Infant formula containing galacto-and fructo-oligosaccharides and

13 Pages·2016·0.52 MB·English

JNS
JOURNAL OF NUTRITIONAL SCIENCE
RESEARCH ARTICLE
Infant formula containing galacto-and fructo-oligosaccharides and
Bifidobacterium breve M-16V supports adequate growth and tolerance in
healthy infants in a randomised, controlled, double-blind, prospective,
multicentre study
M. Abrahamse-Berkeveld1*, M. Alles1, E. Franke-Beckmann2, K. Helm2, R. Knecht2, R. Köllges2,
B. Sandner2, J. Knol1,3, K. Ben Amor1 and A. Bufe2,4
1Nutricia Research, Utrecht, The Netherlands
2NETSTAP e.V., Bochum, Germany
3Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands
4Experimental Pneumology, Ruhr-University Bochum, Bochum, Germany
(Received23April2016–Finalrevisionreceived19July2016–Accepted15August2016)
JournalofNutritionalScience(2016),vol.5,e42,page1of13 doi:10.1017/jns.2016.35
Abstract
Theobjectiveofthepresentstudywastoevaluatethegrowthandtoleranceinhealthy,terminfantsconsumingasynbioticformulawithdailyweightgainas
theprimaryoutcome.Inarandomised,controlled,double-blind,multicentre,interventionstudyinfantswereassignedtoanextensivelyhydrolysedformula
containingaspecificcombinationofBifidobacteriumbreveM-16Vandaprebioticmixture(short-chaingalacto-oligosaccharidesandlong-chainfructo-oligo-
saccharides in a 9:1 ratio; scGOS/lcFOS; synbiotic group), or the same formula without this synbiotic concept for 13 weeks (control group).
Anthropometry, formula intake, tolerance, stool characteristics, blood parameters, faecal microbiota and metabolic faecal profile were assessed.
Medically confirmed adverse events were recorded throughout the study. Equivalence in daily weight gain was demonstrated for the intention-to-treat
(ITT)population(n211).Intheper-protocol(PP)population(n102),the90%CIofthedifferenceindailyweightgainslightlycrossedthelowerequiva-
lencemargin.Duringtheinterventionperiod,themeanweight-for-ageandlength-for-agevalueswereclosetothemedianoftheWHOgrowthstandardsin
bothgroups,indicatingadequategrowth.Thenumberofadverseeventswasnotdifferentbetweenbothgroups.Norelevantdifferenceswereobservedin
bloodparametersindicativeforliverandrenalfunction.At13weeks,anincreasedpercentageoffaecalbifidobacteria(60v.48%)andareducedpercentage
ofClostridiumlituseburense/C.histolyticum(0·2v.2·6%)wereobservedinthesynbioticgroup(n19)comparedwiththecontrolgroup(n27).Inconclusion,
thisstudydemonstratesthatanextensivelyhydrolysedformulawithB.breveM-16VandtheprebioticmixturescGOS/lcFOS(9:1)supportsanadequate
sse infantgrowth.
rP
y
tisre Keywords:Infantgrowth:BifidobacteriumbreveM-16V:Hydrolysedformula:Synbiotics:Randomisedcontrolledtrials
v
in
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irb Exclusive human milk is the preferred feed for all term new- affectintestinalhealth,gutmicrobialcolonisationandimmune
ma born infants and provides a complete supply of nutrients to maturation(1–3). Because human-milk feeding may not always
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yb support growth and development in early life. In addition, bepossible,human-milksubstitutesshouldprovidenutritional
e
niln human milk contains bioactive components that beneficially and functional properties as close as possible to those of
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silb
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3.6 Abbreviations:ITT,intention-to-treat;PP,per-protocol;RMMM,repeated-measuresmixedmodel;scGOS/lcFOS,short-chaingalacto-oligosaccharides/long-chainfructo-
10 oligosaccharides;SCORAD,SCORingAtopicDermatitis.
2
.snj/7 *Correspondingauthor:M.Abrahamse-Berkeveld,[email protected]
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1
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/gro ©TheAuthor(s)2016.ThisisanOpenAccessarticle,distributedunderthetermsoftheCreativeCommonsAttributionlicence(http://creative-
.io commons.org/licenses/by/4.0/),whichpermitsunrestrictedre-use,distribution,andreproductioninanymedium,providedtheoriginalworkis
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humanmilk.Currently,infantswithafamilyhistoryofallergic Materials and methods
diseases that are not (exclusively) breastfed are recommended
Ethics statement
to receive a partially or extensively hydrolysed formula with
confirmed reduced allergenicity(4,5). Supplementation of for- Written informed consent was obtained from all parents be-
mulaewithingredientsinfluencingthedevelopingmicrobiome forerandomisation.Allparticipatingcentresobtainedapproval
andpotentiallythematurationoftheneonatalimmunesystem oftheirindependentlocalethicalreviewboard.Thestudywas
have gained an increasinginterest in the prevention ofallergic conductedincompliancewiththeprinciplesoftheDeclaration
diseases(6,7). ofHelsinkiandGoodClinicalPractice(GCP)regulationsand
Prebiotics, non-digestible food ingredients that selectively with those of the local German laws and regulations.
stimulate the growth and/or activity of intestinal bacteria
that affect health positively(8), are postulated to have a poten-
Study design
tial preventiveeffectonthedevelopmentofallergy.However,
according to the Cochrane review of Osborn & Sinn(9), the This study was performed as a double-blind, placebo-con-
overall level of evidence to substantiate a preventive effect trolled, randomised prospective nutritional intervention
of(any)prebioticsonallergyinhealthyand/orhigh-riskpopu- study. The study was designed as an equivalence study to as-
lations is currently insufficient, possibly due to considerable sess growth and tolerance in infants using a formula supple-
heterogeneity, e.g. type of prebiotic intervention, between mented with a specific combination of pre- and probiotics
studies. A more recent review of Vandenplas et al.(10) con- following 3 months of intervention in healthy infants. The
cludes that prebiotics are safe and since most studies suggest study was registered in the ISRCTN clinical trial database
a trend of beneficial effects, their presence brings formula (registration number ISRCTN23993517).
with respect to functionality closer to their ‘gold standard’,
breast milk. In individual studies, a specific mixture of
Participating centres
short-chain galacto-oligosaccharides and long-chain fructo-
oligosaccharides (scGOS/lcFOS; 9:1 ratio) supplemented to The participating centres were located in Germany and
infant formula was found to modulate the microbiota of selected and coordinated by NETSTAP, which is a German,
infants towards a composition with more bifidobacteria and national network organisation for paediatricians conducting
lesspotentialpathogenicbacteriasuchasclostridia-relatedspe- clinical research (Bochum, Germany).
cies(11–13). Moreover, it reduced the numberofinfectious epi-
sodes in healthy and at-risk infants(14,15) and due to its
Subjects
modulatory effect on the immune system, reduced the risk
of atopic dermatitis and some allergic manifestations during Over a period of 23 months, full-term (≥37 weeks) infants
infancyandchildhoodininfantswithahighriskofdeveloping whose mothers had decided not to exclusively breast-feed be-
allergy(11,14,16). yond the 34th day after birth were enrolled. Inclusion criteria
A3-monthinterventionwithasynbioticformulacombining were that infants had to be younger than age 35d, exclusively
the above-mentioned prebiotic scGOS/lcFOS (9:1) mixture formula-fed and of a normal birth weight for gestational age
withBifidobacteriumbreveM-16V,whichhaspotentialanti-aller- and sex (birth weight between the 10th and 90th percentile
gic properties(17), showed a reduction of the SCORingAtopic according to local standards on weight-for-gestational age
Dermatitis(SCORAD)scoreininfantswithIgE-mediatedato- values; Frank(25)). Exclusion criteria were allergic symptoms,
pic eczema as well as a positive modulation of the gut micro- i.e. atopic dermatitis and wheezing, antibiotics use prior to in-
biota(18). In addition, the prevalence ofasthma-likesymptoms clusion, congenital abnormality or chromosomal disorder that
sserP ainnfdanatssthwmhaomreedcieciavteidonthate1syyenabriootficfoflolorwm-uulpawcaosmlopwareerdinwtihthe cnoatualldinafdfieccattinoonrmfoarlignrhoewritthe,daimpamreunntaoldheifistcoiernycaynsdynpdrer-omoreps,eroir-
ytisrevin othfetihripslsaycnebboiotciocucnotnecrpeparttcso(1u9)l.dWinecraenatsiceipthaeteedsttahbaltisthheedpereffisecnaccye daecfionnegdenaist:al(1in)fiencftainotns.’Aillnpersiosril-edaedfiinngedtopvroiotloactioolnvsioolfattihoensprwoetore-
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e of a well-tolerated extensively hydrolysed whey-based infant col; (2) infants taking other foods/drinks than the study for-
g
dirb formula for the management of allergic disease(20,21). mula; water or sugar-free tea; (3) use of antibiotics during the
ma However,first,thenutritionalsafetyandadequacyoftheadd- intervention; (4) development of atopic dermatitis during
C
yb ition of such a new functional ingredient should be demon- the study according to the Hanifin criteria(26); (5) deviation of
eniln strated following stringent evaluation(22–24). the visit window (>3d); (6) deviation in weight change >1 SD
o d This equivalence study was aimed at evaluating the growth withinthefirst1–2monthsoflifeaccordingtoinfants’individ-
e
hsilb and tolerance in healthy infants consuming an extensively ualtargetgrowthcurve;(7)infantsufferingfromdiarrhoea(≥3
uP hydrolysed formula with the prebiotic scGOS/lcFOS (9:1) liquidstoolsperdfor>1week);(8)>7dofnodeclarablefever
5
3.6 mixture with B. breve M-16V following 3 months of interven- or sudden high fever without any demonstrable cause.
1
02 tion compared with the same formula without this synbiotic
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01 ondary outcomes, the study evaluated other growth para-
.0
1/g meters, tolerance, safety, faecal microbiota, metabolic faecal At enrolment, infants were randomly assigned to receive one
ro.io profile as well as the presence of atopic symptoms. of both formulas for a period of 13 weeks by four-block
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randomisation using research centre and family history of al- Table1. Compositionofthetwointerventionproductsusedinthestudy
lergy as strata (at least one first-degree family member suffer-
Nutrient(per100ml) Synbiotic Control
ing from atopic eczema, rhinitis or asthma). Formulas were
coded with letters (A, B, C, D) by the sponsor, and both Energy
the investigators and the infants’ parents were blinded to the kcal 66 66
kJ 276 276
formulas. For each centre a separate randomisation list with Protein(g;wheyhydrolysate) 1·6 1·6
two stratawas made. After randomisation by the investigator, Fat(g) 3·6 3·6
parents received the assigned formula along with instructions Linoleicacid(g) 0·36 0·36
for preparation. During the intervention period infants were α-Linolenicacid(g) 0·07 0·07
Totalcarbohydrates(g) 6·8 6·8
fed ad libitum exclusively with their allocated formulas starting Glucose(g) 0·1 0·1
onthedayofenrolment(aged0–35d)throughto13weeksof Lactose(g) 2·6 2·6
intervention.Thestudyconsistedofascreeningvisitandfour Maltose(g) 0·5 0·5
Polysaccharides(g) 3·6 3·6
subsequenthospitalvisitsatbaseline,4weeks,8weeks,and13
Transgalacto-oligosaccharides(g) 0·72 –
weeks of intervention. At each visit, parents received a diary Inulin(g) 0·08 –
for 4 weeks to record formula intake and tolerance on a Bifidobacteriumbreve(cfu) 12·6×108 –
daily basis. In this feeding record, the number of feedings Minerals
Ca(mg) 52 52
andtheamountofformulapreparedandconsumedperfeed-
P(mg) 26 26
ingwereregistered.Moreover,toleranceandstoolcharacteris-
Mg(mg) 5 5
tics were registered on a daily basis using a Likert scale(27). In Na(mg) 19 19
addition, parents were asked to record any medication, treat- K(mg) 71 71
Cl(mg) 45 45
ment or use ofother nutrition during the interventionperiod.
Fe(mg) 0·5 0·5
At the hospital visits, the physician monitored the growth of Zn(mg) 0·5 0·5
the infants and the presence of atopic symptoms of the skin Cu(mg) 0·04 0·04
and/or airways. I(μg) 10 10
Se(μg) 1·2 1·2
Three criteria were set as stopping rules for early termin-
Vitamins
ation of the study: (1) case of infection by B. breve; (2) >10 VitaminA(μgRE) 60 60
% of the subjects with deviating weight change (±1 SD) β-Carotene(μg) 4·6 4·6
from individual target growth curve; (3) >10% of the infants VitaminD(μg) 1·5 1·5
dropping out due to diarrhoea (≥3 liquid stools/d for more VViittaammiinnEK((mα-gT)E) 15··12 15··12
than 1 week). VitaminB (mgNE) 0·04 0·04
1
Asaquality-controlprocedure,anindependentstudymoni- VitaminB2(mg) 0·1 0·1
Niacin(mgNE) 0·7 0·7
tor (CRO München) visited each study site prior to the study
VitaminB (mg) 0·04 0·04
and regularly during the study to perform reviews of clinical VitaminB6 (μg) 0·18 0·18
12
research forms and subject diaries, as well as to check the in- VitaminC(mg) 8 8
vestigational products and storage facilities. Folicacid(μg) 11 11
Panthothenicacid(mg) 0·3 0·3
Choline(mg) 7 7
Taurine(mg) 4·6 4·6
Study formulas
cfu,Colony-formingunits;RE,retinolequivalents;TE,tocopherolequivalents;NE,
Both formulas were standard extensively hydrolysed (63% of
niacinequivalents.
proteins<1000Da)wheyprotein-basedpowderproductswith
sserP easdteaqbuliastheednuatnrittii-oanllaelrgseunpicpoprrtoopferitniefsanatnsdininttheendfierdstto6 mproonvtidhes Measurements
ytisrevin poefrli1fe0(020m,21l).aTshimeilfaorrmamuolausntwoerfe6i6sokecnaelr(g2e7t6ickaJ)ndenceorgnyt,ai1n·e6d pTehredpirniminafraynotsutfceodmtheeosftuthdeyfsoturdmyuwlaassfrdoemfinaegdea≤s3w5etiogh1t1g9aidn.
U e g protein, 6·8g carbohydrate, 3·6g lipid, vitamins and miner- Thesecondaryoutcomeswererecumbentlength,headcircum-
g
d
irb als (Table 1). In contrast to the control formula, the synbiotic ference, symptoms of digestive tolerance, faecal microbiota
ma formula contained a specific mixture of short-chain galacto- and metabolic profile, plasma parameters of renal and liver
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yb oligosaccharides and long-chain fructo-oligosaccharides in a function, presence of atopic symptoms and frequency of ad-
e
niln 9:1 ratio (0·8g per 100ml formula) and the probiotic strain verse events.
o d B. breve M-16V (1·3×109colony-forming units per 100ml Theinfantswereweighednakedwhilelyingquietlyonacali-
e
h
silb formula; Morinaga Milk Industry Co., Ltd). The synbiotic bratedelectronicscaleaccurateto10g.Recumbentlengthwas
uP andcontrolformulashadasimilartaste,smellandappearance measuredtothenearest0·1cmwithalengthboard.Headcir-
5
3.6 and were supplied by Nutricia Research. Products were man- cumferencewasmeasuredusinganon-stretchmeasuringtape.
1
02 ufactured according to current good manufacturing practices, The investigators assessed the formula intake, the records of
.sn
j/7 and were packaged and coded at Nutricia Research. The digestive tolerance and the adverse events at each visit.
1
01 studyproductswerestoredatacool,secureandlimitedaccess Average daily intake of the formula (ml/d) was calculated
.01/g storageareaprotectedfromextremesoflight,temperatureand based on the parents’ monthly records. Infants were regarded
ro.io humidity. as compliant if: (1) they had an average daily intake of >400
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ml; (2) the average daily intakewashigher than their age- and for determining total faecal cell counts. Paraformaldehyde-
sex-specified minimum intake requirements; (3) the left-over, fixed faecal samples were hybridised with the specific probes
defined as prepared minus consumed, was below 25%. as described previously(30). Thereafter the samples were
Symptomsofdigestivetoleranceconsistedofdailystoolfre- counted using an automated Olympus AX70 epifluorescence
quency, stool consistency (on a five-point scale: 1=watery, microscope equipped with a Lang LStep13 8 slides-stage
2=soft/pudding like, 3=soft formed, 4=dry formed, 5= (Paes Nederland bv) and an F-View II charge-coupled device
dry/hard pellets) and severity of constipation, diarrhoea, (Soft Imaging System GmbH) and image analysis software.
colic, vomiting, regurgitation, flatulence and nappy rash (0= The percentage of bacterial cells was determined at twenty-
absent,1=mild,2=moderate,3=severe).Inaddition,crying five randomly chosen positions on each well by counting all
frequency and sleeping behaviour were monitored (ten-point cells using a DAPI filter set (SP100; Chroma Technology
scale; rating of 10 for sleeping is very good, rating of 10 for Corp.) and by counting the targeted bacterial group using a
crying is very often). Cy3 filter set (41007; Chroma Technology Corp.).
Any atopic dermatitis symptoms following clinical observa- In addition, pH, concentrations of SCFA and lactate were
tion of the skin were recorded at the monthly visits using the measuredinthefaecalsamples(asdescribedinKnoletal.(31)).
SCORADscore(28).Moreover,possibleairwaysymptomswere
documented. At the last visit (week 13), the SCORAD score
Statistics
of all infants was assessed.
The timing and duration, intensity and a description of the All statistical analyses were performed using SAS 9.2 (SAS
adverse events were documented by the parents in the daily Institute, Inc.). Primary outcome was the difference in weight
diariesinbetweenthestudyvisits.Anadverseeventisconsid- gain per d during 13 weeks of intervention with the objective
eredanyadversechangefrombaseline(pre-intervention)con- to demonstrate growth equivalence. Growth of infants in the
dition, which occurs during the course of the study after the synbiotic and control groups was considered equivalent
intervention has started, whether considered related to the when the two-sided 90% CI of the difference in the means
intervention or not. The investigators afterwards controlled of weight gain laid within the interval from −0·5 to +0·5
the diaries and stated a possible relationship with the study SD(32).Thisisequivalenttothetwoone-sidedtestingapproach.
product. A serious adverse event is defined as any untoward The required sample size for two one-sided statistical testing
medical occurrence resulting in death, is life-threatening (at using α=0·05 in each and a power=0·80 was sixty-nine
thetimeoftheevent),requireshospitalisationorprolongation infants per group. Taking a drop-out rate of 25% into ac-
ofexistinghospitalisationorresultsinpersistentorsignificant- count, a total of 152 had to be enrolled. An ANCOVA
lydisabilityorincapacity.Investigatorswereobligedtoreporta methodwasusedintheequivalenceanalysis,takingstudycen-
seriousadverseeventwithin24hofoccurrencetothemedical tre,riskforallergy,sex,andweightatbaselineascovariates.In
monitor and sponsor of the study. Faecal and blood samples theanalysis,centresthatincluded≤4infantsatweek13inthe
were collected at four of the seventeen sites. Blood samples growth analysis were pooled to one centre.
(twenty-fivefor synbiotic and forty-one for control) were col- Secondary outcomes included calculated anthropometric z-
lected at baseline and at the end of intervention (week 13) to scores using WHO growth standards as reference(33). All an-
determinebloodparametersindicativeforrenalandliverfunc- thropometric results will be given for both per-protocol (PP)
tioning consisting of: number of leucocytes; thrombocytes; and intention-to-treat (ITT) populations; the remaining para-
lymphocytes; monocytes; eosinophils; basophils and erythro- meters will mainly be described for the ITT population. For
cytes; Hb (g/l); haematocrit (%); mean corpuscular volume evaluating the difference from the WHO growth standards,
(fl);meancorpuscularHb(pg);meancorpuscularHbconcen- a repeated-measures mixed model (RMMM) was used per
sserP tprraotitoenin(g(g//l)l;);seaglbmuemnitned(gn/elu);truorpehails(%(%););crneeautitnroinpehi(lms(g%%));;toaltaa-l gagroeuapswthitehfiaxerdanedfofemct.inPtaerracmepettearnsdcosnlospisetinfgorofsucbojnectitnsuaonuds
y
tisrevin ntriannesfaemraisneot(rAanSsAfeTra,seG(OATL;ATU,/Gl);PTγ-;gUlu/talm);yalsptarartnastpeeapmtidinaose- cdaastae woferneoann-naloyrsmedaliutysin(gevathlueattewdo-bsyamthpeleSthatepsitr,o–oWr,ilikn ttehset
U e (U/l); and pre-albumin (mg/l). with α=0·05), the Mann–Whitney test (comparing means)
g
dirb Faecal samples (twenty-four and thirty-six infants in the or the Jonckheere–Terpstra test (ordinal data). Categorical
ma synbiotic group v. control group) were collected at baseline parameters were analysed using Fisher’s exact test. With re-
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yb (before start of the intervention), week 1 and week 13 of the spect to secondary parameters, blood and stool samples
e
niln intervention. Directly after collection two tubes with stool were only collected in a subgroup of subjects recruited at
o d sample were stored at −20°C by the parents and transported fouroftheseventeensites.Powercalculationsbasedondiffer-
e
h
silb to the hospital in a cold storage bag as soon as possible. The ences in albumin levels, assuming 1 SD as a clinically relevant
uP microbial composition of the faecal samples was determined difference and using α=0·05 and a power=0·80, indicated
5
3.6 by fluorescent in situ hybridisation (FISH) using specific 16S a total of sixteen infants per group was required. Assuming
1
02 rRNA-targeted oligonucleotide probes specific for these bac- a drop-out rate of 20%, a total of forty infants needed to
.snj/7 teria groups(19,29) (bifidobacteria, lactobacilli, Bacteroides/Prevo- beincludedinthesubpopulationtomeettherequiredamount
1
01 tella, Clostridium histolyticum/C. lituseburense, Enterobacteriaceae ofinfantsperarm.Duringthestudyitbecameevidentthatfor
.0
1/g and C. coccoides/Eubacterium rectale). The nucleic acid stain someofthefaecalsamplesthecollectedaliquots didnotcon-
ro.io DAPI (4’,6-diamidino-2-phenylindole) (Invitrogen) was used tainenoughfaecestoperformtheplannedanalyses.Moreover,
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forsomeinfants,thestoolsampleswerenotcollected.Hence, Table2. Drop-outnumberandreasonsduringthestudy*
sample collection atthesesites wascontinued tosecureasuf-
ficient amount of samples for the stool sample analysis. Reasonfordrop-out Synbiotic(n) Control(n)
Useofantibiotics 5 4
Hospitalisation 0 1
Refusalofformula 5 6
Results Unsatiated 4 5
Atopicdermatitis 2 0
Subject characteristics Consentwithdrawal 1 2
Crying 0 2
From April 2005 to November 2006 a total of 1282 subjects
Constipation 4 5
werescreened,ofwhich228subjectswererandomisedtoone Diarrhoea 2 1
of the infant formulas. A flowchart showing the enrolment Vomiting 2 9
Abdominalpain 1 2
and disposition of the study subjects is given in Fig. 1. The
Otherreasons 1 2
large number of screening failures was due to the fact that
most of the infants were not exclusively formula fed at the *Fisher’sexacttestwasusedtoevaluatepotentialdifferencesbetweenthetreat-
mentgroups.
time of screening. Of the 228 infants that were recruited,
211 infants received at least some of the study formula and
constituted the ITT study population, excluding seventeen In the PP population, only the higher prevalence of atopic
infants from further analyses (twelve in the control group skin symptoms remained significant (8 v. 0% for synbiotic v.
and five in the synbiotic group). A substantial amount of control; Mann–Whitney test, P=0·042). However, maternal
109 infants was excluded from the PP population (fifty-four weight was higher (75 (SD 16·5) v. 66·9 (SD 12·5)kg; t test,
for the control group and fifty-five for the synbiotic group) P=0·006) and maternal height tended to be higher (1·70
due to protocol violations or dropping-out from the study. (SD0·06)v.1·67(SD0·06)m;ttest,P=0·067))inthesynbiotic
In total, forty-six subjects were recorded as having major groupcomparedwiththecontrolgroup,ultimatelyresultingin
protocol violations and were excluded from the PP groups. ahighermaternalBMIinthesynbioticgroup(26·04(SD5·38)
The protocol violations included erroneous enrolment (not kg/m2) compared with the control group (23·9 (SD 4·02)kg/
meeting criteria; n 17), use of probiotics during the study m2; t test P=0·027)
(n 5), non-compliance (n 10) and/or consumption of mis-
labelled products (n 15). In addition, a total number of sixty-
Formula consumption
three infants dropped out during the study. The reasons for
dropping out did not show a significant difference between Theobservedformulaintakewasinlinewithexpectations,and
the groups (Table 2). With regards to demographics of the infants of the synbiotic and control groups consumed similar,
ITT population, infants of the synbiotic group were more increasing amounts of formula in the first, second and third
often male (Fisher’sexacttest;P=0·027), hada highergesta-
tionalageandweightatbirth(Mann–Whitneytest,P=0·035;t
test,P=0·012)andahigherlength,butnotweight,atbaseline Table3. Birthandbaselinecharacteristicsofsubjectsintheintention-to-
treatpopulation
(Mann–Whitney test; P=0·046) and had a higher prevalence (Meanvaluesandstandarddeviations,orpercentage)
of atopic skin symptoms at baseline (Fisher’s exact test; P=
0·008) compared with infants of the control group (Table 3). Synbiotic(n100) Control(n111)
Mean SD Mean SD
Birthweight(g)‡ 3450* 434 3300 424
sse Gestationalage(weeks)§ 39·4* 1·2 39·0 1·4
rP y Vaginaldelivery(%)‖ 75·8 67·6
tisrevin WSeexig(h%tamtablaes)‖eline(g)‡ 405391·0* 665 384932·2 685
U e Lengthatbaseline(cm)‡ 53·2* 2·2 52·4 2·6
gd Headcircumferenceat 36·6† 1·6 36·2 1·6
irb baseline(cm)‡
m
aC Ageatbaseline(d)§ 23·3 11·7 22·5 11·2
yb Maternalheight(m)‡ 1·67 0·07 1·67 0·06
en Maternalweight(kg)§ 74·4 17·6 70·4 15·7
ilno MaternalBMI(kg/m2)‡ 26·5† 5·6 25·2 5·4
de Paternalheight(m)‡ 1·80 0·08 1·80 0·08
h
silb Paternalweight(kg)§ 81·4 13·1 83·5 13·4
uP Atriskforatopy(%)‖ 39·0 42·3
53 Atopicskinsymptoms 11·0* 1·8
.61 (%infants)‖
0
2
.snj/71 †*VVaalluueewteansdesdigtnoifibceanstilgyndifiifcfaenretlnytdfrifofemretnhtaftrofomrtthheatcfoonrttrhoelgcoronutrpol(Pgr<ou0p·0(50)·.10>P>
01 0·05).
.01 Fig.1. Dispositionofstudysubjects.Atotalofforty-sixinfantswereexcluded ‡Two-samplettest.
/gro.io fsrioxmty-tthhreeepeinr-fparnottsocdorolp(PpePd)-ogurotudpurdinugetthoemsatujodry.pIrToTto,cionltednetivoiantitoontsr;eaat.totalof §‖FMisahnenr–’sWehxitancetytetesst.t.
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month after the start of the intervention (Table 4; t test P= standard were observed to be close to zero in both groups
0·973, P=0·475 and P=0·852, respectively). of the PP population (Table 6; RMMM P=0·331 and P=
0·868 for the control and synbiotic groups, respectively).
Themeanheadcircumferencegain,aswellastheheadcircum-
Infant growth patterns ference at each visit (data not shown) were not significantly
different between the synbiotic and control groups in both
Average weight gain (g/d) during the study period was not the ITT and PP populations (ANCOVA; P>0·10 for all;
statisticallydifferentbetween thesynbioticand controlgroups
Table 5).
(Table 5). Equivalence in weight gain per d could not be
demonstrated for the PP population (Fig. 2). The 90% CI
of the difference in means of weight gain slightly crossed the Adverse events and blood parameters
lower 0·5 SD margin (ANCOVA, 90% CI (−3·3 to +0·8g/ The percentage of children with at least one (serious) adverse
d) and the margin ±3·1g/d, respectively). However, in the
event during the study was similar between treatment groups
ITT population the equivalence in weight gain per d was (15·0 v. 19·8% for synbiotic and control groups; Fisher’s
demonstrated, with an 90% CI of the difference in means exacttest,P=0·372).Exceptfornon-infection-relatedrespira-
that lay well within the equivalence margins (ANCOVA, 90
torytractdiseasesorsymptoms,whichwerehigherinthesyn-
% CI (−3·0 to +0·3g/d) and the margin ±3·1g/d, respect-
bioticcomparedwiththecontrolgroupoftheITTpopulation
ively) despite an even slightly larger point estimate of the dif- (8·0 v. 1·8%; Fisher’s exact test, P=0·049), the frequency of
ference in means compared with the PP population (Fig. 2). specificdiseasesrelatedtotheadverseeventswerenotsignifi-
Inclusion of maternal BMI as a covariate did not affect the
cantly different between treatment groups (data not shown).
outcome of the equivalence analyses in the PP or ITT popu- One infant developed atopic dermatitiswhich was reflected
lation (data not shown). At all visits during the intervention
in a higher eosinophil count after 13 weeks of intervention in
period, the mean body weight of infants in the synbiotic and the synbiotic group of the ITT subpopulation (median 4·2 v.
control groups were similar both for the ITT and the PP 2·9%; Mann–Whitney test, P=0·029); omitting this value
populations(ANCOVA,P>0·10;datanotshown).Theover-
resulted in similar counts compared with the control group.
all weight-for-age development during the interventionperiod
No differences in any of the other blood parameters were
wasin linewith the sex-specific WHO child growth standard,
observed compared with the control group (data not shown).
which represents the growth of exclusively breast-fed infants.
As depicted in Table 6, weight-for-age z-scores of infants in
both groups of the PP population were very close to zero Gastrointestinal tolerance
(RMMMP=0·130andP=0·102forthecontrolandsynbio-
Withrespecttotolerancedata,onlythestoolconsistencyscore
tic groups). In contrast to the synbiotic group, infants of the inthefirst4weeksoftheinterventionperiodwaslowerinthe
control group had an increase in weight-for-age z-score over
synbiotic v. control group of the ITT subpopulation
time, resulting in a significantly different slope compared (Jonckheere–Terpstra test; P=0·035), which was accompan-
with the WHO standard (RMMM, effect for age, P=0·572
ied by a lower severity of nappy rash in the synbiotic group
and P=0·002, respectively). in that particular period (Jonckheere–Terpstra test; P=
IntheITTpopulation,nosignificantdifferencesinlengthor 0·026; Table 7). At the end of the intervention period, stool
lengthgainbetweenthetwogroupswereobserved.InthePP
frequencytendedtobelowerinthesynbioticgroupv.control
population, the mean length gain was not different between group of the ITT subpopulation (Mann–Whitney test, P=
the synbiotic compared with the control group (Table 5; 0·056; Table 7).
ANCOVA, P=0·11), although a lower absolute length was
sse observed in the synbiotic v. the control group at week 4
rP
ytisrevinU e (w05·e06e0·k03)8,(SDa(5n9d0··22w)(eSveD.k5061·2·37) v((6.S2D6·050·1·(2S)(DScDm00·;3·2)A)Nvc.mC6O;3AV·4NA,C(SODPV=0A·03,·)0P3cm1=),; FFfoaareecctaahllesmasmyicnrpboliebositoiotcfag,arSsoCuuFbpApoaanpndudlatlthaioicrtntayot-esfixsixftoyrinthfaenctso(nttwroelntgyr-ofouupr)
gd ANCOVA, P=0·022). During the intervention period the
irbm length-for–age z-scores using the sex-specific WHO child were to be analysed at baseline, at week 1 and at 13 weeks
a after intervention (a total of 180 samples). In total, twenty-
C
yb seven samples were missing and another fourteen samples
e
niln Table4. Dailyformulaintakeofthesynbioticandcontrolgroupsinthe did not contain a sufficient amount of faecal matter to per-
o
d per-protocolpopulation* formallplannedanalysis,resultinginasomewhatlowernum-
e
hsilb (Meanvaluesandstandarddeviations) ber for some time points. At baseline, faecal microbiota and
uP 5 Synbiotic(n45) Control(n57) related stool parameters were not significantly different be-
3.6 tweenthesynbioticv.controlgroupoftheITTsubpopulation
102 Formulaintake(ml/d) Mean SD Mean SD (Tables 8 and 9).
.snj/7 Baseline–week4 672 108 689 111 A statistically significantly higher level of the bifidobacteria
101 Week4–week8 758 110 758 140 populationwasobservedatweek13inthesynbioticcompared
.01/g Week8–week13 796 101 795 100 with the control group (Mann–Whitney test, P=0·014),
ro.io *Statisticalcomparisonswereperformedusingatwo-samplettest. whereas only a trend towards a higher level was found at
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Table5. Weightgain,lengthgainandheadcircumferencegainduringtheinterventionperiod*
(Meanvaluesandstandarddeviations)
ITTpopulation PPpopulation
Interventiongroup... Synbiotic(n100) Control(n111) Synbiotic(n45) Control(n57)
Mean SD Mean SD Mean SD Mean SD
Weightgain(g/d) 27·5 0·7 28·5 0·7 28·7 6·4 29·8 6·0
Lengthgain(cm/week) 0·77† 0·02 0·81 0·02 0·77 0·13 0·82 0·16
Headcircumferencegain(cm/week) 0·36 0·01 0·38 0·01 0·37 0·01 0·39 0·01
ITT,intention-to-treat;PP,per-protocol.
*TheANCOVAmethodwasusedtoevaluatedifferencestakingstudycentre,riskforallergy,sex,andweightatbaselineascovariates.
†Tendencyforalowermeanlengthgaincomparedwithcontrol(P=0·093).
week1inthesynbioticgroup.Asignificantlylowerpercentage be lower in the synbiotic group at week 13 compared with
ofthepotentialpathogensrepresentedbytheC.histolyticum/C. the control group (Mann–Whitney test, P=0·052).
lituseburenseclusterwasobservedinthesynbioticv.thecontrol No differences in faecal acetate or butyrate levels were
groupatbothinterventiontimepoints(Mann–Whitneytest,P observed during the intervention period. Compared with the
=0·003; and Mann–Whitney test, P=0·013, respectively). In control group, faecal propionic acid levels tended to be
addition, the level of C. coccoides/E. rectale cluster tended to lower after 1 week of intervention in the synbiotic group
sse
rP
y
tisre
v
in
U
e
g
d
irb
m
a
C
y
b
e
n
iln
o
d
e
h
silb
u
P
5
3
.6
1
0
2
.sn
j/7
1
0
1
.0
1
/g Fig.2. Equivalencetestingforweightgain(g/d)duringtheinterventionperiodintheper-protocolpopulation(a)andtheintentiontotreatpopulation(b).AnANCOVA
ro
.io methodwasusedintheequivalenceanalysis,takingstudycentre,riskforallergy,sex,andweightatbaselineascovariates.
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Table6. WHOweight-for-agez-scoresandlength-for-agez-scoresintheintention-to-treat(ITT)andper-protocol(PP)populations
(Meanvaluesandstandarddeviations)
ITTpopulation PPpopulation
Interventiongroup... Synbiotic(n100) Control(n111) Synbiotic(n45) Control(n57)
Mean SD Mean SD Mean SD Mean SD
Weight-for-agez-scores
Baseline −0·101 0·854 −0·253 0·896 −0·161 0·729 −0·224 0·905
Week4 −0·148 0·778 −0·270 0·957 −0·178 0·733 −0·175 0·910
Week8 −0·171 0·824 −0·204 0·950 −0·212 0·851 −0·164 0·965
Week13 −0·060 0·824 0·051 1·012 0·100 0·844 0·042 1·006
Length-for-agez-scores
Baseline −0·030 0·948 −0·285 1·033 −0·071 0·869 −0·375 1·138
Week4 −0·035 1·000 −0·206 1·054 −0·073 0·869 −0·172 1·089
Week8 0·041 0·985 −0·044 1·099 0·026 0·905 0·045 1·148
Week13 0·235 1·025 0·321 1·259 0·228 1·031 0·248 1·266
(Mann–Whitney test, P=0·089). The concentrations of the L-lactate levels and although the D-lactate levels were low, a
branched SCFA such as isobutyrate and isovalerate were stat- slightly higher concentration of D-lactate was observed at
istically significantly lower in the synbiotic group compared week 13 in the synbiotic group (Mann–Whitney test; P=
with the control group following 1 week of intervention 0·04). Furthermore, supplementation with the synbiotic for-
(Mann–Whitney test, P=0·002; and Mann–Whitney test, mula resulted in a statistically significantly lower faecal pH
P=0·003, respectively). No differences were observed in from week 1 in the synbiotic group than in the control
Table7. Toleranceparametersofinfantsinthesynbioticandcontrolgroupsintheintention-to-treatpopulation
(Meanvaluesandstandarddeviations,andnumberofinfants)
Synbiotic Control
Parameter Time(weeks) Mean SD n Mean SD n
Stoolfrequency(n/d) 0–4 1·4 0·97 95 1·5 0·93 105
4–8 1·2 0·72 77 1·2 0·65 79
8–13 1·1† 0·59 74 1·2 0·56 74
Stoolconsistencyscore‡ 0–4 2·2* 0·8 95 2·3 0·7 105
4–8 2·1 0·5 77 2·2 0·6 79
8–13 2·1 0·5 74 2·2 0·6 74
Constipationseverity§ 0–4 0·1 0·5 89 0·2 0·6 103
4–8 0·0 0·2 71 0·0 0·1 76
8–13 0·0 0·0 68 0·0 0·1 71
Diarrhoeaseverity 0–4 0·1 0·3 89 0·1 0·3 101
4–8 0·0 0·2 71 0·0 0·1 76
8–13 0·0 0·1 68 0·0 0·1 71
Colicseverity 0–4 0·2 0·6 89 0·2 0·6 103
sse 4–8 0·1 0·4 72 0·2 0·6 79
rP y 8–13 0·0 0·2 68 0·0 0·2 72
tisrevin Vomitingseverity 04––48 00··11 00··33 9702 00··21 00··63 17073
U e 8–13 0·1 0·2 68 0·0 0·2 71
gd Regurgitationseverity 0–4 0·8 0·8 91 0·8 0·7 106
irb 4–8 0·7 0·7 74 0·7 0·7 79
m
aC 8–13 0·7 0·7 71 0·7 0·8 74
yb Flatulenceseverity 0–4 0·9 0·9 91 0·8 0·8 105
en 4–8 0·8 0·8 75 0·7 0·8 79
ilno 8–13 0·6 0·8 71 0·5 0·6 74
de Nappyrashseverity 0–4 0·0 0·3 88 0·1 0·3 103
h
silb 4–8 0·1 0·3 71 0·1 0·2 77
uP 8–13 0·1 0·3 68 0·1 0·3 73
53 Cryingseverity‖ 0–13 4·1 1·9 93 4·1 2·1 101
.61 Sleepingscore‖ 0–13 7·2 1·9 94 7·1 2·0 101
0
2
.snj/71 *†MMeeaannvvaalluueewteansdseidgntoificbaenstliygndiiffifcearnetnlytfdroifmfertehnattffroormthtehactofnotrrothlegrcoounptr(oPlg<r0o·u0p5)(.0·10>P>0·05).
01 ‡Consistencyscores:1=watery;2=soft/puddinglike;3=softformed;4=dryformed;5=dry/hardpellets.
.01 §Severityscores:0=absent;1=mild;2=moderate;3=severe.
/gro.io ‖gaCsrtyroiningtefrsetiqnuaelnscyymapntodmssleecopmingpasrcisoorensswweerreemteesatesduruesdinogntaheteJno-pnockinhtesecrael–eT:earprasttirnagtoefst1,0stfooorlsflreeeqpuinegncisyvceormypgaoroisdo;nasrawteinrgeotefs1t0edfourscinrygintgheisMvaenryno–fWtehni.tnSetyootelcsot.nsistencyand
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Table8. Faecalmicrobiotacomposition(%)overtimeinasubgroup ofinfantsinthesynbioticandcontrolgroupsoftheintention-to-treatpopulation
(Medians,minimum–maximumandnumberofinfants)
Synbiotic Control
Microbiota Time Median Range n Median Range n
Bacteroides/Prevotellagroup Baseline 10−4 10−4–5·6 21 10−4 10−4–6·4 32
Week1 10−4 10−4–0·8 22 10−4 10−4–6·8 30
Week13 10−4 10−4–10−4 19 10−4 10−4–7·4 27
Bifidobacteria Baseline 56·4 10−4–88·8 21 48·5 10−4–77·3 32
Week1 58·6† 10−4–81·0 21 42·6 5·7–79·7 29
Week13 60·1* 27·0–89·4 19 48·3 9·3–76·0 27
Clostridiumhistolyticum/C.lituseburensegroup Baseline 10−4 10−4–19·8 21 10−4 10−4–21·0 32
Week1 10−4* 10−4–5·5 22 2·8 10−4–21·5 30
Week13 0·2* 10−4–11·5 19 2·6 10−4–29·2 27
Enterobacteriaceae Baseline 10−4 10−4–24·9 21 0·2 10−4–18·5 31
Week1 10−4 10−4–43·6 21 2·5 10−4–21·8 28
Week13 10−4 10−4–10·6 19 0·2 10−4–10·6 27
C.coccoides/Eubacteriumrectalegroup Baseline 10−4† 10−4–17·3 20 10−4 10−4–46·2 32
Week1 10−4 10−4–16·3 22 10−4 10−4–29·0 30
Week13 1·9† 10−4–26·3 19 13·7 10−4–43·3 26
Lactobacilli/enterococci Baseline 0·3 10−4–24·0 20 0·5 10−4–20·2 32
Week1 0·1 10−4–11·6 22 1·1 10−4–17·9 30
Week13 0·2 10−4–10·6 19 0·1 10−4–12·8 26
*Medianvaluewassignificantlydifferentfromthatforthecontrolgroup(P<0·05;Mann–Whitneytest).
†Medianvaluetendedtobesignificantlydifferentfromthatforthecontrolgroup(0·10>P>0·05;Mann–Whitneytest).
group and only a trend was observed at week 13 (week 1: and showed a stronger effect of the synbiotic intervention.
Mann–Whitney test, P=0·044; week 13: Mann–Whitney Indeed, the percentage of bifidobacteria in the synbiotic
test, P=0·062). group was statistically significantly higher than in the control
The results of the PP population (data not shown) con- group from the first week of the intervention (Mann–
firmed the above results observed in the ITT population Whitney test, P=0·04) and this effect was maintained until
Table9. SCFA,branched-chainfattyacids,lactate(mmol/kgwetweight)andpHinthestoolsofasubgroupofinfantsintheintention-to-treatpopulation
(Medians,minimum–maximumandnumberofinfants)
Synbiotic Control
Parameter Time Median Range n Median Range n
Aceticacid Baseline 48·50 0–143 20 44·20 0–115 32
Week1 34·00 12–95 21 39·11 0–74 28
Week13 38·50 18–59 16 46·06 18–144 25
Butyricacid Baseline 0·46 0·00–10·73 20 0·00 0·00–8·31 32
Week1 0·00 0·00–3·56 21 1·08 0·00–12·44 28
Week13 3·36 0·00–8·95 16 3·08 0·00–15·09 25
Propionicacid Baseline 8·78 0·00–36·20 20 8·25 0·00–24·38 32
sserP WWeeeekk113 160··6140† 11··9499––2131··1800 1261 98··7800 20··1000––2418··5260 2258
ytisre Valericacid WBaeseeklin1e 00··0000 00··0000––00··9030 2210 00··0000 00··0000––10··4000 2382
v
in Week13 0·00 0·00–0·88 16 0·00 0·00–1·79 25
U
eg D-Lactate Baseline 0·68 0·00–4·92 21 0·60 0·00–6·57 33
dirbm WWeeeekk113 00··0006* 00··0000––11··0625 1292 00··0101 00··0000––01··4985 2279
aC y L-Lactate Baseline 0·47 0·00–14·00 21 0·00 0·00–6·31 33
b e Week1 1·12 0·00–4·25 22 0·60 0·00–11·38 29
niln Week13 0·58 0·00–3·34 19 0·75 0·00–4·14 27
o d Isobutyricacid Baseline 0·00 0·00–2·10 20 0·00 0·00–1·50 32
eh Week1 0·00* 0·00–1·10 21 0·76 0·00–2·40 28
silb Week13 0·00† 0·00–1·90 16 0·88 0·00–2·76 25
u
P 5 Isovalericacid Baseline 0·00 0·00–2·60 20 0·00 0·00–2·92 32
3.6 Week1 0·00* 0·00–1·93 21 1·00 0·00–5·35 28
10 Week13 0·85 0·00–2·69 16 1·41 0·00–5·25 25
2
.sn pH Baseline 5·98 5·17–7·79 21 6·14 5·08–7·72 34
j/7 Week1 6·02* 5·23–8·28 23 6·32 4·95–7·48 30
1
01 Week13 5·92† 5·23–8·01 20 6·39 5·46–7·21 27
.0
1
/gro.io *†MMeeddiiaannvvaalluueewteansdseidgntoificbaenstliygndiiffifcearnetnlytfdroifmfertehnattffroormthtehactofnotrrothlegrcoounptr(oPlg<r0o·u0p5;(0M·1a0nn>–PW>h0it·n0e5y;tMesatn).n–Whitneytest).
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theendoftheintervention.Asignificantlylowerpercentageof power for statistical evaluation of weight gain, a minimum
the potential pathogens represented by the C. histolyticum/C. of sixty-nine infants per treatment group was estimated as
lituseburenseclusterwasobservedinthesynbioticv.thecontrol the required sample size, but the synbiotic group and control
group at both intervention time points (week 1: Mann– group of the PP population only consisted of forty-five and
Whitney test, P=0·043; week 13: Mann–Whitney test, P= fifty-seven infants, respectively. Moreover, weight-for-age
0·058). Faecal pH was also statistically significantly lower in z-scores indicated growth rates not different from WHO
the synbiotic compared with the control group at week 1 growth standards. Since these standards are based on the
(Mann–Whitney test; P=0·007) and week 13 (Mann– growthcurveofexclusivelybreastfedinfants, whichisuniver-
Whitney test; P=0·021). At the end of the intervention, the sally considered to be the most optimal nutrition for infants,
C. coccoides/E .rectale cluster was statistically significantly lower this is indicative of a sufficient nutritional efficacy of both
in the synbiotic than in the control group (Mann–Whitney formulas.
test; P=0·013). AsdescribedinasystematicreviewbyMugambietal.alim-
itednumberofpublicationspreviouslydescribedtheimpactof
differentcombinationsofpre-andprobioticsoninfantgrowth
Atopic symptoms and safety(34–37). Although investigating different mixtures of
Atbaseline,theprevalenceofatopicsymptoms,especiallyato- pre- and probiotics (in a different nutritional composition),
pic skin symptoms, was higher in the synbiotic group com- all three studies concluded that the use of those specific mix-
pared with the control group of the ITT population (12·0 tures is safe, well-tolerated and supports adequate growth in
and 2·7% of the infants, respectively; Fisher’s exact test, healthy,terminfants.Itshouldbenoted,though,thatthepre-
P=0·013).Duringandattheendofthestudy,nodifferences defined equivalence margins for weight gain during 3 months
were statistically significantly confirmed in atopic symptoms ofinterventioninthesestudiesareconsiderablywiderranging
between the two groups (data not shown). from ±3·9 to ±5·4g/d compared with that applied in our
study (±3·1g/d). Interestingly, if we would have applied a
similar (wider) equivalence margin in the present study, we
Discussion
would have demonstrated equivalence in weight gain per d
This is the first longitudinal study to evaluate the effect of an forthePPpopulationinthisstudyaswell.Althoughprevious
extensively hydrolysed formula with the specific combination studies have demonstrated adequate growth of healthy term
oftheprebioticmixturescGOS/lcFOS(9:1)andtheprobiotic infants on formulas containing various mixtures of pre- and
strainB.breveM-16Vongrowthandtoleranceinhealthy,term probiotics, caution should be used drawing conclusions on
infants.Givenpredefinedequivalencemargins,weightgainper thesafetyofthesemixturesingeneral,sincedifferentmixtures
d following 13 weeks of formula feeding was proven to be can have different physiological effects. Moreover, ‘negative’
equivalent between the synbiotic group and control group results areless likelytobepublished and someclinicalstudies
in the ITT population, but not in the PP population. might even have been stopped prematurely. The secondary
However,theweight-for-age z-scores based on WHO growth outcome measures absolute length and length gain were
standards of infants in the two groups of the PP population lower in the synbiotic group compared with the control
were close to zero during the entire intervention period. group in the PP population, but not in the ITT population,
Absolute length gain was slightly decreased in the synbiotic during the intervention period. This is in contrast to the pre-
population, but the length-for-age z-score values were close and probiotics studies mentioned previously that indicated a
to zero according to WHO growth standards throughout the similar length and/or length gain during the intervention per-
study period for both the synbiotic group and control group. iods(34–37).However,thelength-for-agez-scoresofthesynbio-
sse Additional safety and tolerance parameters like head circum- tic group in this study were not different from the WHO
rP ference, gastrointestinal parameters, number and type of ad- growthstandardforboththeITTandPPpopulations,indicat-
y
tisre verse events, blood parameters indicative for renal or liver ing an adequate growth in height.
vin function, gastrointestinal parameters, and atopic symptoms Thepercentageofinfantswithatleastone(serious)adverse
U e were similar between the two groups following 13 weeks of eventwassimilarbetweentreatmentsgroups(15·0and19·8%
g
d
irb intervention. for the synbiotic group and control group). The type of ad-
m
a Theweight gain per d during 13 weeks of intervention was verse events reported is typical for young infants and no rele-
C
yb similarinbothgroupsinthePPpopulation.Sincethe90%CI vant difference in type of adverse events was observed
e
niln of the difference estimate slightly crossed the predefined between treatment groups. Moreover, the addition of the
o de equivalencemarginof−0·5to+0·5SD,equivalenceinweight pre- and probiotic mixture did not affect formula consump-
h
silb gain during 13 weeks of intervention was not demonstrated tion (ml/d), indicating a good acceptance and palatability.
uP between groups in the PP population. However, the point es- Infantsfedtheexperimental formulahad slightlysofterstools
5
3.6 timateofthedifferenceinthePPpopulationwassmallercom- in the first 4 weeks of the intervention, which is in line with
1
02 pared with that observed in the ITT population in which previouslyreportedstool-softeningeffectsofthisspecificpre-
.snj/7 equivalenceingrowthwasdemonstrated.Hence,themainrea- biotic mixture(38). This is considered to be beneficial, since
1
01 sonfortheinability todemonstrateequivalence ismostprob- breast-fed infants have, in general, a higher stool frequency
.01/g ably the lack of power in the PP population due to a much and softer stools compared with cows’ milk-based formula-
ro.io higher than expected drop-out rate. To safeguard 80% fed infants(39).
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