed infants, the nevirapine concentrations were below therapeutic targets (though some infants were found to have plasma concentrations well in excess of adult target concentrations). 3TC concentrations on the other hand were extremely high in the breastmilk — but below optimal plasma concentrations in the infant. Finally, AZT concentrations were extremely low in the breastmilk and infant concentrations were almost always below the limit of quantification (Mirochnick). At the same conference, a team from Mozambique reported similar findings.
Thus, infants who were infected (14 out of 24 of whom were infected either in utero or during delivery, and 12 of these became resistant) were probably indirectly exposed to a sub-optimal ART regimen through the breastmilk.
The most probable reason no protease inhibitor mutations were observed is that nelfinavir does not achieve high concentration in breastmilk — which, in fact, has previously been reported (Colebunders).
These findings add another piece to the puzzle about how to make breastfeeding safer for HIV-exposed infants, along with other reports at the conference which showed that extended nevirapine prophylaxis for the infant could reduce transmission (although again with a risk of resistance).
“There’s also the need to assess risk factors and benefits of antiretrovirals that are less likely to be transmitted through breast milk,” said Dr Zeh. “And differing HIV resistance patterns - depending on the maternal regimen - may have implications for subsequent treatment of infants who become HIV-infected."
However, the practical options for antiretroviral regimens in most resource limited settings are quite limited — and it will not be easy to improve upon these in the near future.
Another point to reiterate is that resistance took time to evolve in most infants. So one possible inference would be that these findings strengthen the case to make an early diagnosis in HIV-infected infants— and to make certain that those who test positive go onto ART as soon as possible (and before resistance has a chance to evolve).
Early diagnosis of HIV was the topic of the most recent issue of HIV and AIDS Treatment in Practice.
Zeh C et al. Emergence of HIV-1 drug resistance among breastfeeding infants born to HIV-infected mothers taking antiretrovirals for prevention of mother-to-child transmission of HIV: the Kisumu Breastfeeding Study, Kenya. 15th Conference on Retroviruses and Opportunistic Infections, Boston, abstract 84LB, 2008.
Mirochnick M et al. Plasma antiretroviral concentrations in breastfeeding infants whose mothers are receiving HAART. Fourteenth Conference on Retroviruses and Opportunistic Infections, Los Angeles, abstract 72, 2007.
Colebunders, R et al. The effect of highly active antiretroviral treatment on viral load and antiretroviral drug levels in breast milk.AIDS. 19(16):1912-1915, 2005.