Lipid-based nutrient supplements do not decrease breast milk intake of

Malawian infants13
Chiza Kumwenda, Kathryn G Dewey, Jaimie Hemsworth, Per Ashorn, Kenneth Maleta, and Marjorie J Haskell
additional energy from LNS may displace breast milk intake,
thereby reducing the benefits of breast milk for infant health.
Breastfed infants self-regulate their energy intake (7), and some
studies have shown an inverse relation between the energy density
of complementary foods and breast milk intake (8, 9). Two studies
have assessed breast milk intake after LNS supplementation: one
at 7 mo of age (10) and the other at 910 mo of age (11); however,
neither study included an unsupplemented control group.
We therefore designed the current study to measure breast milk
intake in a subsample of infants 910 mo of age participating in
a randomized clinical trial to assess the effect of consumption of
different daily quantities and formulations (with and without
milk powder) of LNS on child growth and development in rural
Malawi. Specifically, we assessed the effect of 10, 20, or 40 g
LNS provided for daily use between 6 and 18 mo of age; a
comparison group did not receive any study supplement until
after 18 mo of age (when they received a fortified maize-soy
blend). The target age for this substudy was chosen so that infants in the intervention groups would have received LNS for
$3 mo before the measurement of breast milk intake.

INTRODUCTION

1
From the Department of International Health, University of Tampere School
of Medicine, Tampere, Finland (CK and PA); the Program in International and
Community Nutrition, Department of Nutrition, University of California, Davis,
Davis, CA (MJH and KGD); the Department of Nutrition and Public Health
Intervention Research, London School of Hygiene and Tropical Medicine,
London, United Kingdom (JH); and the Department of Community Health,
College of Medicine, University of Malawi, Blantyre, Malawi (CK and KM).
2
Supported by the Mathile Institute for the Advancement of Human Nutrition, the International Atomic Energy Agency, the University of Tampere,
and the Bill & Melinda Gates Foundation.
3
Address correspondence to MJ Haskell, Program in International and
Community Nutrition, Department of Nutrition, University of California,
Davis, Davis, CA 95616. E-mail: mjhaskell@ucdavis.edu.
4
Abbreviations used: FTIR, Fourier transform infrared spectroscopy; iLiNS,
International Lipid-based Nutrient Supplements; LNS, lipid-based nutrient supplements.
Received September 27, 2013. Accepted for publication December 9, 2013.
First published online December 24, 2013; doi: 10.3945/ajcn.113.076588.

The WHO recommends that infants receive breast milk exclusively until they reach 6 mo of age, with continued breastfeeding along with safe and nutritious complementary foods
thereafter to 2 y of age or beyond (1). In rural Malawi, although
initiation of breastfeeding is nearly universal, infants usually
begin receiving maize porridge at w34 mo of age (2), and the
complementary food diet is usually very low in nutrient and
energy densities (3). To address nutrient gaps, lipid-based nutrient supplements (LNS)4 have been designed to enrich the
local diet with micronutrients, macrominerals, essential fatty
acids, and high-quality protein beginning at 6 mo of age (4).
Preliminary studies in Ghana and Malawi suggest that the addition of small (w20 g/d) quantities of LNS to complementary
foods that are prepared in the home daily may help to prevent
growth faltering in infancy (5, 6). However, there is concern that

SUBJECTS AND METHODS

Study site and subjects

The International Lipid-based Nutrient Supplements (iLiNS)
DOSE trial was carried out in southern Malawi in areas surrounding the Mangochi District Hospital and Namwera Health
Centre. Healthy infants were eligible for enrollment into the main

Am J Clin Nutr 2014;99:61723. Printed in USA. 2014 American Society for Nutrition

617
Supplemental Material can be found at:
http://ajcn.nutrition.org/content/suppl/2014/02/14/ajcn.113.0
76588.DCSupplemental.html

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ABSTRACT
Background: The potential for small-quantity lipid-based nutrient
supplements (LNS) to promote growth and development after 6 mo
of age is currently being investigated. Because infants self-regulate
energy intake, consumption of LNS may reduce breast milk intake and
potentially decrease the beneficial effects of breast milk.
Objective: The objective was to test the hypothesis that the breast
milk intake of 9- to 10-mo-old rural Malawian infants receiving LNS
would not be lower than that of infants receiving no supplementation.
Design: This was a substudy of the International Lipid-based Nutrient Supplements (iLiNS) DOSE trial, in which 6-mo-old infants
were randomly assigned to receive 10, 20, or 40 g LNS/d containing
56, 117, or 241 kcal/d, respectively, or no LNS until 18 mo of age. A
subset was randomly selected to estimate breast milk intake at 910
mo of age with the dose-to-mother deuterium oxide dilution method.
The noninferiority margin was ,10% of total energy requirements.
Results: Baseline characteristics (n = 376) were similar across
groups. The mean (6SD) daily breast milk intake of unsupplemented infants was 730 6 226 g. The differences (95% CIs) in
mean intake of infants provided with 10, 20, or 40 g LNS/d, compared with controls, were +62 (218, +143), +30 (240, +99), and +2
(268, +72) g/d, respectively. Nonbreast milk oral water intake did
not differ by group (P = 0.39) and was inversely (r = 20.22, P ,
0.01) associated with breast milk intake.
Conclusion: In this rural Malawian population, breast milk intake at
910 mo of age was not reduced by supplementation with complementary foods with 1040 g LNS/d. This study was a substudy within
the iLiNS DOSE trial, which was registered at clinicaltrials.gov as
NCT00945698.
Am J Clin Nutr 2014;99:61723.

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KUMWENDA ET AL

Study protocol
Breast milk intake was estimated by using the dose-to-mother
H2O dilution method developed by Coward et al (12) and later
modified by Haisma et al (13, 14). The method is suitable for
estimating average daily breast milk intake in community settings (12) and also provides estimates of water intake from
sources other than breast milk (13). Maternal and infant saliva
samples were collected for analysis of deuterium enrichment,
following a standard protocol (14). On study day 0 (baseline),
mother-infant pairs came to the study clinic, where maternal and
infant morbidity during the previous 7 d was assessed by using
a questionnaire. If the mother reported that she or her infant
experienced fever or symptoms of diarrhea or vomiting within
the past 7 d, the pair was requested to report back to the clinic
1 wk later. For healthy mother-infant pairs, maternal and infant
weights were measured, followed by saliva sample collection
from both the mother (two 2-mL samples) and infant (one 2-mL
sample). Then an oral dose of 30 g 2H2O (99.8% purity; Cambridge Isotopes Laboratories) was administered to mothers soon
after the baseline saliva sample was collected. The 2H2O doses
were weighed to the nearest 0.0001 g into a sterile Nalgene
bottle (60 mL) by using an analytic scale (Adventurer Pro
Balances-AV64 Pine Brook).
After 2H2O was consumed, 50 g drinking water was added to the
bottle without removing the straw, and the mother was asked to
2

drink all of the water to ensure that all the 2H2O was ingested.
Subsequent saliva samples were collected from the mother-infant
dyad in their home on study days 1, 2, 3, 4, and 13. On study day
14, mother-infant pairs came to the study clinic for final saliva
sample collection and weight measurement. Fieldworkers also collected weekly information on maternal and infant morbidity during
the study period by administering questionnaires to the mother.
Anthropometric measurements
Anthropometric measurements were taken by trained fieldworkers, who were supervised and retrained every 3 mo; all
measurements were done in triplicate. Mothers were weighed in
light clothing to the nearest 0.01 kg with an electronic scale
(SECA 846; Chasmors Ltd), and their height was measured to the
nearest 0.1 cm with a stadiometer (Harpenden; Holtain Ltd). Infants
were weighed nude to the nearest 0.01 kg with an electronic scale
(SECA 735; Chasmors Ltd). The study scales were calibrated every
morning in compliance with the main trials standard operating
procedures for anthropometric measurements.
Saliva collection
Maternal saliva was collected by carefully placing a small
piece of cotton wool in the mothers mouth for about 2 min until
it was fully soaked with saliva. The mother transferred the
soaked cotton wool directly to the empty barrel of a 20-mL
disposable syringe (to prevent contamination of the cotton).
Saliva was squeezed out of the soaked cotton wool into a vial
labeled with participant number, date, and time of sample collection. The procedure was repeated by using a new piece of
cotton wool, until a total of 4 mL saliva was collected. Infant
saliva was collected by tightly wrapping a piece of cotton wool
around the tip of a wooden stick and placing the stick in the infants
mouth, between the lower gum and cheek for w12 min until the
cotton wool was soaked with saliva. Cotton wool was separated
from the stick and placed into a 20-mL disposable syringe for
collection of saliva as described for mothers. In infants the procedure was repeated until a total of w2 mL saliva was collected.
Saliva samples from the field were carried back to the study
clinic in soft cooler boxes on frozen ice packs. For both mothers
and infants, the baseline and postdose saliva samples were stored
separately in labeled plastic sealed bags to prevent any crosscontamination of samples and to protect samples from evaporation or exposure to environmental moisture. All saliva samples
were stored at 2208C until analyzed for deuterium enrichment.
Infant breast milk intake (g/d), nonbreast milk oral water intake, and total water intake were calculated by fitting the deuterium enrichment data to a 2-compartment steady state model
of water turnover in the mother-infant dyad by using the solver
function in Microsoft Excel (12, 13).
Deuterium enrichment analysis
Deuterium enrichment in saliva was measured by Fourier
transform infrared spectroscopy (FTIR 8400 Series; Shimadzu
Corporation) at the College of Medicine, Mangochi Laboratory,
following standard protocols (14). Before analysis, frozen saliva
was thawed at room temperature. Calibration curves were prepared by using a set of standards with known deuterium enrichment and comparing the calculated (known) concentration
with the measured concentration. Each day, before proceeding

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trial if they were 5.506.49 mo of age, resided in the 2 study

catchment areas, would be available during the 12-mo study
period, and were not concurrently participating in any other
clinical trial. Participants in the main trial were randomly assigned to 6 groups: 3 groups receiving milk-containing LNS (10,
20, or 40 g/d), 2 groups receiving LNS without milk (20 or 40 g/d),
and the control group, which did not receive LNS between 6 and
18 mo of age (the iLiNS-DOSE trial). All mothers were encouraged to breastfeed their infants on demand during the 12-mo
intervention period and to offer a variety of locally available
complementary foods.
In the current substudy, the 2 intervention groups receiving
20 g LNS/d (with or without milk) were combined, and the 2 intervention groups receiving 40 g LNS/d were combined, making
4 groups varying only in the energy received from LNS (0, 56,
117, or 241 kcal/d). Initially, infants were randomly assigned
concurrently to the breast milk intake study during enrollment into
the main trial by using a predetermined randomization scheme.
However, as the substudy progressed, we noted higher than anticipated participant attrition; thus, additional mother-infant pairs
were randomly selected from the main trial participants.
Mother-infant pairs were eligible for the breast milk intake
substudy if the infant was enrolled in the main iLiNS-DOSE trial,
infant age was between 9.0 and 10.0 mo, the mother was breastfeeding the infant on demand, and the mother and infant would be
available for the full study period of 2 wk. Exclusion criteria were as
follows: 1) the mother was breastfeeding more than one infant and
2) the mother and/or infant had a severe illness warranting hospital
referral. Informed consent from the mother (for mother and infant
participation) was obtained at enrollment, for both the main
iLiNS-DOSE trial and the breast milk intake substudy. Both the
main trial and the substudy protocols were approved by the Institutional Review Boards of the College of Medicine, University
of Malawi, and the Pirkanmaa Hospital District, Finland.

BREAST MILK INTAKE IN RURAL MALAWIAN INFANTS

619

with the saliva analysis, the accuracy of the FTIR measurements

was assessed by measuring a standard that contained 1000 ppm
deuterium. Saliva samples were analyzed if the measured deuterium of the standard by FTIR was within 1% (9901010 ppm)
of the known enrichment. The within- and between-day CVs for
the deuterium standard were #0.59%.
LNS used in iLiNS-DOSE trial
The LNS that was used in the iLiNS-DOSE trial was produced
and packaged by Nutriset S.A.S. (each package weighed 140 g).
Raw ingredients included vegetable oil, peanut paste, dried
skimmed milk, maltodextrin, sugar, and a mineral and vitamin
mix (see Supplemental Table 1 under Supplemental data in the
online issue). LNS was delivered to caregivers at 2-wk intervals
by the study team for the main trial. The infants guardians were
provided with small spoons (5 g) and advised to mix one
spoonful (5 g) of LNS with 2 tablespoons of porridge prepared
in the home. Participants in the 10-, 20-, and 40-g groups were
told to provide 2, 4, or 8 spoonfuls of LNS/d, respectively. Every

2 wk, the study team collected empty LNS cups and leftover
LNS, and a new supply was provided to the mother. For a subsample of infants also enrolled in a substudy of dietary intake
(n = 568), the intake of LNS was estimated by using the interactive 24-h dietary recall method (15) on 2 different days.
During the dietary intake assessment visits, the data collectors
carried additional packages of LNS as models so that caregivers
could demonstrate how much LNS they added to the infants
porridge. The purpose of collecting dietary data was to estimate
energy intake from all nonbreast milk sources of energy (LNS
+ complementary foods) and to test whether there was a difference in energy intake between the LNS and control groups. The
dietary data will be published separately.
Sample size calculation and statistical analysis
The original sample size calculation was based on an assumed
average breast milk intake at 911 mo of age of 616 6 172 g/d (16);
using this assumption, a sample size of 89 mother-infant pairs per
group was needed to detect a group difference in milk intake of

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FIGURE 1. Participant flow. *The numbers of additional infants selected are higher in the LNS-20 g and LNS-40 g groups than in the control and LNS-10 g
groups because there were 2 main trial intervention groups within each of the 2 former groups from which to randomly select infants: milk- and nonmilkcontaining LNS groups. iLiNS, International Lipid-based Nutrient Supplements; LNS, lipid-based nutrient supplements.

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KUMWENDA ET AL

TABLE 1
Baseline infant and maternal characteristics1
Study group

Infant characteristics
Proportion of males [n/N (%)]
Weight (kg)
Age (mo)
Maternal characteristics
Height (cm)2
Weight (kg)
Age (y)
Education (y)3
BMI (kg/m2)

Control (n = 79)

LNS-10 g/d (n = 75)

LNS-20 g/d (n = 98)

LNS 40 g/d (n = 107)

39/82 (48)
8.0 6 1.1
9.9 6 0.5

39/75 (52)
8.1 6 1.0
9.9 6 0.5

49/105 (47)
8.1 6 1.1
9.8 6 0.5

49/112 (44)
8.0 6 1.0
9.8 6 0.5

0.81
0.85
0.66

155.4 6
52.7 6
26.0 6
4
21.8 6

155.2 6
53.3 6
27.2 6
3.5
22.1 6

0.34
0.78
0.86
0.77
0.90

154.1 6
52.1 6
26.0 6
5
21.9 6

4.9
6.7
6.2
2.8

155.2 6
52.4 6
26.3 6
4
21.8 6

5.8
9.0
7.7
3.3

5.7
7.8
6.1
2.8

5.2
8.6
6.2
3.2

Continuous values are means 6 SDs and were compared by ANOVA; the categorical variable was compared by chi-square test. LNS, lipid-based
nutrient supplements.
2
Height was measured at enrollment into the main trial.
3
Values are medians.
1

those whose data were and were not included in the analyses
(P . 0.06). Breast milk intake was measured in mother-infant
dyads with 7 time point saliva samples for both the mother and the
infant. Incomplete saliva collection (n = 2736 per group) was
a result of missed clinic visits by the mothers or inability to
complete home visits because of the unavailability of the motherinfant pairs. One infant excluded from the breast milk intake
measurement because of not breastfeeding was included in the
final analysis, with an assumed zero breast milk intake (0 g/d). The
infant stopped breastfeeding 2 wk after starting the intervention at
6 mo. Because the reason for stopping breastfeeding is not known,
we could not rule out an effect of the intervention.
Among the unsupplemented control infants, the mean (6SD)
intakes of breast milk and other fluids were 730 6 226 and 378
6 234 g/d, respectively. The range and distribution of breast
milk intakes were similar between the control and the intervention groups (Figure 2). Compared with the controls, the
mean intake of breast milk was 62, 30, and 2 g/d higher among
infants supplemented with 10, 20, or 40 g LNS/d, respectively
(Table 2). In all breast milk intake comparisons between the

RESULTS

The study was conducted between March 2010 and November

2011. In total, 595 iLiNS-DOSE trial mother-infant pairs were
approached for enrollment into this substudy. The flow of participants is shown in Figure 1. No clinically significant differences in participant characteristics were found at baseline
between the study groups (Table 1). No significant differences
were found between groups in the proportion of participants
not included in the analysis for the reasons shown in Figure 1
(P = 0.17). In addition, no statistically significant differences in
maternal or infant baseline characteristics were found between

FIGURE 2. Box-whisker plots of breast milk intakes by group. The outer

bounds for the control group are 161 and 1512 g/d. For the LNS-10 g/d, LNS-20
g/d, and LNS-40 g/d groups, the outer bounds are 225 and 1895 g/d, 125 and
1580 g/d, and 0 and 1679 g/d, respectively. LNS, lipid-based nutrient supplements.

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$86 g/d (a = 0.05, b = 0.80, effect size = 0.5); the number was
increased to 100 per group to account for attrition, which was estimated as 12%. A difference in breast milk intake of #86 g/d represents ,10% of total daily energy needs of infants at 911 mo of
age [86 g of breast milk intake represents w5560 kcal, which is
89% of the total energy needs of 686 kcal/d at that age (16) and
was therefore chosen as the noninferiority margin]. We assumed
that a reduction in breast milk intake of this magnitude would not
result in negative health consequences to the infant.
Data analyses were done by using Stata software (version 12.1;
StataCorp). Variables were first assessed for normality to determine whether any transformations were necessary. Baseline
characteristics were compared between groups by using ANOVA
for continuous variables and a chi-square test for proportions. The
noninferiority of differences in breast milk intake of each of the LNS
groups compared with the control group was assessed by using
2-sided 95% CIs. Noninferiority was deemed established if the
lower bound of the 2-sided 95% CIs of the LNS group was above the
set noninferiority margin, a difference in breast milk of 86 g between
the LNS and control groups. Nonbreast milk oral water intake and
total water intake in each of the intervention groups were compared
with the intakes of the control group by using a 2-sample t test and
by using Holms method to adjust for multiple comparisons (17).
The primary analysis was conducted on the basis of intention to
treat. The interaction between baseline anthropometric indexes
(weight-for-length, weight-for-age, and length-for-age z scores)
and intervention group was assessed by using a likelihood ratio
test. A secondary, per-protocol analysis was conducted by using
data on actual LNS intake, as described above.

BREAST MILK INTAKE IN RURAL MALAWIAN INFANTS

621

TABLE 2
Breast milk intakes of infants in the control and intervention groups: intention-to-treat analysis1
Planned daily dose of LNS
Intervention group

Control (n = 79)
2

Breast milk intake (g/d)

Difference (95% CI) in means between intervention
and control groups
Nonbreast milk oral water intake (g/d)2
P for intervention compared with control3
Difference (95% CI) in means between intervention
and control groups
Total water intake (g/d)2
P for intervention compared with control3
Difference (95% CI) in means between intervention
and control groups

730 6 225.6

377.6 6 234

1148.1 6 310.1

LNS-10 g/d (n = 75)

LNS-20 g/d (n = 98)

792 6 279.7
62 (218.5, 142.9)

760 6 239.2
30 (239.9, 99.3)

413.9 6 281.5
0.39
36.3 (246.0, 118.5)

371.4 6 238.8
0.86
26.3 (276.9, 64.3)

1249.9 6 410.0
0.08
101.8 (213, 217.1)

1172.4 6 320.3
0.61
24.3 (269.9, 118.5)

LNS-40 g/d (n = 107)

732 6 249.5
2 (267.8, 72.3)
413.0 6 374.8
0.51
31.5 (262.9, 126.0)
1186.0 6 471.6
0.54
37.9 (284.5, 160.3)

LNS, lipid-based nutrient supplements.

Values are means 6 SDs.
3
Values were compared by 2-sample t test.
2

that the latter could be affected by reverse causation (ie, women

with lower milk volume feed more LNS to their infants).
Our results are consistent with those of 2 previous studies
showing no differences in breast milk intake between infants
provided with LNS and those given traditional corn-soy blends.
The first study was conducted in rural Malawi in 7-mo-old infants
(10) who were supplemented with LNS at doses of 25 and 50 g/d with
an energy density of 5 kcal/g for 1 mo. In the other study,
conducted in the Democratic Republic of Congo (11), infants
were supplemented with 50 g ready-to-use lipid-based complementary food with an energy density of 5.5 kcal/g. In our study,
LNS had a similar energy density, 5.5 kcal/g. Two studies that
evaluated the effect of the overall energy density of the complementary food diet have reported an effect on breast milk
intakes. In India, increasing the energy density of cereal-legume
based complementary foods by adding vegetable oil reduced

DISCUSSION

Our objective was to test the hypothesis that mean breast milk
intake (g/d) at 910 mo of age would not be reduced by providing LNS at a daily dose of 10, 20, or 40 g/d among rural
Malawian infants. The intention-to-treat results are consistent
with this hypothesis, ie, breast milk intake was not lower among
infants receiving LNS compared with the group not receiving
LNS. Furthermore, on the basis of the per-protocol analysis, no
difference in breast milk intake was observed between infants
reportedly consuming no LNS and those reportedly consuming
110, 1120, or .20 g LNS/d, although, for the latter group, we
could not conclusively reject the possibility of a difference
based on the 95% CI. The intention-to-treat analysis is a stronger test of our hypothesis than is the per-protocol analyses, given

FIGURE 3. Mean differences in breast milk intakes between the LNS

intervention groups and the control group (intention-to-treat analysis). Differences were calculated as mean breast milk intake for each LNS group
minus the mean for the control group. Error bars indicate 95% CIs. All CIs
lie to the right of the noninferiority margin (286 g), which is denoted by the
dotted line and the black triangle; thus, the LNS groups were not significantly inferior to the control group, but also not superior as each CI crosses
the zero line. LNS, lipid-based nutrient supplements.

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intervention groups and the control group, the entire 95% CIs
exceeded the preset noninferiority margin (Figure 3).
The percentage of infants whose breast milk intake was below
the global reported average intake of 616 g/d at this age (16) was
26% and did not differ by group (P = 0.79). No interaction effect
on breast milk intake was observed between group assignment
and baseline weight-for-age z score (P = 0.95), length-for-age z
score (P = 0.73), or weight-for-length z score (P = 0.69).
The mean nonbreast milk oral water and total fluid intakes
were similar between the groups (Table 2). For all groups
combined, an inverse relation was found between the infants
daily intake of breast milk and nonbreast milk oral water
(r = 20.22, P , 0.01).
The per-protocol analysis supported noninferiority in breast
milk intake among those who received 110 or 1120 g LNS/d compared with those who received 0 g LNS/d (Table 3). For the
comparison between infants who consumed .20 g LNS/d and
those with no LNS intake, the CI included both the preset
noninferiority margin and zero, which made the finding inconclusive. The differences in sample sizes in the intention-to-treat
and per-protocol analyses are a result of the fact that the latter
categorized infants based on reported LNS intakes rather than
group assignment.

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KUMWENDA ET AL

TABLE 3
Breast milk intakes of infants who consumed different daily doses of LNS: per-protocol analysis1
Actual daily intake of LNS
LNS intake (n)
Breast milk intake (g/d)
Difference (95% CI) in means between each group
and the infants with zero LNS intake
Nonbreast milk oral water intake (g/d)
Raw P value for intervention compared with control3
Difference (95% CI) in means between each group
and the infants with zero LNS intake
Total water intake (g/d)
Raw P value for intervention compared with control3
Difference (95% CI) in means between each group
and the infants with zero LNS intake

0 g/d (n = 107)

110 g/d (n = 81)

1120 g/d (n = 57)

751 6 222.4

787 6 281.4
36 (236.8, 108.2)

786 6 259.6
35 (241.5, 111.3)

387.3 6 256.2

435.5 6 411.1
0.33
48.2 (248.2, 144.6)

364.3 6 292.7
0.60
223.0 (2110.2, 64.2)

1180.6 6 316.6

1267.5 6 535.6
0.17
86.9 (236.5, 210.4)

1190.7 6 393.2
0.87
10.1 (2101.6,121.8)

.20 g/d (n = 40)

698 6 190.0
253.0 (2131.5, 25.4)
386.7 6 151.6
0.99
20.6 (285.8, 84.7)
1127.1 6 220.7
0.33
253.4 (2161.0, 54.2)

1
The data for the per-protocol analysis are from infants (n = 285) for whom dietary-assessment data were available; thus, n is not equal to 359. LNS,
lipid-based nutrient supplements.
2
Mean 6 SD (all such values).
3
Values were compared by 2-sample t test.

respectively, of nonbreast milk oral water. Our results also

confirm that none of the participating infants was still exclusively breastfed, because all had positive values for nonbreast
milk oral water intake, which is a proxy for complementary food
intake (22). Exclusive breastfeeding beyond 6 mo is associated
with poor nutritional outcomes in infants (23).
Our study had many strengths. Unlike similar studies (10, 11),
ours had a large sample size and was therefore appropriately
powered to test a noninferiority hypothesis. We also included
a true control group that did not receive any study supplements,
which allowed us to assess the independent effect of LNS on
breast milk intake. Furthermore, we obtained dietary intake data
(data not reported) from all study groups to ascertain the amounts
of complementary food and LNS intake, which allowed us to
perform the per-protocol analysis to confirm the intention-to-treat
findings. In addition, our study used an objective method to
measure breast milk intake and thus increased the validity of the
findings. The analytic error was minimized by calibrating the
FTIR machine every day before using it and measuring its
precision on a daily basis. The study team had access to facilities
for maintaining a cold chain in the field, which minimized the
potential for error due to differences in sample handling.
One limitation of our study was that we did not directly observe the supplement intake of our study participants. However,
the per-protocol analysis of the data agrees with the intentionto-treat results, although the sample size was smaller for the
former than for the latter because dietary data were not collected
for all of the infants in this substudy. Because of the higher
attrition than anticipated, we implemented a second phase of
recruitment, which could have introduced selection bias. However, the chance of bias was minimized by randomly selecting
participants from all treatment groups and keeping the study
implementation team blind to group assignment. The attrition
rate was higher than estimated because the mother-infant dyads
were not always available to provide saliva samples at their
assigned time points during the 14-d sampling period, and/or
they were not available for body weight measurements. Because
their saliva and/or body weight data were incomplete, breast
milk intake could not be measured reliably and they were not
included in the final analysis.

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breast milk intake in 6-to 10-mo-old infants (18). In Bangladesh,

increasing the dietary energy density from 0.5 to 1.5 kcal/g
significantly reduced breast milk intake by w11% in infants
918 mo of age (9). However, in our study, LNS were only one
part of the complementary food diet, and even though LNS themselves are energy-dense foods, their addition to home-prepared
foods results in an overall diet that is not necessarily high in
energy density. Thus, our findings are not inconsistent with the
available evidence and indicate that home fortification of complementary foods with LNS does not negatively affect breast
milk intake among 9- to 10-mo-old infants.
The overall mean breast milk intake (752 g/d) in our study
participants exceeded the global estimate of average infant breast
milk intake of 616 g/d (16) in developing countries at this age.
Breast milk intakes higher than the reported global average have
also been reported among 9- to 10-mo-old Congolese infants
(705 g/d) receiving ready-to-use complementary food (11) and
Zambian infants 9 mo of age (635 g/d), whose cereal-legume
complementary foods were treated with a-amylase to increase
energy density (19). The higher average breast milk intake
among our study infants, compared with those from Zambia and
the Democratic Republic of Congo, may be partly attributable to
rural compared with urban differences (11). In all 3 studies,
breast milk intake was measured by using deuterium dilution
methods, whereas the global average intake estimates are based
on data obtained by using the test-weighing method, which has
been shown to underestimate breast milk intake (20). Another
possible explanation for higher-than-expected breast milk intakes is that national and community breastfeeding promotion
campaigns and programs may have resulted in increased breastfeeding in sub-Saharan countries (20, 21). Last, it is possible that
the provision of LNS or improved complementary foods increases
overall appetite, including appetite for breast milk. In Zimbabwe,
breastfeeding frequency increased significantly from 9.9 6 0.7 to
10.8 6 0.7 breastfeedings/24-h period in infants 612 mo of age
who received 20 g LNS/d for 2 wk (21).
The mean nonbreast milk oral water intake in this study was
389 g/d, which is within the range of what has been reported in
the sub-Saharan region. In the Democratic Republic of Congo
(11) and Zambia (19), infants consumed 333 and 451 g/d,

BREAST MILK INTAKE IN RURAL MALAWIAN INFANTS

These results contribute to the knowledge base demonstrating

that complementary foods in rural settings of developing
countries can be fortified with LNS without negatively affecting
breast milk intake.
We thank Christine Slater (Division of Human Health, International
Atomic Energy Agency) for sharing the laboratory standard operating procedures for the preparation of standards and analysis of deuterium oxide
enrichment in the saliva samples. We are also grateful to the iLiNS-DOSE
participants, data collection team, and site supervisors in Mangochi and Namwera for their support and help during the implementation of the study; Mary
Arimond (iLiNS Project Manager) for her guidance and leadership; Mark J
Manary for his advice on the collection of biospecimens; and the iLiNS Project Steering Committee for oversight and guidance.
The authors responsibilities were as followsKGD, MJH, PA, and KM:
designed the substudy, supervised the research, and contributed to the manuscript; CK: conducted the research, analyzed the data, and wrote the draft
manuscript; and JH: supervised the dietary intake assessment. All authors
read and approved the final manuscript. None of the authors had a conflict of
interest.

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