ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2009, p. 1170–1176
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 53, No. 3
Antiretroviral Concentrations in Breast-Feeding Infants of Mothers
Receiving Highly Active Antiretroviral Therapy?
Mark Mirochnick,1* Timothy Thomas,2Edmund Capparelli,3Clement Zeh,2Diane Holland,3
Rose Masaba,2Prisca Odhiambo,2Mary Glenn Fowler,4Paul J. Weidle,5
and Michael C. Thigpen5
Boston Medical Center, Boston University School of Medicine, Boston, Massachusetts1; Centers for Disease Control and Prevention/Kenya
Medical Research Institute, Kisumu, Kenya2; University of California, San Diego Pediatric Pharmacology Research Unit, 4094 4th
Avenue, Room 201, San Diego, CA 92103-09793; Makerere University-Johns Hopkins University Research Collaboration,
P.O. Box 23491,Kampala, Uganda4; and Division of HIV/AIDS Prevention, National Center for HIV, Viral Hepatitis,
STD, and TB Prevention, CDC, Mailstop E-45, 1600 Clifton Road, Atlanta, Georgia 303335
Received 20 August 2008/Returned for modification 24 November 2008/Accepted 17 December 2008
There are limited data describing the concentrations of zidovudine, lamivudine, and nevirapine in nursing
infants as a result of transfer via breast milk. The Kisumu Breastfeeding Study is a phase IIb open-label trial
of prenatal, intrapartum, and postpartum maternal treatment with zidovudine, lamivudine, and nevirapine
from 34 weeks of gestation to 6 months postpartum. In a pharmacokinetic substudy, maternal plasma, breast
milk, and infant dried blood spots were collected for drug assay on the day of delivery and at 2, 6, 14, and 24
weeks after delivery. Sixty-seven mother-infant pairs were enrolled. The median concentrations in breast milk
of zidovudine, lamivudine, and nevirapine during the study period were 14 ng/ml, 1,214 ng/ml, and 4,546 ng/ml,
respectively. Zidovudine was not detectable in any infant plasma samples obtained after the day of delivery,
while the median concentrations in infant plasma samples from postpartum weeks 2, 6, and 14 were 67 ng/ml,
32 ng/ml, and 24 ng/ml for lamivudine and 987 ng/ml, 1,032 ng/ml, and 734 ng/ml for nevirapine, respectively.
Therefore, lamivudine and nevirapine, but not zidovudine, are transferred to infants via breast milk in
biologically significant concentrations. The extent and effect of infant drug exposure via breast milk must be
well understood in order to evaluate the benefits and risks of maternal antiretroviral use during lactation.
The administration of antiretroviral agents during preg-
nancy and around delivery has been shown to be effective in
reducing mother-to-child transmission of human immunodefi-
ciency virus (HIV) (6). However, transmission of HIV from
mother to child after birth via breast milk remains a major
problem in areas of the world where formula feeding is not
safe, affordable, or practical (5, 23). The extension of maternal
highly active antiretroviral therapy (HAART) through the pe-
riod of breast feeding has been proposed as one strategy to
reduce breast milk HIV transmission by reducing plasma and
breast milk HIV concentrations and/or by providing prophy-
laxis to the infant through ingestion of antiretrovirals present
in breast milk (33). Previous human studies have shown that
antiretrovirals administered to nursing mothers are present in
their breast milk, but the extent of antiretroviral transfer from
mother to infant via breast milk and the resulting infant anti-
retroviral drug exposure have not been well delineated (4, 7,
19, 21, 26, 27). The aim of this study was to describe antiret-
roviral concentrations in maternal plasma, breast milk, and
infant dried blood spots during the administration of combi-
nation antiretroviral therapy to nursing mothers during the
first 6 months after delivery.
(This study was presented in part at the 14th Conference on
Retroviruses and Opportunistic Infections, Los Angeles, CA,
27 February 2007.)
MATERIALS AND METHODS
The Kisumu Breastfeeding Study is a phase IIb open-label clinical trial in
Kisumu, Kenya, sponsored by the Centers for Disease Control and Prevention
(CDC), the Kenya Medical Research Institute (KEMRI), and the Kenya Min-
istry of Health (31). Pregnant HIV-infected women enrolled in the trial received
HAART with lamivudine and zidovudine plus either nevirapine or nelfinavir for
prevention of mother-to-child HIV transmission starting at 34 to 36 weeks of
gestation, continuing through labor and delivery, and for 6 months postpartum.
This analysis presents data on women who received nevirapine-based HAART,
consisting of lamivudine and zidovudine administered as a fixed-dose combina-
tion (Combivir) in 1 tablet twice a day and nevirapine (Virammune) at 200 mg
once daily for 14 days, followed by 200 mg twice a day. Infants received single
doses of 2 mg/kg of body weight of nevirapine within 72 h of birth. Mothers were
instructed to exclusively breast feed their infants and then to start weaning at 51⁄2
months postpartum. Maternal plasma, breast milk, and infant dried blood spots
were collected from a nonrandom subset of sequentially enrolled subjects par-
ticipating in a breast milk substudy. Informed consent for participation in the
main study and for participation in the substudy was obtained from all substudy
mothers. The protocol was approved by the CDC, KEMRI, Boston University
Medical Center, and University of California at San Diego human study com-
Maternal plasma and breast milk samples and infant dried blood spots were
collected within 24 h of delivery and at four postpartum study visits (2, 6, 14, and
24 weeks after delivery). Dried blood spot samples were collected based on
procedures described by Mei et al. (15) All infant single nevirapine doses were
administered after the day-of-delivery sample had been collected. All study
nevirapine was dispensed in pill bottles with Medication Event Monitoring Sys-
tem caps (Aardex, Ltd., Union City, CA), which were used to determine the
dosing times for the last three maternal antiretroviral doses prior to sampling;
timing was confirmed by pharmacy staff for both nevirapine and zidovudine/
lamivudine through interview of the women. The three most-recent infant feed-
* Corresponding author. Mailing address: Boston Medical Center,
850 Harrison Avenue, Yawkey 2N-06, Boston, MA 02118. Phone:
(617) 414-3754. Fax: (617) 414-7297. E-mail: email@example.com.
?Published ahead of print on 29 December 2008.
ings were also recorded during the same interview. Maternal and infant clinical
and demographic characteristics were recorded in the main study database.
Drug assays from breast milk and maternal plasma were performed as previ-
ously described (27). Zidovudine concentrations in maternal plasma, breast milk,
and infant dried blood spots were determined by enzyme immunoassay (EIA) or
radioimmunoassay. The initial samples were assayed for zidovudine by EIA until
part way through the study, when EIA plates became unavailable, and subse-
quent samples were assayed by radioimmunoassay. Cross-validation was done to
establish the equivalency of the two assays, and proficiency testing samples
(plasma from AIDS Clinical Trials Group [ACTG] and dried blood spots from
CDC) were run for both assays. For dried blood spots, the zidovudine immuno-
assay methods were a modification of the method described by Mei et al. (16).
The lower limits of quantitation for zidovudine in plasma, breast milk, and dried
blood spots were 15, 5, and 30 ng/ml, respectively.
Liquid chromatography-mass spectroscopy (LC-MS) was used to measure
lamivudine and nevirapine in maternal plasma and dried blood spots. Due to
limitations in the availability of LC-MS equipment, LC-MS or high-performance
LC was used to measure lamivudine and nevirapine in breast milk samples. The
lower limits of quantitation for plasma, breast milk, and dried blood spots by
LC-MS were 7, 17, and 16 ng/ml for lamivudine and 40, 17 and 43 ng/ml for
nevirapine, respectively. The lower limit of quantitation for both lamivudine and
nevirapine in breast milk by high-performance LC was 34.4 ng/ml. Assay profi-
ciency was demonstrated by using samples provided by the ACTG for plasma and
breast milk assays and the CDC for dried blood spot assays (10).
Descriptive statistics were calculated for demographic and clinical data for
mothers and infants. Antiretroviral concentration and milk-to-plasma ratio data
are presented as medians and interquartile ranges (IQR). Since maternal
plasma, breast milk, and infant dried blood spot antiretroviral concentrations
from different sampling days were collected at different times after the admin-
istration of maternal doses, they were treated independently for analysis and
were compared using the Kruskal-Wallis test, which assumes independent sam-
ples. Linear mixed-effects regression analysis was used to analyze changes in
maternal plasma and breast milk concentrations over a dosing interval. Calcu-
lations were performed by using Microsoft Excel 2003 (Microsoft, Redmond,
WA) and SAS version 9.1 (SAS Institute, Cary, NC). Infant daily doses from
breast milk were estimated assuming a daily breast milk intake of 150 ml/kg/day
(2, 12). Observed infant nevirapine concentrations were compared with those
predicted using the estimated daily nevirapine dose and a previously developed
population model of nevirapine pharmacokinetics in infants during the first year
of life (17).
Maternal plasma, breast milk, and infant dried blood spots
were collected from 67 HIV-infected mothers and their in-
fants. The median maternal age was 24.5 years (range, 18.1 to
37.0 years), and the median maternal weight was 57.5 kg
(range, 44.3 to 73.1 kg). There were 28 female infants and 39
male infants. The median infant birth weight was 3,000 g
(range, 2,200 to 4,000 g).
The dried blood spot concentrations for each antiretroviral
at each study visit are presented numerically in Table 1 and
graphically in Fig. 1. Maternal plasma and breast milk concen-
trations are presented numerically in Table 2 and graphically in
Fig. 2 and 3. Zidovudine concentrations were assayed in non-
randomly selected groups of 82 infant dried blood spots, 45
TABLE 1. Zidovudine, lamivudine, and nevirapine concentrations and IQRs in dried blood spots from infants in the Kisumu Breastfeeding
Study, Kenya, 2004 to 2007a
Median concn (ng/ml) (IQR) ?no. of samples? at:
DeliveryWk 2 Wk 6Wk 14 Wk 24
24 (BQL-76) ?16?
67† (48–168) ?22?
2,963† (1,726–5,082) ?22?
BQL (BQL-BQL) ?24?
32*† (23–44) ?39?
987*† (790–1,269) ?39?
BQL (BQL-BQL) ?22?
24*† (16–40) ?39?
1,032*† (811–1,375) ?39?
BQL (BQL-BQL) ?13?
20*† (15–29) ?34?
734*† (560–1,017) ?34?
BQL (BQL-BQL) ?7?
BQL* (BQL-BQL) ?19?
303* (BQL-444) ?19?
aFor comparative purposes, the IC50for wild-type HIV-1, subtype B, for lamivudine is ?0.6 to 21 ng/ml and for nevirapine is ?17 ng/ml. ?, P ? 0.05 compared to
results for day of delivery sample; †, P ? 0.05 compared to results for 24-week sample.
FIG. 1. Infant concentrations (conc) of zidovudine (ZDV), lamivu-
dine (3TC), and nevirapine (NVP) on day of delivery (DEL) and at 2,
6, 14, and 24 weeks postpartum, Kisumu Breastfeeding Study, Kenya,
2004 to 2007. For comparative purposes, the IC50for wild-type HIV
type 1, subtype B, is ?0.6 to 21 ng/ml for lamivudine and ?17 ng/ml for
VOL. 53, 2009INFANT ANTIRETROVIRAL CONCENTRATIONS FROM BREAST MILK1171
maternal plasma samples, and 35 breast milk samples. Sixteen
dried blood spots obtained on the day of delivery were assayed,
and eight had quantifiable amounts of zidovudine, with a me-
dian zidovudine concentration of 24 ng/ml (IQR, below quan-
tifiable limit [BQL] to 76 ng/ml). The zidovudine concentra-
tions in the remaining 66 infant dried blood spots, collected at
the 2-, 6-, 14-, and 24-week time points were all below the assay
limit of detection. Due to the preponderance of dried blood
spot samples with zidovudine concentrations below the assay
limit of quantification, no further specimens were assayed for
Lamivudine and nevirapine assay data for an individual visit
were included in the final data set if the samples from all three
matrices (maternal plasma, breast milk, and infant dried blood
spots) were adequate for assay and if the maternal nevirapine
and lamivudine concentrations were detectable, indicating that
the mother had been adherent with the antiretroviral regimen.
A total of 153 visits from 58 (of 67 enrolled) mother-infant
pairs met these criteria. The median infant dried blood spot
lamivudine concentration was higher on the day of delivery (67
ng/ml) than at week 2 (32 ng/ml), week 6 (24 ng/ml), week 14
(20 ng/ml), or week 24 (BQL). The median infant dried blood
spot nevirapine concentration was higher on the day of delivery
(2,963 ng/ml) than at week 2 (987 ng/ml), week 6 (1,032 ng/ml),
week 14 (734 ng/ml), or week 24 (303 ng/ml).
FIG. 2. Maternal plasma and breast milk concentrations (conc) of
zidovudine (ZDV), lamivudine (3TC), and nevirapine (NVP) plotted
against time since last maternal dose, Kisumu Breastfeeding Study,
Kenya, 2004 to 2007. Dashed lines are regression lines for maternal
plasma data, and solid lines are regression lines for breast milk data.
FIG. 3. Zidovudine (ZDV), lamivudine (3TC), and nevirapine
(NVP) breast milk (BM)-to-maternal plasma ratio plotted against time
since last maternal dose, Kisumu Breastfeeding Study, 2004 to 2007.
The filled circles represent samples with zidovudine breast milk con-
centrations below the limit of detection and represent the highest
possible breast milk/plasma ratio. The P values represent the proba-
bility that the slope of the line is different than zero as calculated by
linear mixed-effects regression analysis.
1172 MIROCHNICK ET AL.ANTIMICROB. AGENTS CHEMOTHER.
There were no differences across study visits for median
maternal plasma or breast milk concentrations of any of the
antiretrovirals. The median maternal plasma zidovudine con-
centration across all study visits was 23 ng/ml (IQR, 12 to 59
ng/ml). The median breast milk zidovudine concentration was
9 ng/ml (IQR, BQL to 26 ng/ml), and the median ratio of the
breast milk-to-maternal plasma zidovudine concentrations was
0.44 (IQR, 0.23 to 0.65). The median estimated infant daily
dose of zidovudine (based on an estimated daily breast milk
intake of 150 ml/kg/day) was 1.35 ?g/kg.
The median maternal plasma lamivudine concentration was
508 ng/ml (IQR, 290 to 800 ng/ml). The median breast milk
lamivudine concentration was 1,214 ng/ml (IQR, 862 to 1,651),
and the median ratio of the breast milk-to-maternal plasma
lamivudine concentrations was 2.56 (IQR, 1.79 to 3.89). Over
the course of the dosing interval, the rate of decline of the
lamivudine concentration in breast milk was slower than in
maternal plasma, resulting in an increasing lamivudine breast
milk-to-plasma ratio (Fig. 2 and 3). Assuming a daily breast
milk intake of 150 ml/kg/day, the median estimated infant daily
dose of lamivudine was 182 ?g/kg.
The median maternal plasma nevirapine concentration was
6,087 ng/ml (IQR, 4,895 to 7,518 ng/ml). The median breast
milk nevirapine concentration was 4,546 ng/ml (IQR, 3,480 to
5,715 ng/ml), and the median ratio of the breast milk-to-ma-
ternal plasma nevirapine concentrations was 0.75 (IQR, 0.64 to
0.89). Assuming a daily breast milk intake of 150 ml/kg/day, the
median estimated infant daily dose of nevirapine was 682 ?g/
kg. The distribution of the number of days required to receive
a cumulative 2-mg/kg dose of nevirapine, the infant prophy-
lactic dose administered as part of the single-dose nevirapine
regimen, is presented in Fig. 4 (9). The observed infant nevi-
rapine concentrations are plotted against those predicted using
our estimates of daily infant nevirapine doses and our previous
infant pharmacokinetic model in Fig. 5 (17).
Our data confirm that the antiretroviral drugs zidovudine,
lamivudine, and nevirapine administered to pregnant and nurs-
ing women can be measured in the infants’ plasma shortly after
birth and in breast milk expressed while nursing. Once the
immediate newborn period has passed, lamivudine and nevi-
rapine remain present in biologically significant concentrations
in the infants’ plasma, while zidovudine is not detected above
the lower limit of quantitation of the assay used. Three previ-
ous studies have described concentrations of zidovudine, lami-
vudine, and nevirapine associated with the administration of
HAART to breast-feeding mothers (5, 8, 27). Only one of
these studies measured infant antiretroviral concentrations,
and in that study, infant treatment with prophylactic daily
zidovudine dosing prevented an evaluation of zidovudine ex-
posure due to breast milk alone (27). The data reported here
are the first from a cohort of women receiving nevirapine-
based HAART and their infants studied on the day of delivery,
as well as at several subsequent times over the first 6 months of
Our data demonstrate that the magnitude of infant drug
concentrations from exposure to maternally administered drug
differs for each individual antiretroviral agent studied, as well
as with the time postpartum. For all antiretrovirals, infant
FIG. 4. Proportion of breast-feeding infants receiving a cumulative
breast milk nevirapine dose of 2 mg/kg plotted against number of days
of breast feeding (estimated from day-of-delivery breast milk nevirap-
ine concentrations), Kisumu Breastfeeding Study, Kenya, 2004 to 2007.
TABLE 2. Zidovudine, lamivudine, and nevirapine concentrations in maternal plasma and breast milk and breast milk-to-plasma ratios in the
Kisumu Breastfeeding Study, Kenya, 2004 to 2007
Median concn (ng/ml) (IQR) ?no. of samples? in:
Breast milk/plasma ratio
Maternal plasmaBreast milk
23 (12–59) ?45?
508 (290–800) ?153?
6,087 (4,895–7,518) ?153?
9 (BQL-26) ?35?
1,214 (862–1,651) ?153?
4,546 (3,480–5,715) ?153?
0.44 (0.23-.65) ?31?
2.56 (1.79–3.89) ?153?
0.75 (0.64–0.89) ?153?
FIG. 5. Observed and model-predicted infant dried blood spot
(DBS) nevirapine (NVP) concentrations (conc) at weeks 2, 6, and 14
postpartum, Kisumu Breastfeeding Study, Kenya, 2004 to 2007.
VOL. 53, 2009 INFANT ANTIRETROVIRAL CONCENTRATIONS FROM BREAST MILK1173
concentrations were highest on the day of delivery compared
to the other postnatal sampling times, consistent with trans-
placental passage of the antiretrovirals from mother to fetus
prior to delivery. The infant concentrations of zidovudine,
lamivudine, and nevirapine at delivery observed in this study
are consistent in magnitude with those seen in other studies
reporting infant antiretroviral concentrations at delivery fol-
lowing chronic maternal dosing during pregnancy and labor
(18, 19, 24). In our current study, infant lamivudine and nevi-
rapine concentrations declined over the study period despite
constant breast milk concentrations, consistent with previously
observed developmental increases in nevirapine and lamivu-
dine clearances over the first 6 months of life (17, 32). The
decreased drug concentrations at the 24-week visit may have
been affected by the initiation of weaning prior to the collec-
tion date, though we do not have precise information on when
weaning began for each woman.
The infant drug concentrations at the week 2 through week
14 postnatal visits reflect drug exposure from breast feeding
during adherent maternal HAART administration and exclu-
sive breast feeding. At these times, maternal plasma zidovu-
dine concentrations were low, reflecting the very short (?1 h)
half-life of zidovudine in adults and the resulting rapid clear-
ance of an administered dose from plasma during a dosing
interval. The median breast milk-to-plasma ratio for zidovu-
dine was around 50%. While the standard daily zidovudine
infant dose used for prevention of mother-to-child transmis-
sion in the first 4 to 6 weeks of life is 8 mg/kg (or 8,000 ?g/kg),
we estimated a median daily infant zidovudine dose from
breast milk of 1.35 ?g/kg, more than 1,000 times lower than the
standard prophylactic dose (3). Given this extremely small
breast milk dose, it is not surprising that zidovudine was not
detectable in any of the infant samples obtained after the day
of delivery. A limitation of our study is that very few of our
samples were obtained within the first 2 h after maternal dos-
ing, when maternal plasma and breast milk zidovudine con-
centrations are highest. We also did not assay for intracellular
concentrations of phosphorylated zidovudine in the infants due
to the large amount of blood required for this assay. The active
form of zidovudine is its intracellular triphosphorylated me-
tabolite, which has a longer half-life than unmetabolized
plasma zidovudine. It is possible that some zidovudine may
reach the infant from breast milk, entering and persisting in-
side the cells while plasma concentrations quickly fall below
the limit of quantitation of our assay, especially if nursing takes
place soon after maternal dosing.
The lamivudine plasma concentrations in the mothers were
consistently higher than those of zidovudine, which is consis-
tent with the longer plasma half-life of 5 to 7 h for lamivudine.
The lamivudine concentrations in breast milk were generally
greater than those in plasma, which is consistent with previous
findings (8, 27). Lamivudine concentrations in breast milk de-
clined more slowly than in plasma, leading to a gradual in-
crease in the lamivudine milk-to-plasma ratio as the dosing
interval progressed. The change in the lamivudine milk-to-
plasma ratio over the course of the dosing interval demon-
strates why breast milk and maternal plasma drug concentra-
tion data must be analyzed taking into account the time since
maternal dosing (34). The estimated infant median daily dose
of lamivudine from breast milk was 182 ?g/kg, which is ap-
proximately 2% of the recommended daily treatment dose of
lamivudine of 8 mg/kg divided into two doses in children over
3 months of age. Exposure to this amount of lamivudine via
breast milk, as measured from weeks 2 to 24, resulted in a
median infant lamivudine concentration of 23 ng/ml, which is
just above the upper limit of the range of the lamivudine 50%
inhibitory concentration (IC50) for wild-type HIV (0.6 to 21
Nevirapine has a long half-life (20 to 30 h) with chronic
dosing in adults. Maternal nevirapine concentrations were in
the range typically seen with chronic nevirapine therapy in
adults (10). The median nevirapine breast milk-to-plasma ratio
was just over 70% and remained constant over the maternal
dosing interval. The median estimated daily dose of nevirapine
administered to the infants from breast milk was just over 600
?g/kg/day. Studies investigating the efficacy of direct adminis-
tration of nevirapine to breast-feeding infants to prevent breast
milk HIV transmission are ongoing (13, 20). The dose used in
these studies is 4 mg/kg (or 4,000 ?g/kg) once a day, and our
estimated breast milk dose is about 15% of this dose (29).
Using a previously developed model of nevirapine pharmaco-
kinetics in infants, the predicted nevirapine concentrations
were somewhat lower than those observed in the current study.
This analysis suggests that our estimated dose of presumed
daily breast milk intake derived from U.S. data may be con-
servative, underestimating the actual daily nevirapine dose
from breast milk in these Kenyan infants. During weeks 2, 6,
and 14, the median infant nevirapine concentration was 896.9
ng/ml, well above the median HIV IC50of 17 ng/ml for nevi-
rapine but below the suggested target trough nevirapine con-
centration of 3,000 ng/ml (1; Nevirapine package insert, re-
vised April 2007 [Boehringer Ingelheim]). The maternal
plasma, breast milk, and infant concentrations and the breast
milk/plasma ratio observed for nevirapine in this study are very
similar to those recently reported in a study of efavirenz trans-
fer from breast milk. In that study, the median maternal
plasma efavirenz concentration was 6,030 ng/ml, the median
breast milk efavirenz concentration was 3,450 ng/ml, the mean
efavirenz breast milk/plasma ratio was 0.54, and the median
infant efavirenz concentration was 870 ng/ml, just below the
suggested target trough efavirenz concentration of 1,000 ng/ml
(1, 26). These data suggest that the transfer of either non-
nucleoside reverse transcriptase inhibitor from maternal breast
milk to infants would have similar potential for beneficial or
The infant nevirapine concentrations we observed in the
day-of-delivery samples reflect transplacental passage of nevi-
rapine and were roughly equivalent to those in maternal
plasma at the time of delivery. Based on the range of breast
milk nevirapine concentrations observed in this study, we es-
timate that by day 3 of life, 50% of infants ingested from breast
milk a total nevirapine dose exceeding the 2-mg/kg infant post-
natal prophylactic dose and by day 4 of life, the breast milk
nevirapine dose exceeded 2 mg/kg in over 70% of infants. As a
result of this combined transplacental and breast milk nevirap-
ine exposure over the course of the first week of life, nursing
infants whose mothers received a chronic prenatal and post-
natal nevirapine HAART regimen will have nevirapine con-
centrations that exceed those seen in infants exposed to the
mother-infant single-dose prophylactic perinatal regimen. Ad-
1174MIROCHNICK ET AL.ANTIMICROB. AGENTS CHEMOTHER.
ministration of the single-dose postnatal infant prophylactic
nevirapine may be unnecessary in these infants if the mother is
adherent to combination therapy before and after delivery.
However, there was no evidence that the nevirapine dose given
to the infants in this study was harmful, and programmatically
it may be difficult to determine which mothers are adherent
In countries with high per capita incomes where triple-com-
bination antiretroviral regimens, elective cesarean section, and
safe formula feeding are readily available and acceptable to
pregnant women, rates of mother-to-child transmission as low
as 0.6% have been reported (22). In resource-limited settings
where some or all of these interventions are not available, the
use of shorter, less-intensive antiretroviral regimens has been
shown to result in smaller but still very significant reductions in
antenatal and intrapartum mother-to-child HIV transmission
(7). In resource-limited settings where formula feeding is not
safe or practical and breast feeding substantially improves in-
fant survival, prevention of postnatal HIV transmission via
breast milk remains a major challenge (33). Although exclusive
breast feeding has been shown to be associated with lower
rates of HIV transmission than mixed feeding, the risk of
transmission is not eliminated. Recent studies of the strategy
of exclusive breast feeding for 4 to 6 months followed by early
cessation of breast feeding and the use of replacement feeding
and complementary foods have been associated with increased
risks after weaning of morbidity and mortality from infectious
diseases and malnutrition (30). The development of practical
and effective strategies to allow continued breast feeding while
minimizing the risk of mother-to-child HIV transmission is
One proposed strategy for the prevention of breast milk
HIV transmission is the administration of HAART to nursing
mothers. Treatment of nursing mothers with zidovudine, lami-
vudine, and nevirapine from 28 weeks of gestation through 1
month postpartum has been shown to reduce breast milk HIV
viral RNA loads at delivery and at the end of the first post-
partum week compared to the levels in untreated women,
although breast milk HIV DNA may be not be suppressed (8,
28). Several studies of the efficacy of maternal HAART in
preventing breast milk HIV transmission are under way. How-
ever, animal and human studies have demonstrated that anti-
retroviral agents are transferred into breast milk (5, 19, 21, 25).
Because of the frequency of feeding and the reduced clearance
of antiretrovirals in infants, even low concentrations of anti-
retrovirals in breast milk may result in biologically significant
antiretroviral concentrations in the nursing infant (14). An
understanding of infant drug exposure resulting from antiret-
rovirals received via breast milk is necessary before the use of
antiretrovirals in nursing women becomes widespread.
Our data clearly show that lamivudine and nevirapine, but
not zidovudine, are transmitted in biologically significant con-
centrations via breast milk to nursing infants when their moth-
ers receive these drugs. The resulting infant antiretroviral drug
exposure may have benefits, such as prevention of HIV infec-
tion or partial suppression of HIV replication in infants who
become HIV infected. Alternatively, this drug exposure could
result in potential drug side effects or, in infants who become
HIV infected, the emergence of HIV drug resistance. Recent
data from the parent Kisumu Breastfeeding Study demonstrate
the emergence of HIV type 1 genotypic resistance mutations to
nucleoside reverse transcriptase inhibitors (primarily to lami-
vudine but, to a lesser degree, to zidovudine as well) and
nonnucleoside reverse transcriptase inhibitors among children
who are HIV infected at birth or during the first 6 months
while breast feeding and whose mothers received treatment
with zidovudine, lamivudine, and nevirapine during pregnancy
and while nursing (35). While enhanced prophylactic strategies
such as this are likely to reduce perinatal and postpartum HIV
infections among infants, the extent of infant drug exposure via
breast milk and the effects on infants of this exposure, espe-
cially the emergence of resistance mutations in those infants
who become HIV infected despite maternal treatment, are
important considerations for HIV treatment programs provid-
ing maternal HAART during breast feeding for prevention of
mother-to-child transmission and for pediatric treatment pro-
grams that treat HIV-infected infants who have been exposed
to these drugs via breast feeding.
We thank the Kisumu Breastfeeding Study participants, as well as
the rest of the Kisumu Breastfeeding Study Team, the Kenya Medical
Research Institute (KEMRI), and the KEMRI/CDC Field Station. We
also thank Glaxo-Smith-Kline and Boehringer Ingelheim for providing
the study medications.
Funding was provided by the U.S. Centers for Disease Control and
Prevention (CDC) and grants from the National Institute of Child
Health and Human Development, National Institutes of Health
(1R21HD051470-01 and 5U10 HD031318-14). CDC staff participated
in the design, data collection, analysis, and interpretation of the data;
the writing of the report; and the decision to submit the paper for
M.M., T.T., E.C., P.J.W., and M.C.T. participated in the conception,
design, conduct, and data analysis of the project and the writing of the
manuscript; C.Z., R.M., and P.O. participated in the design and con-
duct of the project and the writing of the manuscript; D.H. participated
in the conduct and data analysis of the project and the writing of the
manuscript, and M.G.F. participated in the conception and design of
the project and the writing of the manuscript. The authors declare no
conflicts of interest.
The findings and conclusions in this article are those of the authors
and do not necessarily represent the views of the U.S. Centers for
Disease Control and Prevention. Use of trade names is for identifica-
tion purposes only and does not constitute endorsement by the U.S.
Centers for Disease Control and Prevention or the Department of
Health and Human Services.
The protocol for use of human subjects was approved by the Insti-
tutional Review Boards of the Kenya Medical Research Institute, U.S.
Centers for Disease Control and Prevention, Boston University Med-
ical Center, and University of California at San Diego.
1. Back, D., T. Blaschke, C. Boucher, et al. 2006. Optimizing TDM in HIV
clinical care. http://www.hivpharmacology.com.
2. Bailey, B., and S. Ito. 1997. Breast-feeding and maternal drug use. Pediatr.
Clin. N. Am. 44:41–54.
3. Boucher, F. D., J. F. Modlin, S. Weller, A. Ruff, M. Mirochnick, S. Pelton, C.
Wilfert, R. McKinney, Jr., M. J. Crain, M. M. Elkins, et al. 1993. Phase I
evaluation of zidovudine administered to infants exposed at birth to the
human immunodeficiency virus. J. Pediatr. 122:137–144.
4. Coates, J. A., N. Cammack, H. J. Jenkinson, A. J. Jowett, M. I. Jowett, B. A.
Pearson, C. R. Penn, P. L. Rouse, K. C. Viner, and J. M. Cameron. 1992.
(?)?2?-Deoxy-3?-thiacytidine is a potent, highly selective inhibitor of human
immunodeficiency virus type 1 and type 2 replication in vitro. Antimicrob.
Agents Chemother. 36:733–739.
5. Colebunders, R., B. Hodossy, D. Burger, T. Daems, K. Roelens, M. Coppens,
B. Van Bulck, Y. Jacquemyn, E. Van Wijngaerden, and K. Fransen. 2005.
The effect of highly active antiretroviral treatment on viral load and antiret-
roviral drug levels in breast milk. AIDS 19:1912–1915.
6. Coutsoudis, A., F. Dabis, W. Fawzi, P. Gaillard, G. Haverkamp, D. R. Harris,
VOL. 53, 2009INFANT ANTIRETROVIRAL CONCENTRATIONS FROM BREAST MILK1175
J. B. Jackson, V. Leroy, N. Meda, P. Msellati, M. L. Newell, R. Nsuati, J. S. Download full-text
Read, and S. Wiktor. 2004. Late postnatal transmission of HIV-1 in breast-
fed children: an individual patient data meta-analysis. J. Infect. Dis. 189:
7. De Cock, K. M., M. G. Fowler, E. Mercier, I. de Vincenzi, J. Saba, E. Hoff,
D. J. Alnwick, M. Rogers, and N. Shaffer. 2000. Prevention of mother-to-
child HIV transmission in resource-poor countries: translating research into
policy and practice. JAMA 283:1175–1182.
8. Giuliano, M., G. Guidotti, M. Andreotti, M. F. Pirillo, P. Villani, G. Liotta,
M. C. Marazzi, M. G. Mancini, M. Cusato, P. Germano, S. Loureiro, S.
Ceffa, M. Regazzi, S. Vella, and L. Palombi. 2007. Triple antiretroviral
prophylaxis administered during pregnancy and after delivery significantly
reduces breast milk viral load: a study within the Drug Resource Enhance-
ment Against AIDS and Malnutrition Program. J. Acquir. Immune Defic.
9. Guay, L. A., P. Musoke, T. Fleming, D. Bagenda, M. Allen, C. Nakabiito, J.
Sherman, P. Bakaki, C. Ducar, M. Deseyve, L. Emel, M. Mirochnick, M. G.
Fowler, L. Mofenson, P. Miotti, K. Dransfield, D. Bray, F. Mmiro, and J. B.
Jackson. 1999. Intrapartum and neonatal single-dose nevirapine compared
with zidovudine for prevention of mother-to-child transmission of HIV-1 in
Kampala, Uganda: HIVNET 012 randomised trial. Lancet 354:795–802.
10. Havlir, D., S. H. Cheeseman, M. McLaughlin, R. Murphy, A. Erice, S. A.
Spector, T. C. Greenough, J. L. Sullivan, D. Hall, M. Myers, et al. 1995.
High-dose nevirapine: safety, pharmacokinetics, and antiviral effect in pa-
tients with human immunodeficiency virus infection. J. Infect. Dis. 171:537–
11. Holland, D. T., R. DiFrancesco, J. D. Connor, and G. D. Morse. 2006.
Quality assurance program for pharmacokinetic assay of antiretrovirals:
ACTG proficiency testing for pediatric and adult pharmacology support
laboratories, 2003 to 2004: a requirement for therapeutic drug monitoring.
Ther. Drug Monit. 28:367–374.
12. Kent, J. C., L. Mitoulas, D. B. Cox, R. A. Owens, and R. E. Hartmann. 1999.
Breast volume and milk production during extended lactation in women.
Exp. Physiol. 84:435–447.
13. Kumwenda, N. I., D. R. Hoover, L. M. Mofenson, M. C. Thigpen, G. Kafu-
lafula, Q. Li, L. Mipando, K. Nkanaunena, T. Mebrahtu, M. Bulterys, M. G.
Fowler, and T. E. Taha. 2008. Extended antiretroviral prophylaxis to reduce
breast-milk HIV-1 transmission. N. Engl. J. Med. 359:119–129.
14. McNamara, P. J., and M. Abbassi. 2004. Neonatal exposure to drugs in
breast milk. Pharm. Res. 21:555–566.
15. Mei, J. V., J. R. Alexander, B. W. Adam, and W. H. Hannon. 2001. Use of
filter paper for the collection and analysis of human whole blood specimens.
J. Nutr. 131:1631S–1636S.
16. Mei, J. V., W. H. Hannon, T. L. Dobbs, C. J. Bell, C. Spruill, and M. Gwinn.
1998. Radioimmunoassay for monitoring zidovudine in dried blood spot
specimens. Clin. Chem. 44:281–286.
17. Mirochnick, M., E. Capparelli, K. Nielsen, J. Pilotto, P. Musoke, A. Shetty,
and K. Luzuriaga. 2006. Nevirapine pharmacokinetics during the first year of
life: a population analysis across studies, abstr. E-PAS2006:59:5523.135.
Abstr. Pediatr. Acad. Soc. Meet., San Francisco, CA.
18. Mirochnick, M., S. Siminski, T. Fenton, M. Lugo, and J. L. Sullivan. 2001.
Nevirapine pharmacokinetics in pregnant women and in their infants after in
utero exposure. Pediatr. Infect. Dis. J. 20:803–805.
19. Moodley, J., D. Moodley, K. Pillay, H. Coovadia, J. Saba, R. van Leeuwen, C.
Goodwin, P. R. Harrigan, K. H. Moore, C. Stone, R. Plumb, and M. A.
Johnson. 1998. Pharmacokinetics and antiretroviral activity of lamivudine
alone or when coadministered with zidovudine in human immunodeficiency
virus type 1-infected pregnant women and their offspring. J. Infect. Dis.
20. Moorthy, A., A. Gupta, R. Bhosale, S. Tripathy, J. Sastry, S. Kulkarni, M.
Thakar, R. Bharadwaj, A. Kagal, A. V. Bhore, S. Patil, V. Kulkarni, V.
Venkataramani, U. Balasubramaniam, N. Suryavanshi, C. Ziemniak, N.
Gupte, R. Bollinger, and D. Persaud. 2009. Nevirapine resistance and breast-
milk HIV transmission: effects of single and extended-dose nevirapine pro-
phylaxis in subtype C HIV-infected infants. PLoS ONE 4:e4096.
21. Musoke, P., L. A. Guay, D. Bagenda, M. Mirochnick, C. Nakabiito, T.
Fleming, T. Elliott, S. Horton, K. Dransfield, J. W. Pav, A. Murarka, M.
Allen, M. G. Fowler, L. Mofenson, D. Hom, F. Mmiro, and J. B. Jackson.
1999. A phase I/II study of the safety and pharmacokinetics of nevirapine in
HIV-1-infected pregnant Ugandan women and their neonates (HIVNET
006). AIDS 13:479–486.
22. Naver, L., S. Lindgren, E. Belfrage, K. Gyllensten, K. Lidman, M. Gisslen,
A. Ehrnst, M. Arneborn, and A. B. Bohlin. 2006. Children born to HIV-1-
infected women in Sweden in 1982-2003: trends in epidemiology and vertical
transmission. J. Acquir. Immune Defic. Syndr. 42:484–489.
23. Nduati, R., G. John, D. Mbori-Ngacha, B. Richardson, J. Overbaugh, A.
Mwatha, J. Ndinya-Achola, J. Bwayo, F. E. Onyango, J. Hughes, and J.
Kreiss. 2000. Effect of breastfeeding and formula feeding on transmission of
HIV-1: a randomized clinical trial. JAMA 283:1167–1174.
24. O’Sullivan, M. J., P. J. Boyer, G. B. Scott, W. P. Parks, S. Weller, M. R.
Blum, J. Balsley, Y. J. Bryson, et al. 1993. The pharmacokinetics and safety
of zidovudine in the third trimester of pregnancy for women infected with
human immunodeficiency virus and their infants: phase I acquired immuno-
deficiency syndrome clinical trials group study (protocol 082). Am. J. Obstet
25. Ruprecht, R. M., A. H. Sharpe, R. Jaenisch, and D. Trites. 1990. Analysis of
3?-azido-3?-deoxythymidine levels in tissues and milk by isocratic high-per-
formance liquid chromatography. J. Chromatogr. 528:371–383.
26. Schneider, S., A. Peltier, A. Gras, V. Arendt, C. Karasi-Omes, A. Mu-
jawamariwa, P. C. Ndimubanzi, G. Ndayisaba, and R. Wennig. 2008. Efa-
virenz in human breast milk, mothers’, and newborns’ plasma. J. Acquir.
Immune Defic. Syndr. 48:450–454.
27. Shapiro, R. L., D. T. Holland, E. Capparelli, S. Lockman, I. Thior, C.
Wester, L. Stevens, T. Peter, M. Essex, J. D. Connor, and M. Mirochnick.
2005. Antiretroviral concentrations in breast-feeding infants of women in
Botswana receiving antiretroviral treatment. J. Infect. Dis. 192:720–727.
28. Shapiro, R. L., T. Ndung’u, S. Lockman, L. M. Smeaton, I. Thior, C. Wester,
L. Stevens, G. Sebetso, S. Gaseitsiwe, T. Peter, and M. Essex. 2005. Highly
active antiretroviral therapy started during pregnancy or postpartum sup-
presses HIV-1 RNA, but not DNA, in breast milk. J. Infect. Dis. 192:713–
29. Shetty, A. K., H. M. Coovadia, M. M. Mirochnick, Y. Maldonado, L. M.
Mofenson, S. H. Eshleman, T. Fleming, L. Emel, K. George, D. A. Katzen-
stein, J. Wells, C. C. Maponga, A. Mwatha, S. A. Jones, S. S. Abdool Karim,
and M. T. Bassett. 2003. Safety and trough concentrations of nevirapine
prophylaxis given daily, twice weekly, or weekly in breast-feeding infants
from birth to 6 months. J. Acquir. Immune Defic. Syndr. 34:482–490.
30. Sinkala, M., L. Kuhn, C. Kankasa, P. Kasonde, C. Vwalika, M. Mwiya, N.
Scott, K. Semrau, G. Aldrovani, D. Thea, and Z. E. B. S. Group. 2007. No
benefit of early cessation of breastfeeding at 4 months on HIV-free survival
of infants born to HIV-infected mothers in Zambia: the Zambia Exclusive
Breastfeeding Study, abstr. 74LB. 14th Conf. Retrovir. Opportun. Infect.,
Los Angeles, CA.
31. Thomas, T., R. Masaba, R. Ndivo, C. Zeh, C. Borkowf, M. Thigpen, et al.
2008. PMTCT of HIV-1 among breastfeeding mothers using HAART: the
Kisumu breastfeeding study, Kisumu, Kenya, 2003–2007, abstr. 45aLB. Ab-
str. 15th Conf. Retrovir. Opportun. Infect., Boston, MA.
32. Tremoulet, A. H., E. V. Capparelli, P. Patel, E. P. Acosta, K. Luzuriaga, Y.
Bryson, D. Wara, C. Zorrilla, D. Holland, and M. Mirochnick. 2007. Popu-
lation pharmacokinetics of lamivudine in human immunodeficiency virus-
exposed and -infected infants. Antimicrob. Agents Chemother. 51:4297–
33. Wilfert, C. M., and M. G. Fowler. 2007. Balancing maternal and infant
benefits and the consequences of breast-feeding in the developing world
during the era of HIV infection. J. Infect. Dis. 195:165–167.
34. Wilson, J. T., R. D. Brown, J. L. Hinson, and J. W. Dailey. 1985. Pharma-
cokinetic pitfalls in the estimation of the breast milk/plasma ratio for drugs.
Annu. Rev. Pharmacol. Toxicol. 25:667–689.
35. Zeh, C., P. Weidle, L. Nafisa, H. Musuluma, J. Okonji, E. Anyango, P.
Bondo, R. Masaba, M. Thigpen, and T. Thomas. 2008. 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, abstr. 84LB. 15th Conf. Retrovir.
Opportun. Infect., Boston, MA.
1176 MIROCHNICK ET AL.ANTIMICROB. AGENTS CHEMOTHER.