Probiotics, prebiotics infant formula use in preterm or low birth weight infants: a systematic review

1475-2891-11-58 1475-2891 Review Probiotics, prebiotics infant formula use in preterm or low birth weight infants: a systematic review MugambiNMarynkmugambi@hotmail.com MusekiwaAlfredalfred.musekiwa@gmail.com LombardMartanimartani@sun.ac.za YoungTaryntyoung@sun.ac.za BlaauwReneérb@sun.ac.za

Division of Human Nutrition, Faculty of Medicine and Health Sciences, Stellenbosch University, P.O Box 19063, Tygerberg 7505, South Africa

Wits Reproductive Health & HIV Institute (WRHI), Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

Centre for Evidence-Based Health Care, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa

Nutrition Journal 1475-2891 2012 11 1 58 http://www.nutritionj.com/content/11/1/58 10.1186/1475-2891-11-5822928998 2510201126720122882012 2012Mugambi et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Probiotic Prebiotic Preterm infant Low birth weight infant

Abstract

Background

Previous reviews (2005 to 2009) on preterm infants given probiotics or prebiotics with breast milk or mixed feeds focused on prevention of Necrotizing Enterocolitis, sepsis and diarrhea. This review assessed if probiotics, prebiotics led to improved growth and clinical outcomes in formula fed preterm infants.

Methods

Cochrane methodology was followed using randomized controlled trials (RCTs) which compared preterm formula containing probiotic(s) or prebiotic(s) to conventional preterm formula in preterm infants. The mean difference (MD) and corresponding 95% confidence intervals (CI) were reported for continuous outcomes, risk ratio (RR) and corresponding 95% CI for dichotomous outcomes. Heterogeneity was assessed by visual inspection of forest plots and a chi² test. An I² test assessed inconsistencies across studies. I²> 50% represented substantial heterogeneity.

Results

Four probiotics studies (N=212), 4 prebiotics studies (N=126) were included. Probiotics: There were no significant differences in weight gain (MD 1.96, 95% CI: -2.64 to 6.56, 2 studies, n=34) or in maximal enteral feed (MD 35.20, 95% CI: -7.61 to 78.02, 2 studies, n=34), number of stools per day increased significantly in probiotic group (MD 1.60, 95% CI: 1.20 to 2.00, 1 study, n=20). Prebiotics: Galacto-oligosaccharide / Fructo-oligosaccharide (GOS/FOS) yielded no significant difference in weight gain (MD 0.04, 95% CI: -2.65 to 2.73, 2 studies, n=50), GOS/FOS yielded no significant differences in length gain (MD 0.01, 95% CI: -0.03 to 0.04, 2 studies, n=50). There were no significant differences in head growth (MD −0.01, 95% CI: -0.02 to 0.00, 2 studies, n=76) or age at full enteral feed (MD −0.79, 95% CI: -2.20 to 0.61, 2 studies, n=86). Stool frequency increased significantly in prebiotic group (MD 0.80, 95% CI: 0.48 to 1.1, 2 studies, n=86). GOS/FOS and FOS yielded higher bifidobacteria counts in prebiotics group (MD 2.10, 95% CI: 0.96 to 3.24, n=27) and (MD 0.48, 95% CI: 0.28 to 0.68, n=56).

Conclusions

There is not enough evidence to state that supplementation with probiotics or prebiotics results in improved growth and clinical outcomes in exclusively formula fed preterm infants.

Background

Growth is a major challenge for premature and low birth weight infants (born < 37 weeks gestation or with a birth weight of < 2500 g). They have several factors that put them at risk for nutritional deficiencies resulting in poor growth. Decreased nutrient stores result in low body stores of glycogen, fat, protein, fat soluble vitamins, calcium, phosphorus, magnesium and trace minerals. Preterm infants require increased energy and nutrients for rapid growth and may need a 10 fold increase in weight gain in order to achieve optimum catch up growth 1 2 . To achieve optimum growth for the preterm infant, the goals are to continue the process of intra-uterine growth in an extra-uterine environment until 40 weeks post conception, foster catch-up growth and nutrient accumulation in the post discharge period 3 4 5 6 . A weight gain of 15 to 20 g/ kg/day, length of 0.75 to 1.0 cm/week and head circumference 0.75 cm/week is required. This is difficult to achieve and requires between 130 – 135 kcal / kg /day to maintain this growth rate 3 . Furthermore, infants lose weight after birth (up to 6% to 8% for extreme low birth weight infants) and they often do not regain the weight for up to 1 to 2 weeks 5 . Daily growth monitoring (weight gain, linear and head circumference) then becomes vital.

Preterm infants have immature physiological systems due to an underdeveloped gastrointestinal barrier function as reflected by increased intestinal permeability. As a result, potentially pathogenic bacteria translocate from the intestinal lumen and cause systemic infections 7 . Reducing intestinal permeability is associated with gut maturation which promotes growth and avoids severe infections 4 . In addition, digestive and absorptive capabilities are decreased due to low concentration of lactase, pancreatic lipase and bile salts. Gastrointestinal motility and stomach capacity are decreased which limits feeding volume and gastric emptying. A coordinated suck and swallow is not developed until 32 to 34 weeks gestation. Introduction of enteral feeding maybe delayed due to increased risk of aspiration 1 2 8 9 . Preterm infants in neonatal intensive care units (NICUs) develop a different intestinal microbiota compared to healthy breast fed infants. This is due to decreased exposure to the maternal microbiota, increased exposure to organisms that colonize NICUs, multiple courses of antibiotics and delays in feeding 8 9 .

Humans have consumed probiotics in the form of fermented food, dairy products and more recently infant and toddler formula. Probiotics are defined as “live microorganisms” which when administered in adequate amounts confer a health benefit to the host 10 . The main probiotic organisms used worldwide belong to the genera Lactobacillus and Bifidobacteria and are found in the gastrointestinal micro flora 10 11 . Prebiotics are found in fruit and vegetable components, they are non- digestible food ingredients that benefit the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon and improving the host’s health 12 13 . The most widely studied prebiotics are inulin, fructo-oligosaccharide (FOS) and galacto-oligosaccharide (GOS) which are plant storage carbohydrates in vegetables, cereals and fruit. FOS and inulin are added to different foods as fat and sugar replacements to improve texture or for their functional benefits 12 14 15 16 . Probiotics and prebiotics are added to infant formula to promote an intestinal microbiota resembling that of breastfed infants which have a greater concentration of bifidobacteria and less pathogenic bacteria than formula fed infants 10 17 .

There are a number of ways in which probiotics improve health. Health benefits conferred by probiotic bacteria are strain specific and not species or genus specific 10 . Probiotic bacteria improve health by affecting the immune system in different ways. They increase cytokine production such as Interleukin-6 (IL-6), Interferon- gamma (IFN-γ), Tissue Necrosis Factor – alpha (TNF-α), Interleukin-1beta (IL-1β) and Interleukin-10 (IL-10) 18 . Some strains increase phagocytic activity of peripheral blood leukocytes (monocytes, polymorphonuclear cells). Other strains strengthen the mucosal barrier function by promoting the production of mucosal antibodies and reducing the trans mucosal transfer of antigens. This reduces the intestinal permeability which in turn promotes growth 19 20 21 22 . Probiotics bacteria also enhance production of low molecular weight antibacterial substances produced by epithelial cells and production of short chain fatty acids, the main energy source for colonocytes. This maintains the integrity of colon mucosa 19 23 24 25 26 .

Prebiotics are resistant to digestive enzymes and pH extremes found in the human gastrointestinal tract. They transit through the upper gastrointestinal tract and reach the colon intact where they are selectively fermented by indigenous bacteria, especially bifidobacteria and lactobacilli 12 15 26 27 . Beneficial bacteria (including bifidobacteria and lactobacilli) possess enzymes needed to metabolize prebiotics, while other bacteria (such as E coli, clostridia and salmonella) do not 15 27 . Consumption of prebiotics by preterm formula fed infants results in an increase of beneficial microorganisms in the colon, decreasing harmful bacteria to the levels found in breastfed infants. This improves the gastrointestinal mucosal barrier (decreasing intestinal permeability) which prevents infections and eventually results in improved growth 27 28 . In general the aim of adding probiotics and prebiotics to preterm infant formula is to improve growth, development and decrease infections by promoting an intestinal microbiota resembling that of breastfed infants 9 29 30 .

The effects of probiotics on the intestinal microbiota of premature infants have been varied due to differences on gestational age and products administered. Effects of probiotics on weight gain have also been varied. Administration of Bifidobacteria breve led to improved weight gain while Saccharomyces bourladii did not 9 . With premature infants optimal strains and dose regimens are yet to be examined closely 8 . The effects of prebiotics on the growth of premature infants are not clear. If prebiotic supplementation reduces the risk of Necrotizing Enterocolitis (NEC) or improves feed tolerance in very low birth weight infants is yet to be established 8 9 . Recent systematic reviews (published from 2005 to 2009) on the use of probiotics or prebiotics in preterm infants have focused on prevention of NEC and / or sepsis, impact on diarrhea 31 32 33 34 . These reviews focused on studies that used breast milk and mixed feeds (formula combined with breast milk). This review included infants given only infant formula and focused on growth with clinical outcomes that were not adequately addressed by previous reviews.

The Human Research Ethics Committee at the University of Stellenbosch, South Africa reviewed the review protocol (unpublished), ruled that all data to be collected for this review was from the public domain and was therefore exempt from ethical approval.

Objective

To assess if addition of probiotics or prebiotics to preterm infant formula led to improved growth and clinical outcomes in preterm or low birth weight infants.

Methods

Eligibility criteria

All randomized controlled trials (RCTs), irrespective of language, which compared the use of preterm infant formula containing probiotic(s) or prebiotic(s) to conventional preterm infant formula without or with placebo amongst preterm infants born <37 weeks gestation, low birth weight infants with <2.5 kg at birth and hospitalized, receiving formula feeds and / or parenteral feed were considered. Studies published as abstracts were included if sufficient information could be obtained to assess study quality and obtain relevant study findings.

Outcome measurements

Primary outcomes included: Short term growth parameters (assessed for entire study duration approximately 4 weeks): weight gain (grams/day or grams/week), linear growth (centimeters/week), head growth (cm/week). Secondary outcomes included: Complications: Incidence of NEC (defined as suspected or confirmed positive Bell stage II or more), Sepsis (defined as signs or symptoms of infection and positive blood culture), Other infections (example bacteraemia defined as blood cultured positive for bacteria), Mortality / death. Adverse events during entire study duration: Number of days on parenteral, number of days to full enteral nutrition, maximal enteral feed (millilitres/day, millilitres/kilogram/day, millilitres /kilogram). Feed intolerance: Incidence of vomiting, gastric aspirates, abdominal distension. Stool characteristics: Stooling frequency and stool consistency as firm, loose or watery. Changes in intestinal permeability as measured by ratio of Lactulose / mannitol in urine or other sugar absorption tests (such as lactulose / L – rhamnose ratio, D- xylose, 3-O2- methyl-D- glucose tests). Gastrointestinal (GI) micro flora: number of colony forming units (cfu) of bifidobacteria, lactobacillus and pathogens post intervention).

Search method for identification of studies

A literature search in all languages was conducted on electronic databases which included The Cochrane Central Register for Controlled Trials 2009, Scopus (1990 to 19/01/2010), EBSCO host (1960 to 15/11/2009), OVID (1950 to 01/12/2009), SPORT Discus (1960 to 19/01/2010), Web of Science (1970 to 19/01/2010), Science Direct (1950 to 30/11/2009), EMBASE (1980 to 01/12/2009), CINAHL (1981 to 19/01/2010), PUBMED / MEDLINE (1966 to 10/04/2010), Latin American Caribbean Health Sciences literature (LILACS), (1965 to 19/01/2010), NLM Gateway (1950–1966). RCTs published in non-English language journals were translated by independent translators who were familiar with the subject matter. The search strategy used to search PUBMED is shown on Table 1. This search strategy was modified to search other electronic databases.

Table 1

Search (probiotic* OR prebiotic*) AND (infant formula* OR infant feeding OR formula OR formula milk) AND (preterm or premature or low birth weight babies) AND (randomized controlled trial* OR controlled clinical trial* OR random allocation*) Limits: Human

Search (probiotic* infant formula* OR prebiotic* infant formula* OR prebiotic* OR probiotic*) AND (infant formula* OR infant feeding) AND (premature OR preterm) AND (randomized controlled trial* OR controlled clinical trial OR random allocation* OR double blind method OR single-blind method OR clinical trial OR placebo* OR random* OR research design OR comparative study OR follow-up studies OR prospectiv* OR volunteer* OR control* (singl* OR doubl* OR trebl* OR tripl*) NEAR (blind* OR mask*) Limits: Human

We conducted a hand search on abstracts of major conference proceedings such as the Pediatric Academic Society meetings (www.pas-meetings.org, www.abstracts2view.com), cross checked references cited in RCTs and in recent reviews (published from 2005 to 2009) for additional studies not identified by electronic searches and specialty journals which were not included in any database such as Pediatrika, Chinese Journal of Microecology and International Journal of Probiotics and Prebiotics.

To identify on-going and unpublished trials, we contacted experts in the field, manufacturers of infant formula containing probiotics and prebiotics, we searched web sites of companies that have conducted or were conducting RCTs on probiotics and prebiotics e.g. Pfizer (www.pfizerpro.com/clinicaltrials), Chris Hansen Laboratory (www.chr-hansen.com/research_development/documentation.html). We also searched prospective trial registries such as World Health Organisation (WHO) International Clinical Trials Registry Platform Search Portal (www.who.int/trialsearch), Clinical Trials.gov register (www.clinicaltrials.gov), Current Controlled Trials metaRegister of Controlled Trials [mRCT] (www.controlled-trials.com/mrct) and www.clinicaltrialresults.org.

Selection of studies

Two reviewers (MM, ML) independently reviewed all abstracts, citations and identified potentially eligible studies. The full reports of eligible studies were retrieved by one reviewer (MM) and the pre-specified selection criteria applied independently by two reviewers (MM, ML) using a study eligibility form. (Figure 1) If more than one publication of a study existed, all reports of the study were grouped together under one study name. Any disagreements between the reviewers were resolved through discussion. If disagreements could not be resolved a third party was consulted. Trial authors were contacted if eligibility was unclear.

Figure 1

Study eligibility form

Study eligibility form.

Assessment of quality of evidence

Two reviewers (MM, ML) independently assessed the risk of bias of included studies as described in the Cochrane Handbook for Systematic Reviews for Interventions according to the following 6 components. 1) sequence generation; 2) allocation concealment; 3) blinding; 4) incomplete outcome data; 5) selective outcome reporting; and 6) other sources of bias 35 . Where necessary, trial authors were contacted for clarification on the methodology of their studies. Any disagreements regarding risk of bias were resolved through discussion between MM, ML and RB.

Data extraction and management

Two reviewers (MM, ML) independently extracted data using a pre tested data extraction form. The reviewers (MM, ML) cross checked data and resolved any differences through discussion. One reviewer (MM) entered the data in Review Manager (RevMan 5) and the other reviewer (ML) validated the data. Trial authors were contacted for missing data or for clarification.

Data synthesis and management

Results for probiotic and prebiotic studies were analysed separately. For continuous outcomes the mean difference (MD) and corresponding 95% confidence intervals (CI) were calculated. For dichotomous outcomes, the risk ratio (RR) and corresponding 95% CI were calculated. Trial authors were contacted if there was missing data in their reports. Available case analysis was used where there was missing data. The potential impact of the missing data on the results of the review is addressed in the discussion section. Heterogeneity of the trials used in the review was assessed by visually inspecting the forest plots to detect overlapping confidence intervals and by performing a chi² test. A p<0.1 was considered statistically significant. An I-square test (I²) was used to test for inconsistencies across studies. If the I² exceeded 50% and visual inspection of the forest plot supported these results, this represented substantial heterogeneity.

If the included studies were not clinically diverse and had similar outcome measures, a Meta - analysis was carried out in Review Manager software (RevMan 5) by one review author (AM). For continuous data, if heterogeneity was low, an inverse variance fixed-effect method was used. If heterogeneity was high, an inverse variance random-effects method was used. For dichotomous data, if heterogeneity was low, a Mantel-Haenszel fixed-effects method was used. If heterogeneity was high, a Mantel- Haenszel random-effects method was used. The source of heterogeneity was explored through subgroup analysis with respect to the type of intervention. If studies were too diverse, no Meta-analysis was conducted and a narrative synthesis was provided. We had intended to perform sensitivity analysis with respect to study quality in order to investigate the robustness of our findings but this could not be done mainly because most of the meta-analysis had too few studies (mostly two) to warrant sensitivity analysis. In some cases, all the studies in the meta-analysis had similar study quality thus rendering sensitivity analysis inappropriate.

Results

Results of the search and description of studies

Electronic search of available databases yielded 151 citations. After reading titles, abstracts, the duplicate reports were removed and 35 potentially relevant articles were identified. A hand search yielded 4 more articles. The full text reports were retrieved and reviewed for eligibility. One study was published in two other reports. The three studies were considered as one study since they reported the same identical study and are referred to as Boehm 2002 in this review 36 37 38 . Eight published studies (four probiotic and four prebiotic studies) 36 39 40 41 42 43 44 45 and five on-going studies were included in this review 46 47 48 49 50 51 . The process followed is shown in Figure 2. Table 2 gives a list of 27 studies which were excluded for: use of breast milk or mixed feeds (18 studies), no use of probiotic or prebiotic (2 studies), being a follow –up study, not RCT (3 studies), duplicate publishing (1 study); using different inclusion criteria with different outcomes (2 studies) and type of feed was unspecified (1 study). No eligible studies were excluded for failure to report the review’s pre-specified outcomes.

Figure 2

Process followed in the selection of studies

Process followed in the selection of studies.

Table 2

Reasons for exclusion of studies

Use of breast milk or mixed feeds (breast milk and formula)

use of probiotic, prebiotic

Follow up - study, Not RCT

Duplicate publishing

Different inclusion criteria and outcomes

Type of feed unspecified

Agarwal 2003 52

Lin H-C 2008 53

Riskin 2009 54

Andrews 1969 55

Chou I-C 2009 56

Stansbridge 1993 57

Cukrowska 2002 58

Karvonen 2002 51

Bin-Nun 2005 59

Manzoni 2006 60

Rouge 2009 61

Taylor 2009 62

Hoyos 1999 63

Wang 2007 64

Dani 2002 65

Millar 1993 66

Samanta 2005 67

Lidesteri 2003 68

Kitajima 1997 69

Mohan 2006 70

Westerbeek 2008 71

Lee 2007 72

Mohan 2008 73

Westerbeek 2010 74

Lin H-C 2005 75

Patole 2005 76

Yong Gu 2009 77

A summary of the included probiotic, prebiotic and on-going studies are shown in Tables 3, 4 and 5. The included probiotic studies (N=212) were conducted in Greece, Italy and United States of America (USA). Treatment duration was 30 days using different probiotics. All four probiotic studies reported short term growth parameters (weight gain) which were recorded daily during the entire study duration [Table 3]. None of the probiotic studies reported data on: other types of infections, use of parenteral nutrition, feed intolerance (gastric aspirate [ml], abdominal distension) and stool consistency. The included prebiotic studies (N=126) were conducted in conducted in Greece, Italy, and Germany. Treatment duration ranged from 14 days to 28 days. All four prebiotic studies reported short term growth parameters (weight gain, length, head growth) which were recorded at different intervals during the entire study duration [Table 4]. None of the prebiotic studies reported data on: complications (NEC, sepsis, other types of infections, death / mortality), use of parenteral nutrition, feed intolerance (vomiting, gastric aspirate [ml], abdominal distension) and changes in intestinal permeability.

Table 3

Costalos 2003 [ 39

Indrio 2008 [ 42

Reuman 1986 [ 41

Stratiki 2007 [ 40

Location of study

Athens, Greece

University of Bari, Policinico, Italy

Gainesville, Florida, USA

Alexandra Regional Hospital, Greece

Participants - inclusion criteria

28 - 32 weeks gestation

3- 5 days old, appropriate for gestational age, preterm infants with normal agpar scores

Premature infants, <2000g at birth, less than 72 hours old (>24 old to <72 hours old)

27 to 37 weeks gestation, in stable state

Number of study participants

Study group=51 , Placebo = 36

Study group = 10 , Placebo = 10

Study group = 15, Placebo = 15

Study group = 41, Placebo = 34

Probiotic bacteria used

Saccharomyces Bourlardii

Lactobacillus Reuteri ATCC 55730

Lactobacillus acidophilus

Bifidobacteriumlactis

Dose of probiotic

10⁹cfu at 50mg/kg every 12 hours

1 X 10⁸cfu/day

9 X 10⁶cfu/ml formula

2 X 10⁷cfu/g milk powder

Placebo

Maltodextrin

Indistinguishable placebo

Conventional preterm formula

Dose of placebo

50 mg /kg / 12 hours

Not reported

Treatment initiation

1st week of life as soon as enteral feed was tolerated

At 3–5 days of life

1st 72 hours of life

1st 2 days of life

Treatment duration

30 days

Not specified

30 days

Reported Outcomes

Growth parameters

Weight gain

Weight gain, Linear growth, Head circumference

Timing and duration of measurement of growth parameters

Measured daily for 30 days

Measured daily, duration not specified

Weight gain: measured daily, Lineargrowth (measured weekly), Head circumference (measured weekly)

Feed tolerance

Number of days to full enteral feed, Maximal enteral feed, vomiting

Maximal enteral feed

Number of days to full enteral feed, Maximal enteral feed

Stool characteristics

Stooling frequency

Complications

NEC, Sepsis

Mortality / death

NEC, Sepsis

Intestinal permeability

Changes in Intestinal permeability

Changes in gastrointestinal microflora

cfu of bifidobacteria, lactobacillus, pathogens

cfu of bifidobacteria

Table 4

Boehm 2002 [ 36

Indrio 2009 [ 43

Kapiki 2007 [ 45

Mihatsch 2006 [ 44

Location of study

Milan, Italy

University of Bari, Policinico, Italy

Athens, Greece

Ulm University, Germany

Participants - entry criteria

<32 weeks gestation

Healthy preterm newborns

≤ 36 weeks gestation

< 1500 g birth weight

Number of study participants

Study group = 15, Placebo = 15

Study group = 10 , Placebo = 10

Study group = 36, Placebo = 20

Study group = 10, Placebo = 10

Prebiotic used

GOS 90%, FOS 10%

scGOS, lcFOS at ratio 9:1

FOS

GOS, FOS

Dose of prebiotic

1g/dl

0.8 g/dl

0.4g/100ml

1g/dl

Placebo

Maltodextrin

Dose of placebo

1 g/dl

0.8 g/dl

0.4 g

1.8 / 90 ml

Treatment initiation

When enteral feed ≥ 80 mls /kg/day was tolerated

Not clear

Exclusively formula fed at start of study

At full enteral feed at start of study

Treatment duration

28 days

15 days

14 days

15 days

Reported Outcomes

Growth parameters

Weight gain, linear growth

Weight gain, linear growth, head growth

Weight gain

Timing and duration of measurement of growth parameters

Measured on days 1, 7, 14, 28

Measured before start of study, days 3, 5, 15

Measured on days 1, 7, 14

Weight gain: reported as “Average weight gain during study.”

Feed tolerance

Number of days to full enteral feed, maximal enteral feed

Number of days to full enteral feed

Number of days to full enteral feed, maximal enteral feed

Stool characteristics

Stooling frequency, consistency

Stool viscosity, Stooling frequency, consistency

Changes in gastrointestinal microflora

cfu bifidobacteria

cfu bifidobacteria, pathogens

Table 5

Jacobs 2007 [ 46

Lozano 2008 [ 47

Al-Hosni 2010 [ 48

Patole 2009 [ 49

Underwood 2009 [ 50

Location of study

Australia

Colombia

USA

Australia

USA

Participants - inclusion criteria

<32 weeks gestation, <1500 g birth weight, 1–3 days old

Birth weight <2000 grams, < 48 hours of age, admission in NICU, Hemodynamic-ally stable

Extremely Low Birth weight infants: < 1000 grams, 1 to 14 old, intention to start enteral feeds

32 weeks Gestation and 6 days, <1500g birth weight, ready to commence on enteral feeds for up to 12 hours

< 500grams birth weight, age less than 33 weeks gestation, exclusively formula fed

Probiotic bacteria used

Bifidobacteriuminfantis, BifidobacteriumBifidus, Streptococcus thermophilus

Lactobacillus reuteri DSM 17938

Lactobacillus rhamnosus GG, Bifidobacteriuminfantis

Lactobacillus acidophilus 375 million, bifidobacteriumbifidum, bifidobacteria longus

1. ProlactPlus

2. GOS

3. Bifidobacteriuminfantis

4. Bifidobacteriumanimalis

Dose

1X10⁹

1X108 CFU in 5 drops of oil suspension 1/ day until discharge.

L rhamnosus: 500 million cfu, B.infantis: 500 million cfu

L. acidophilus:375 m organisms, B bifidum, B. longus: 125 million organisms

1. week 1 95:5 to week 5 75:25

2. week: 0.25g/dL, to week 5: 2.0 g/dL

3. week 1: 5X10⁷, to week 5: 4.2 X10⁹

4. week 1: 5X10⁷, to week 5: 4.2 X10⁹

Start date of study

July- 2007

August 2008

February 2008

June 2009

Reported Outcomes

Sepsis,

Sepsis

Average weight gain

Sepsis

Fecal microflora

NEC

Growth velocity

NEC

Death

Feed tolerance

All-cause mortality

Frequency of events

Volume of feed/day

Time to reach full feeds (150 mls/kg/day)

Length of hospital admission

Gut colonisation by probiotic

Number of antibiotic courses

Days to full enteral feeds

Risk of bias

The quality of the included studies was assessed across six domains using guidelines from the Cochrane Handbook for Systematic Reviews of Interventions 35 (Figure 3).

Figure 3

Methodological quality of included studies

Methodological quality of included studies.

Random sequence generation: Three trials described clearly the methods used for random sequence generation 40 41 44 . Mihatsch used computer generated random lists with variable block sizes 44 . Stratiki used balance block randomization using random numbers 40 and Reuman used random numbers list combined with the last digit of the patients’ medical record 41 . The method used for random sequence generation was not clearly described 5 studies 36 39 42 43 45 .

Allocation Concealment: In two trials treatment allocation was adequately concealed 33 40 . In the Stratiki trial, treatment allocation was conducted by a third party who was not involved in the study (Nutritional service) 40 . Mihatsch used precoded sachets in sealed envelopes 44 . In one study treatment allocation was not adequately concealed because the method used was alternation, matching of infants by birth weight and gestational age 41 . In the rest of the studies, allocation concealment was not clearly demonstrated or described 36 39 42 43 .

Blinding: Blinding of study participants, care providers and assessors was clearly done in 4 trials 39 40 41 44 . In the other 4 trials, there was not enough information given on the blinding method to make a judgement 36 42 43 45 .

Incomplete outcome data: Reported outcome data was satisfactory for all the eight included studies. Five studies had no missing outcome data 36 41 42 43 44 . In other three studies, the missing outcome data was balanced across the intervention groups with similar reasons reported 39 40 45 .

Selective reporting (reporting bias): In all eight studies, the pre-specified outcomes in the methods section were reported in the results section 36 39 40 41 42 43 44 45 .

Other potential sources of bias: Only one trial had a baseline imbalance which was a potential source of bias. Costalos had 51 infants enrolled in the treatment group and 36 infants in the placebo group. No explanation was presented whether the imbalance was due to a problem at randomization stage 39 . All other studies appeared to be free from other potential sources of bias.

Effects of interventions

Probiotics versus control

Four studies investigated the effect of probiotic administration versus no probiotic (control group) 39 40 41 42 .

Primary outcomes: short term growth parameters

Weight gain

All four studies reported on weight gain 39 40 41 42 . Results from two studies (n=34) were pooled in a meta-analysis 41 42 . There was no statistically significant difference in weight gain (g/day) between the probiotic and control groups (MD 1.96, 95% CI: -2.64 to 6.56). No statistically significant heterogeneity was observed (Chi²=0.18, p=0.67, I²=0%) (Figure 4)

Figure 4

Effect of probiotic administration on weight gain (g/day)

Effect of probiotic administration on weight gain (g/day).

Two studies 39 40 reported their results using medians and could not be pooled in a meta - analysis. Costalos 2003 reported no statistically significant difference in weight gain (g/week) between the probiotic and control groups (p>0.05) [median (Interquartile range) of 163.5 (17.7) for the probiotic group (n=51) compared to 155.8 (16.5) for the control group (n=36)] 39 . Stratiki 2007 also reported no statistically significant difference in weight gain (g/day) between the probiotic and control groups (p=0.144) [median (range) of 28.3 (12 to 38) for the probiotic group (n=41) compared to 30 (10 to 40) for the control group (n=34)] 40 .

Linear growth

Only one study reported this outcome but found no statistically significant difference in length gain (cm/week) between the probiotic and control groups (p=0.124) [median (range) of 1.4 (0 to 3) for the probiotic group (n=41) compared to 1.5 (0 to 3.5) for the control group (n=34)] 40 .

Head growth

Only one study reported this outcome but found no statistically significant difference in head growth (cm/week) between the probiotic and control groups (p=0.124) [median (range) of 1.1 (0.45 to 1.9) for the probiotic group (n=41) compared to 0.9 (0 to 2) for the control group (n=34)] 40 .

Secondary outcomes

Complications

Necrotizing enterocolitis [NEC]

Two studies (n=162) reported on NEC and their results were pooled in a meta-analysis 39 40 . Administration of probiotics failed to significantly reduce the risk of NEC compared to controls (RR 0.42, 95% CI: 0.15 to 1.16). No significant heterogeneity was observed (Chi²=1.06, p=0.30, I²=6%) (Figure 5).

Figure 5

Effect of probiotic administration on NEC

Effect of probiotic administration on NEC.

Sepsis

Two studies (n=162) reported on sepsis and their results were pooled in a meta-analysis 39 40 . Administration of probiotics failed to significantly reduce the risk of sepsis compared to controls (RR 0.40, 95% CI: 0.11 to 1.45. No significant heterogeneity was observed (Chi²=1.18, p=0.28, I²=15%). (Figure 6)

Figure 6

Effect of probiotic administration on sepsis

Effect of probiotic administration on sepsis.

Other infections

No study reported on this outcome.

Mortality

Only one study 42 reported on mortality. The risk ratio for this one study (n=30) was calculated and it showed that the probiotics failed to significantly reduce the risk of death compared to the control (RR 0.33, 95% CI: 0.04 to 2.85).

Number of days on parenteral nutrition

No study reported on this outcome.

Number of days to full enteral feed

Two studies reported this outcome but their results could not be pooled in a meta-analysis because they reported the outcome in terms of medians and ranges 39 40 . Costalos 2003 reported no statistically significant difference in the number of days to full enteral feeding between the two groups (p>0.1) [median (IQR) of 9.3 (2.7) for the probiotic group (n=51) and 9.9 (4.5) for the control group (n=36)] 32 . Stratiki 2007 also reported no statistically significant difference in the number of days to full enteral feeding [median (range) of 10 (0 to 52) for the probiotic group (n=41) and 10 (0 to 30) for the control group (n=34)] 40 .

Maximal enteral feed

All four studies reported on this outcome 39 40 41 42 . Results from two studies (n=34) were pooled in a meta-analysis as they both reported the average amount of feeding (ml/day) in terms of mean (SD) 41 42 . There was no statistically significant difference in the mean amount of feeding (ml/day) between the probiotic and control groups (MD 35.20, 95% CI: -7.61 to 78.02) No statistically significant heterogeneity was observed between the studies (Chi²=1.65, p=0.20, I²=39%).

Costalos 2003 reported no statistically significant difference in the milk intake (ml/kg/day) at maximal enteral feeding (p>0.1) [median (IQR) of 155 (15) for the probiotic group (n=51) versus 148 (13) for the control group (n=36)] 39 . Stratiki 2007 also reported no statistically significant difference in the maximal milk intake (ml/kg/day) (p=0.624) [median (range) of 210 (165 to 250) for the probiotic (n=41) group versus 192 (120 to 250) for the control group (n=34)] 40 .

Feed tolerance: vomiting, gastric aspirate, abdominal distension

Two studies (n=107) reported on vomiting and were pooled in a meta-analysis 39 42 . There was no statistically significant difference in the frequency of vomiting between the probiotic and control groups (RR 0.78, 95% CI: 0.18 to 3.37). No statistically significant heterogeneity was observed (Chi²=0.41, p=0.52, I²=0%).

In all four probiotic studies, there were no reported incidences of gastric aspirates, abdominal distension or diarrhea. Authors were further contacted for clarification and one responded 42 and stated categorically that none of these symptoms were observed.

Stool characteristics

Stool frequency

Only one study (n=20) reported stool frequency as the number of episodes of evacuations per day in terms of mean (SD) 42 . The mean difference for this one study was calculated and it showed that probiotic consumption resulted in a statistically significant larger number of stools per day compared to the control group (MD 1.60, 95% CI: 1.20 to 2.00).

Stool consistency

No study reported on the effects of probiotics on stool consistency.

Changes in intestinal permeability

Two studies reported this outcome but their results could not be pooled in a meta-analysis 39 40 . The studies used two different tests to test for intestinal permeability. Costalos 2003 used a 1-hour D-Xylose blood test and reported no statistically significant difference between the two groups (p>0.1) [median (IQR) of 1.5 (0.4) millimols/L for the probiotics (n=51) and 1.35 (0.3) mmol/L for the control (n=36)] 39 . Stratiki 2007 used a lactulose/mannitol (L/M) urine test and reported no statistically significant difference in the L/M ratios between the probiotic and control groups (p=0.073) but the values for median (range) were presented in a figure from which they could not be accurately extracted 40 .

Changes in gastrointestinal micro flora

Bifidobacteria

Two studies reported on bifidobacteria but their results could not be pooled in a meta-analysis 39 40 . Costalos 2003 reported a significantly higher log viable Bifidobacteria counts per gram of positive infants in the probiotics group compared to the controls (p<0.001) [median (IQR) of 2.65 (0.083) for the probiotics group (n=51) and 2.27 (0.075) for the control group (n=36)] 39 . Stratiki 2007 reported bifidobacteria in terms of log 10 cfu/g wet feces but found no statistically significant difference between the two groups (p=0.075) [median (range) of 9.7 (7.5-10.3) for the probiotics group (n=41) and 8.9 (7.2-10.2) for the control group (n=34)] 40 .

Lactobacillus

Only one study reported on lactobacillus 39 . This study reported no statistically significant difference in the log viable bacterial lactobacillus counts per gram of positive infants between the two groups (p>0.05) [median (IQR) of 1.57 (0.285) for the probiotics group (n=51) and 1.42 (0.287) for the control group (n=36)].

Pathogens

Only one study reported this outcome (enterococci, bacteroides, and staphylococci) in terms of the median (IQR) of log viable bacterial counts per gram of positive infants 39 (Table 6). The study reported significantly higher counts of Enterococci (p<0.05) and Staphylococci (p<0.001) in the probiotic group compared to the controls. However, the study found no statistically significant difference in the counts of bacteroides between the two groups (p>0.05).

Table 6

Costalos 2003 39

Median (IQR)

Pathogens

Probiotic

Control

n= 51

n=36

Enterococci

2.14 (0.359)

2.19 (0.138)

Bacteriodes

2.17 (0.164)

2.25 (0.363)

Staphylococci

1.23 (0.869)

0.6 (0.281)

Prebiotic versus control

Four studies investigated the effect of prebiotics administration versus no prebiotics (control group) 36 43 44 45 .

Primary outcomes: short-term growth parameters

Weight gain

All four studies reported on weight gain 36 43 44 45 . Results from three studies (n=106) were pooled in a meta-analysis 36 43 45 . Moderate heterogeneity was observed between the studies (Chi²=4.04, p=0.13, I²=51%). An investigation of heterogeneity by subgroup analysis with respect to the prebiotic type used (GOS/ FOS versus FOS only) yielded statistically significant subgroup differences (Chi²=4.04, df=1, p=0.04, I²=75.2%) implying that prebiotic type may be the source of heterogeneity. There was no statistically significant heterogeneity between the two studies in the GOS/ FOS subgroup (Chi²=0.01, df=1, p=0.94, I²=0%) 36 43 . The results for the GOS/FOS subgroup yielded no significant difference in weight gain (g/ day) between the two groups (MD 0.04, 95% CI: -2.65 to 2.73, n=50, 2 studies) while the other FOS subgroup yielded a significantly higher weight gain in controls compared to the prebiotics (MD −4.60, 95% CI: -8.24 to −0.96, n=56, 1 study). (Figure 7) Sensitivity analysis with respect to study quality could not be done because all three studies were of poor quality since the methods used for sequence generation, allocation concealment and blinding were all not clear.

Figure 7

Effect of prebiotic administration of weight gain (g/day)

Effect of prebiotic administration of weight gain (g/day).

Mihatsch 2006 reported no statistically significant difference in weight gain (g/kg/day) between the two groups (p=0.4) [median (range) of 17.6 (8.1 to 23.4) for the prebiotic group (n=10) compared to 13 (9.3 to 21.9) for the control group (n=10)] 44 .

Linear growth

Three studies reported on length gain 36 43 45 . Meta-analysis of the results from these three studies (n=106) revealed significant heterogeneity between the three studies (Chi² = 139.41, df = 2, p < 0.00001, I² = 99%). An investigation of heterogeneity by subgroup analysis with respect to the prebiotic type used (GOS/ FOS versus FOS only) yielded statistically significant subgroup differences (Chi²=139.41, df=1, p<0.00001, I²=0%) implying that prebiotic type may be the source of heterogeneity. There was no statistically significant heterogeneity between the two studies in the GOS/ FOS subgroup (Chi²=0.17, df=1, p=0.68, I²=0%). 36 43 . The results for the GOS/FOS subgroup yielded no statistically significant difference in length gain (cm/ week) between the two groups (MD 0.01, 95% CI: -0.03 to 0.04, n=50, 2 studies) while the other FOS subgroup yielded a significantly higher length gain (cm/ week) in prebiotics compared to the controls (MD 0.30, 95% CI: 0.27 to 0.33, n=56, 1 study). (Figure 8) Sensitivity analysis with respect to study quality could not be done because all three studies were of poor quality since the methods used for sequence generation, allocation concealment and blinding were all not clear.

Figure 8

Effect of prebiotic administration of linear growth (cm/week)

Effect of prebiotic administration of linear growth (cm/week).

Head growth

Two studies reported on head growth (cm/week) 43 45 . Meta-analysis of the results from these two studies (n=76) failed to yield statistically significant difference in head growth (MD −0.01, 95% CI: -0.02 to 0.00). No significant heterogeneity was detected between the two studies (Chi² = 0.10, p =0.75, I² = 0%).

Secondary outcomes

Complications

No prebiotic study reported on Necrotizing Enterocolitis (NEC), Sepsis, other infections and mortality.

Feeding tolerance

Number of days on parenteral nutrition

No study reported on parenteral nutrition.

Age at full enteral feed

Two studies reported on age at full enteral feeds 36 45 . Meta-analysis of the results from these two studies (n=86) did not find statistically significant difference in the age at full enteral feed (MD −0.79, 95% CI: -2.20 to 0.61). No significant heterogeneity was detected between the two studies (Chi² =1.16, p =0.28, I² = 14%).

Maximal enteral feed

Two studies reported on this outcome but their results could not be pooled in a meta-analysis 36 44 . Boehm 2002 reported the feeding volume (ml/kg/day) in terms of the mean (SD) and therefore a mean difference was calculated. There was no statistically significant difference in feeding volume between the prebiotics group (n=15) and control groups (n=15) (MD −4.10, 95% CI: -18.16 to 9.96) 36 .

Mihatsch 2006 reported no statistically significant difference in the average formula intake within the study period (ml/kg/d) between the two groups (p=0.35) [median (range) of 156 (127 to 165) for the prebiotic group (n=10) compared to 151 (117 to 169) for the control group (n=10)] 44 .

Feed tolerance: vomiting, gastric aspirate, abdominal distension, diarrhea

All four studies reported this outcome 36 43 44 45 . In all 4 studies (n=126), there were no observed incidences of feed intolerance. There was no vomiting, gastric aspirate removed, no abdominal distension or diarrhea reported. All infants tolerated the preterm formula with prebiotic or control. From further communication with study authors, 2 study authors 43 44 responded that none of these outcomes were observed.

Stool characteristics

Stool frequency

Three studies reported on stool frequency 36 44 45 . Two studies reported the results in form of mean (SD) of the number of stools per day (number/ day) 36 45 . Meta-analysis of results from these two studies (n=86) showed a significantly higher stool frequency in the prebiotic group compared to the control group (MD 0.80, 95% CI: 0.48 to 1.1). No significant heterogeneity was detected between the two studies (Chi² =0.13, p =0.72, I² = 0%) (Figure 9).

Figure 9

Effect of prebiotic administration on stool frequency

Effect of prebiotic administration on stool frequency.

Mihatsch 2006 reported no statistically significant difference in stool frequency between the two groups (p=0.059) [median (range) of 3.6(1.7 to 6.9) stools/day in prebiotic group (n=10) compared to 2.6 (2 to 4.9) stools/day in control group (n=10)] 44 .

Stool consistency

Three studies reported on stool consistency but using three different scales of measurement 36 44 45 . Although two studies 36 45 both measured consistency in form of a scale ranging from 1 to 5 and reported their results as mean (SD), they could not be pooled in a meta-analysis because their scales were going in opposite directions; Boehm 2002 (1=watery, 2=soft, 3=seedy, 4=formed, 5=hard) 36 . Kapiki 2007 (5=watery, 4=loose, 3=soft, 2=firm, hard=1) 45 . The mean differences for these two studies were therefore calculated separately.

In Boehm 2002, the stools from the prebiotic group (n=15) were significantly more watery as compared to the control group (n=15). (MD −0.91, 95% CI: -1.41 to −0.37) 36 . In Kapiki 2007, the stools from the prebiotic group (n=36) were significantly harder as compared to the control group (n=20). (MD −0.34, 95% CI: -0.66 to −0.02) 45 .

Mihatsch 2006 reported a statistically significantly lower stool viscosity at day 14 (Newtons) for the prebiotics compared to controls (p=0.006) [median (range) of 31.8 (1.9 to 67.3) in the prebiotic group (n=10) compared to 157.5 (24.1 to 314.0) in the control group (n=10)] 44 .

Changes in intestinal permeability

No prebiotic study reported on changes in intestinal permeability.

Changes in gastrointestinal micro flora

Bifidobacteria

Two studies reported on this outcome 36 45 . Meta-analysis of these two studies (n=84) revealed statistically significant heterogeneity between the two studies (Chi² =7.63, p =0.006, I² = 87%). An investigation of heterogeneity by subgroup analysis with respect to the prebiotic type used (GOS/ FOS versus FOS only) yielded statistically significant subgroup differences (Chi² =7.63, p =0.006, I² = 86.7%) implying that prebiotic type may be the source of heterogeneity. The results for the GOS/FOS subgroup yielded significantly higher bifidobacteria counts in prebiotics compared to controls (MD 2.10, 95% CI: 0.96 to 3.24) 36 . The other FOS subgroup also yielded significantly higher bifidobacteria counts in prebiotics compared to controls (MD 0.48, 95% CI: 0.28 to 0.68) 45 (Figure 10).

Figure 10

Effect of prebiotic administration on total counts of Bifidobacteria

Effect of prebiotic administration on total counts of Bifidobacteria.

Lactobacilli

Only one study 36 reported this outcome but the actual values were not given.

Pathogens [Post-intervention]

Two studies reported on this but their results could not be pooled in a meta-analysis 36 45 . Boehm 2002 reported the sum of clinically relevant pathogens at the end of the intervention period in the form of mean (SD) log cfu/g stool. The values were used to calculate the mean difference which showed that the sum of the studied pathogens was significantly lower in the prebiotic group (n=12) compared to the control group (n=13). (MD −0.43, 95% CI: -0.79 to −0.07) 36 .

Kapiki 2007 reported this outcome (staphylococci, E. coli, bacteroides, and enterococci) in terms of mean (SD) log 10 CFU/g wet feces 45 . Mean differences for each of these pathogens were calculated. There was no statistically significant difference in the number of staphylococci (MD 0.00, 95% CI: -0.17 to 0.17) between the two groups but there were significantly fewer E. coli (MD −1.69, 95% CI: -1.85 to −1.53) and enterococci (MD −0.80, 95% CI: -0.99 to −0.61) in the prebiotic group (n=36) compared to the control group (n=20). With regards to bacteroides, there were significantly more bacteroides in the prebiotic group (n=36) compared to the control group (n=20) (MD 0.50, 95% CI: 0.36 to 0.64) 45 .

Discussion

The objective of this review was to assess if addition of probiotics or prebiotics to preterm infant formula led to improved growth and clinical outcomes in preterm or low birth weight infants. Studies that used breast milk or mixed feeds (breast milk and infant formula) were excluded. All RCTs evaluated probiotics or prebiotic use in preterm infants, were of small sample size, varied in enrolment criteria, intervention, treatment initiation and duration.

Summary of main findings

Probiotics

This review was under powered to detect clinically important differences in primary outcomes (weight gain, linear growth, head growth) because of the few number of studies, small sample size (n=34) and poor methodological quality of studies. This review found no significant effect on weight gain from use of probiotics added to infant formula. There was also no significant probiotic effect on linear and head growth from the one study measuring these two outcomes. Probiotic supplementation failed to significantly reduce the risk of complications such as NEC, sepsis and death compared to control group. Outcomes such as number of days on parenteral nutrition and other infections were not reported. There was no significant difference in the amount of feed volume (ml/day) and frequency of vomiting between study groups. Preterm infant formula with probiotics was well tolerated as no gastric aspirates, abdominal distension or diarrhea was reported. Effects of probiotics on stool characteristics were under reported. Results from one study showed probiotics supplementation did result in a larger number of stools per day.

Effects on intestinal permeability could not be evaluated since two different laboratory tests (lactulose / mannitol ratio and D- xylose tests) were reported and the results could not be pooled. Sugar absorption tests (such as lactulose / mannitol ratio) are a direct measure of intestine integrity which reflects gut maturation and in research; they demonstrate the effects of experimental therapy 78 79 . Monitoring changes in intestinal permeability in preterm infants is essential since there is evidence that initiation of enteral feeds decreases intestinal permeability 78 80 . However, this could not be established in this review. Other outcomes such as age at full enteral feeds and intestinal micro flora (pathogens) could not be evaluated as medians (inter quartile ranges) were reported. No probiotic study reported any data on low birth weight infants therefore no conclusions could be made on this population.

The included probiotic studies had short treatment duration of 30 days. This confirms the European Society for Pediatric, Gastroenterology, Hepatology and Nutrition (ESPGHAN) statement that there is a “lack of published evidence on clinical benefits from long term use of probiotic containing infant formula” 81 . This review confirms that there is a need for long term follow-up RCTs on preterm infants. Live probiotic bacteria were used in the trials. There have been few reports of bacteraemia from probiotic use in the biomedical literature 82 83 84 . There were no cases of sepsis reported as a result of probiotic consumption in the included studies. In recent reviews, the time to reach full enteral feeds was earlier in the preterm infants given probiotics with breast milk or mixed feeds. This review could not evaluate this outcome. Well-designed RCTs with similar feeding regimes are needed to evaluate this outcome.

Prebiotics

This review was under powered to detect clinically important differences in primary outcomes (weight gain, linear growth, head growth) because of few number of studies, small sample size (n=106) and poor methodological quality of studies. Addition of prebiotic combinations of GOS /FOS or FOS alone to preterm infant formula did not have any significant effect on weight gain. Addition of GOS / FOS to preterm infant formula did not have any effect on linear growth. However, addition of FOS alone did have a significant effect on linear growth. Neither GOS / FOS combination nor FOS alone had any effect on head growth.

None of the prebiotic studies reported on NEC, sepsis, other infections, mortality (death), parenteral nutrition or changes in intestinal permeability; therefore these outcomes could not be evaluated. Prebiotics did not have any significant effect on the age at which infants reached full enteral feeds or volume of feed tolerated. Prebiotic preterm formula was well tolerated because there were no reports of vomiting, gastric aspirates, abdominal distension or diarrhea. Prebiotic supplementation did result in a higher stooling frequency compared to control. Effects on stool consistency were inconclusive as results from one study resulted in more watery stools in the prebiotic study group compared to control group, in a second study, the prebiotic group experienced harder stools compared to control group. The third study results were presented in medians (range) therefore no conclusions could be made. In preterm infants, frequent watery stools may signify intolerance, a transient lactase deficiency or another pathological state which always require further investigation 6 .

Prebiotics did have a significant effect on intestinal micro flora. Addition of GOS / FOS combination or FOS alone significantly increased counts of bifidobacteria. Effects on lactobacillus counts could not be evaluated as actual figures were not available. The sum of studied pathogens and some selected pathogens (E- coli, enterococci) were significantly fewer in the prebiotic group compared to control group. There was no effect on staphylococci levels while bacteroides were significantly higher in the probiotic group compared to control group. No prebiotic study reported any data on low birth weight infants; therefore no evaluations could be made.

The prebiotic studies were of short duration ranging from 14 to 28 days. The dose of the prebiotic used (GOS, FOS) varied from 0.4 g/dl o 1g/dl. The European Committee on Food recommends that prebiotics added to formula milk do not exceed 0.8 g/100 ml. The rationale for prebiotic doses not exceeding 1g/ml in clinical trials is an attempt to maximize the bifidogenic effect with minimal intolerance as exhibited by, abdominal distension 85 . The preterm infants tolerated the prebiotic formula as there were no symptoms of feed intolerance reported.

Prebiotic supplementation did have some short term benefits: increased stooling frequency and bifidobacteria counts, fewer pathogens in the prebiotic group compared to control group. However, large RCTS with long term follow -up are needed to find out if these short term benefits translate into improved general health and reduced morbidities in preterm infants. Due to the short duration of prebiotic studies, routine supplementation with prebiotics in preterm infants cannot be recommended.

Quality of the evidence and potential biases

In this review, the quality of the evidence was compromised by several factors: Sample size: included studies were of small individual sample size, number of study participants ranged from 20 to 87 in the probiotic studies, 20 to 56 in prebiotic studies. Intervention: Different types of probiotic and prebiotics, doses and treatment duration were used. Methodological quality: Inadequate information was published to assess methodological quality of the studies. Information was missing on sequence generation, allocation concealment, blinding, incomplete outcome data, selective reporting and free of other bias domains. The significance of any relationship between methodological quality and study outcomes could not be verified since no subgroup analysis with respect to study quality could be done as a result of either too few studies in a meta-analysis or having all studies with similar quality in a meta-analysis. Not all the reviews pre- specified outcomes were addressed by the included studies.

At the conclusion of the review process and preparation of the manuscript (for this review), one on- going study was terminated due to being under powered 47 . One study was completed and data analysis commenced. The results from this study could not be included in this review 48 . The other three studies were still on-going 46 49 50 . The reviewers used thorough comprehensive search strategies adopted for the available databases. All attempts were made to minimize publication bias. All steps of this review were conducted independently by the reviewers.

Agreements and disagreements with other reviews

No significant difference was found in contrast with past reviews and that the potential reasons are lack of power, poor quality of studies or a lack of effect in formula fed infants. This review did agree with some aspects of past reviews. Prebiotics did have an impact on GI micro flora (increased bifidobacteria counts, reduction in certain pathogens); feed tolerance (no reported gastric aspirates, abdominal distension).

Conclusion

There is not enough evidence to state that supplementation of preterm infant formula with probiotics or prebiotics does result in improved growth and clinical outcomes in preterm infants. Therefore this review does not support the routine supplementation of preterm formula with probiotics or prebiotics.

Implications for research

For clear recommendations to be made, long term large RCTs on exclusively formula fed preterm and low birth weight infants are required to investigate the effects of probiotics and prebiotics supplementation in preventing NEC, sepsis, death/mortality; changes in intestinal micro flora and intestinal permeability; explore the efficacy of different doses of the same probiotic on clinical outcomes because available studies used different probiotic doses; similarly, explore the efficacy of different doses of the same prebiotic on clinical outcomes because available studies used similar prebiotics with different doses and treatment duration.

Abbreviations

Cfu: Colony forming units; CI: Confidence interval; cm: Centimetres; ESPGHAN: European society for pediatric gastroenterology: hepatology and nutrition; FOS: Fructo-oligosaccharide; GI: Gastrointestinal; GOS: Galacto-oligosaccharide; IQR: Inter quartile range; IFN-γ: Interferon – gamma; IL-6: Interleukin – 6; IL-10: Interleukin – 10; IL-1β: Interleukin – 1beta; kg: Kilogram; L/M: Lactulose mannitol; MD: Mean difference mmol: millimols; ml: Millilitres; NEC: Necrotizing enterocolitis; TNF-α: Tissue necrosis factor – alpha; RCTs: Randomized controlled trials; RR: Risk ratio; SD: Standard deviation; USA: United States of America; WHO: World Health Organisation.

Competing interests

The authors declared that they have no competing interests.

Authors’ contributions

The authors contributed the following: MM: Developed review protocol, selected RCTs, carried out data extraction; assessment of risk of bias in included studies, developed, edited and critically reviewed the manuscript. ML: Selected RCTs, carried out data extraction, assessment of risk of bias in included studies, critically reviewed the manuscript. AM: Carried out the statistical analysis, interpretation of results and critically reviewed the manuscript. TY: Assisted in designing the review and critically reviewed the manuscript. RB: Assisted in designing the review and critically reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgments

This review was supported through a grant from the University of Stellenbosch, Faculty of Health Sciences, South Africa. The funders played no role in study design, data collection, analysis and interpretation, report writing or conclusions reached in this review.

From Pediatric NutritionAndersonDMHandbook of Pediatric NutritionJames and Bartlett Publishers, Sudbury, MassachusettsSamour PQ, Helm KK320055371Neonatal MedicineLissauerTClaydenGIllustrated Text book of PediatricsElsevier, Mosby32007145168NutritionGeorgieffMKAvery’s Neonatology pathophysiology and management of the new bornLippincott Williams and Wilkins, PhiladelphiaMacDonald MG, Seshia MMK, Mullet MD6200538038123102597LissauerTFanaroffAThe preterm infant: growth and nutrition. In Neonatology at a glanceBlackwell Publishing, Malden, Mass20067677Optimizing growth in the preterm infantUhingMRDasUGClin Perinatol20093616517610.1016/j.clp.2008.09.01019161873Enteral nutritionAndersonMSJohnsonCBTownsendSFHayWHandbook of neonatal intensive careMosby, MosbyMerenstein GB, Gardner SL52002314316Effect of enteral IGF-1 supplementation on feeding tolerance, growth and gut permeability in enterally fed premature neonatesCorpeleijinWEvan VlietIde Gast-BakkerDHvan der SchoorSRDAllesMSHoijerMTibboelDvan GoudoeverJBJ Pediatr Gastrotenterol Nutr20084618419010.1097/MPG.0b013e31815affecEnteral feeding for very low birth weight infants: reducing the risk of necrotising enterocolitisChauhanMHendersonGMMcGuireWArch Dis Child Fetal Neonatal Ed200893F162F16618006565A randomized placebo - controlled comparison of 2 prebiotic/probiotic combinations in preterm infants: Impact on weight gain, intestinal microbiota and fecal short chain fatty acidsUnderwoodMASalzmandNHBennettSHBarmanMMillsDAMarcobalATancrediDJBevinsCLShermanMJ Pediatr Gastroenterol Nutr20094821622510.1097/MPG.0b013e31818de195274341819179885Functional cultures and health benefitsShahNPInt Dairy J2007171262127710.1016/j.idairyj.2007.01.014Probiotics and their fermented food products are beneficial for healthParvezSMalikKAKangSAKimHYJ Appl Microbiol20061001171118510.1111/j.1365-2672.2006.02963.x16696665Inulin and oligofructose. New Scientific DevelopmentsGibsonGRNathalieDNutr Today200843545910.1097/01.NT.0000303311.36663.39Fibre and effects on probiotics (the prebiotic concept)GibsonGRClin Nutr2004122531Bacterial metabolism and health related effects of galacto-oligosaccharides and other prebioticsMacfarlaneGTSteedHMacfarlaneSJ Appl Microbiol200810430534418215222Towards a healthier diet for the colon: the influence of fructooligosaccharides and lactobacilli on intestinal healthLosadaMOllerosTNutr Res200222718410.1016/S0271-5317(01)00395-5Inulin, Oligofructose and immunomodulationWatzlBGirrbachSMonikaRBr J Nutr200593Suppl 1S49S5515877895FAO/WHOGuidelines for evaluation of probiotics in food,2002 http/www.who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf Probiotics enhance anti- effective defences in the gastrointestinal tractGillHSBest Pract Res Clin Gastroenterology20031775577310.1016/S1521-6918(03)00074-XProbiotic lactobacilli and VSL#3 induce enterocyte beta-defensin 2SchleeMHarderJKotenBStangeEFWehkampJFellermannKClin Exp Immunol200815152853510.1111/j.1365-2249.2007.03587.x227696718190603Probiotics and prebiotics in gastrointestinal disordersFedorakRNMadsenKKCurr Opin Gastroenterol20042014615510.1097/00001574-200403000-0001715703637Live probiotics protect intestinal epithelial cells from the effects of infection with entero invasive Escherichia Coli (EIEC)Resta-LenertSBarrettKEGut20035298899710.1136/gut.52.7.988177370212801956Effects of specific lactic acid bacteria on the intestinal permeability to macromolecules and the inflammatory conditionHeymanMTerpendKMenardSActa Pediatr200594Suppl 4493436The mechanism of action of probioticsBoirvantMStroberWCurr Opin Gastroenterol200723670692Oral administration of two probiotic strains Lactobacillus gasseri CECT5714 and Lactobacillus coryniformis CECT5711, enhances the intestinal function of healthy adultsOlivaresMDiaz-RoperoMPGómezNLara-VillosladaFSierraSMaldonadoJAMartinRLopez-HuetasERodriguezJMXausJInt J Food Microbiol200610710411110.1016/j.ijfoodmicro.2005.08.01916271414Lactobacillus planatarum 299v enhances the concentrations of fecal short chain fatty acids in patients with recurrent clostridium difficile associated diarrheaWultMHagslattMLJOdenholtIBerggrenADig Dis Sci2007522082208610.1007/s10620-006-9123-317420953Gum Arabic establishes prebiotics functionality in healthy human volunteers in a dose dependent mannerCalameWWeselerARViebkeCFlynnCSiemensmaADBr J Nutr20081001269127510.1017/S000711450898144718466655Acacia gum is a bifidogenic dietary fibre with high digestive tolerance in healthy humansCherbutCMichelCRaisonVKravtchenkoTSeverineMMicrob Ecol Health Dis200315435010.1080/08910600310014377Studies with Inulin type fructans on intestinal infections, permeability and inflammationGuarnerFJ Nutr20071372568S2571S17951504Tolerance and safety of lactobacillus paracasei ssp paracasei in combination with Bifidobacterium animalis ssp lactis in a prebiotic-containing infant formula: a randomised controlled trialVliegerAMRobrochAvan BuurenSKiersJRijkersGBenningaMATebiesebekeRBr J Nutr200910286987510.1017/S000711450928906919331702The delivery of probiotics and prebiotic to infantsKullenMJBettlerJCurr Pharm Des200511557410.2174/138161205338235915638752Probiotics for prevention of necrotizing enterocolitis in preterm infantsAlFalehKAnabreesJBasslerDAl-KharfiTCochrane Database Sys Rev2011Art. No.: CD005496Issue 3 10.1002/14651858.CD005496.pub3Probiotics for Necrotizing Enterocolitis: A Systematic ReviewBarclayARStensonBSimpsonJHLawrenceTWilsonDJ Pediatr Gastroenterol Nutr20074556957610.1097/MPG.0b013e318134469418030235Probiotics for prevention of necrotising enterocolitis in preterm neonates with very low birth weight: a systematic review of randomised controlled trialsDeshpandeGRaoSPatoleSLancet200736995731614162010.1016/S0140-6736(07)60748-X17499603Probiotics for the prevention of pediatric antibiotic-associated diarrheaJohnstonBCGoldenbergJZVandvikPOSunXGuyattGHCochrane Database Sys Rev2011Art. No.: CD004827Issue 11 10.1002/14651858.CD004827.pub3HigginsJPTGreenSCochrane Handbook for Systematic Reviews of InterventionsJohn Wiley & Sons, Chichester (UK)2008Supplementation of a bovine milk formula with an oligosaccharide mixture increases counts of faecal bifidobacteria in preterm infantsBoehmGArch Dis Child Fetal Neonatal Ed200286F178F18110.1136/fn.86.3.F178172140811978748Increase of faecal bifidobacteria due to dietary oligosaccharides induces a reduction of clinically relevant pathogen germs in the feces of formula-fed preterm infantsKnolJBoehmGLidestriMNegrettiFJelinekJAgostiMStahlBMoscaFAct Paediatr2005Suppl 4493133Prebiotic concept for infant nutritionBoehmGFanaroSJelinekJStahlBMariniAActa Paediatr200392Suppl 4416467Enteral feeding of premature infants with Saccharomyces BoulardiiCostalosCEarly Hum Dev200374899610.1016/S0378-3782(03)00090-214580749The effect of a bifidobacter supplemented bovine milk on intestinal permeability of preterm infantsStratikiZCostalosCSevastiadouSKastanidouOSkouroliakouMGiakoumatouAPetrohilouVEarly Hum Dev20078357557910.1016/j.earlhumdev.2006.12.00217229535Lack of effect of Lactobacillus on gastrointestinal bacterial colonization in premature infantsReumanPDDuckworthDHSmithKLKaganRBucciarelliRAyoubEPediatr Infect Dis1986566366810.1097/00006454-198611000-000133099269The effects of probiotics on feeding tolerance, bowel habits and gastrointestinal motility in preterm new-bornsIndrioFRiezzoGRaimondiFBisceguaMCavalloLFrancaillaRJ Pediatr200815280180610.1016/j.jpeds.2007.11.00518492520Prebiotics improve gastric motility and gastric electrical activity in preterm new-bornsIndrioFRiezzo RaimondiFFrancavillaRMontagnaOValenzanoMCavalloLBoehmGJ Pediatr Gastroenterol Nutr20094925826110.1097/MPG.0b013e3181926aec19561548Prebiotic oligosaccharides reduce stool viscosity and accelerate gastrointestinal transport in preterm infantsMihatschWAHoegelJPohlandtFActa Paediatr20069584384810.1080/0803525050048665216801182The effect of a fructooligosaccharide supplemented formula on gut flora of preterm infantsKapikiACostalosCOikonomidouCTriantafyllidouALoukatouEPertrohilouEarly Hum Dev20078333533910.1016/j.earlhumdev.2006.07.00316978805JacobsSThe use of probiotics to reduce the incidence of sepsis in premature infantsAustralian New Zealand Clinical Trials RegistryACTRN126070001444415 26/02/2007 [www.anzctr.org.au]LozanoJMRojasMProphylactic Probiotics in Premature infantsClinical trials registry, NCT00727363 2008 [www.clinicaltrials.gov]Probiotics-supplemented feeding in extremely low birth weight infantsAl-HosniMDuenasMFerrelliKHowardDSollRPediatric Academic Society Conference Proceedings at Vancouver Convention Centre,2010Abstract Number 1670.8: Course number 1670.(www.pas-meetings.org; www.abstract2view.com)PatoleSA randomized placebo controlled trial on the safety and efficacy of a probiotic product in reducing all case mortality and definite Necrotising Enterocolitis in preterm very low birth weight neonatesAustralian New Zealand Clinical Trials Registry, ACTRN12609000374268 27/05/2009 (www.anzctr.org.au)UnderwoodMThe impact of oligosaccharides and bifidobacteria on the intestinal micro flora of premature infantsClinical trials registryNCT00810160 05/11/2009. [www.clinicaltrials.gov]KarvonenAVSinkiewiczGConnolyEVesikariTSafety and colonization of the probiotic Lactobacillus reuteri ATCC 55730 in new born and premature infantsBio Gaia AB Research Laboratories, Stockholm, Sweden2002(Unpublished data)Effects of oral Lactobacillus GG in enteric micro flora in low birth weight neonatesAgarwalRSharmaNChaudhryRDeorariAPaulVGewolbIHPanigrahiPJ Pediatr Gastroenterol Nutr20033639740210.1097/00005176-200303000-0001912604982Oral Probiotics prevent necrotizing enterocolitis in very low birth weight preterm infants: A multicenter, randomized controlled trialLinHCHsuCHChenHLChungMYHsuJFLienRITsaoLYChenCHSuBHPediatrics200812269370010.1542/peds.2007-300718829790The effects of Lactulose supplemented enteral feedings in premature infants: A pilot studyRiskinAHochwaldOBaderDSrugoINaftaliGKugelmanACohenEMorFKaufmanBShaoulRJ Pediatr201015620921410.1016/j.jpeds.2009.09.00619879595Low birth weight infants fed a new carbohydrate- free formula with different sugarsAndrewsBFAm J Clin Nutr1969228458505797051Lack of effects of oral probiotics on growth and neuro developmental outcomes in preterm very low birth weight infantsChouICKuoHTChangJSWuSFChiuHYSuBHLinHCJ Pediatr201015639339610.1016/j.jpeds.2009.09.05119914635Effects of feeding premature infants with Lactobacillus GG on gut fermentationStansbridgeEMWalkerVHallMASmithSLMillarMRBaconCChenSArch Dis Child19936948849210.1136/adc.69.5_Spec_No.48810295908285751Specific proliferative and antibody responses of premature infants to intestinal colonization with non-pathogenic probiotic E-coli strain nissle 1917CukrowskaBLodinova-ZadnikivaREndersCSonnenbornUSchulzeJTlaskalová-HogenováHScan J Immunol20025520420910.1046/j.1365-3083.2002.01005.xOral probiotics prevent necrotising enterocolitis in very low birth weight neonatesBin-NunABromikerRWilschanskiMKaplanMRudenskyBCaplanMHammermanCJ Pediatr200514719219610.1016/j.jpeds.2005.03.05416126048Oral supplementation with Lactobacillus caseii subspecies rhamnosus prevents enteric colonization by candida species in preterm infant: A randomized studyManzoniPMostertMLeonessaMLPrioloCFarinaDMonettiCLatinoMAGomiratoGClin Infec Dis2006421735174210.1086/504324Oral Supplementation with probiotics in very low birth preterm infants: A randomized, double blind, placebo controlled trialRougeCPiloquetHButelMJBergerBRochatFFerrarisLDesRCLegrandAde la CochetièreMFN’GuyenJMVodovarMVoyerMDarmaunDRozéJCAm J Clin Nutr2009891828183510.3945/ajcn.2008.2691919369375Intestinal Permeability in Preterm infants by feeding type: Mother’s milk versus formulaTaylorSNBasileLAEbelingMWagnerCBreastfeed Med20094111510.1089/bfm.2008.0114293254419196035Reduced Incidence of necrotizing enterocolitis associated with enteral administration of lactobacillus acidophilus and bifidobacterium infantis to neonates in an intensive care unitHoyosABInt J Infect Dis1999319720210.1016/S1201-9712(99)90024-310575148Effects of Oral Administration of bifidobacterium breve on fecal lactic acid and short chain fatty acids in low birth weight infantsWangCShojiHSatoHNagataSOhtsukaYShimizuTYamashiroYJ Pediatr Gastroenterol Nutr20074425225710.1097/01.mpg.0000252184.89922.5f17255840Probiotics feeding in prevention of urinary tract infection, bacterial sepsis and necrotizing enterocolitis in preterm infants. A prospective double blind studyDaniCBiadaioliRBertiniGMartelliERubaltelliFBiol Neonate20028210310810.1159/00006309612169832Enteral feeding of premature infants with Lactobacillus GGMillarMRBaconCWalkerVHallMAArch Dis Child19936948348710.1136/adc.69.5_Spec_No.48310295898285750Prophylactic probiotics for prevention of necrotizing enterocolitis in very low birth weight new bornsSamantaMSarkarMGhoshPGhoshJKSinhaMKChatterjeeSJ Trop Pediatr20095512813118842610Oligosaccharides might stimulate calcium absorption in formula fed preterm infantsLidestriMAgostiMMariniMActa Paediatr200392Suppl 441919212650307Early Administration of Bifidobacterium breve to preterm infants: Randomised controlled trialKitajimaHSumidaYTanakaRYukiTFujimuraMArch Dis Child199776F101F107Effects of Bifidobacterium lactis Bb12 supplementation on intestinal microbiota of preterm infants: A double blind, placebo controlled randomized studyMohanRKoebnickCSchildtJSchildtSMuellerMPossnerMRadkeMBlautMJ Clin Microbio2006444025403110.1128/JCM.00767-06Design of a randomised controlled trial on immune effects of acidic and neutral oligosaccharides in the nutrition of preterm infants: Carrot studyWesterbeekEAMvan ElburgRMVan den BergAVan den BergJTwiskJWRFetterWPFLafeberHNBMC Pediatr200884610.1186/1471-2431-8-46257942418947426Effects of Probiotics on enteric flora and feeding tolerance in preterm infantsLeeSJChoSJParkEANeonatology20079117417910.1159/00009744917377402Effects of Bifidobacterium lactis Bb12 supplementation on body weight, fecal pH, acetate, lactate, calprotectin and IgA in Preterm infantsMohanRKoebnickCSchildtJMuellerMRadkeMBlautMPediatr Res20086441842210.1203/PDR.0b013e318181b7fa18552710Neutral and acidic oligosaccharides in preterm infants: a randomized double-blind, placebo controlled trialWesterbeekEAMVan den BergJPLafeberHNFetter-WilemPFBoehmGTwiskWRVan ElburgRMAm J Clin Nutr20109167968610.3945/ajcn.2009.2862520032496Oral probiotics reduce the incidence and severity of necrotizing enterocolitis in very low birth weight infantsLinHCSuBHChenACLinTWTsaiCHYehTFOhWPediatrics20051151415629973Does Carboxy methylcellulose have a role in reducing time to full enteral feeds?PatoleSKMullerRInt J Clin Pract20055954454815857350Effect of Bifid triple viable on feeding Intolerance in preterm infants with very low birth weight. [Clinical study on the effect of probiotic preparation on feeding intolerance in preterm infants with very low birth weight. (from Chinese translation)]YongGFangHShuang-GenMGuo-ChengXChinese Journal of Microecology200921451452Intestinal permeability in relation to birth weight and gestational and postnatal agevan ElburgRMFetterWPFBunkersCMHeymansHSAArch Dis Child Fetal Neonatal Ed200388F52F5510.1136/fn.88.1.F52175599712496227Assessment of intestinal permeability in (premature) neonates by sugar absorption testsCorpeleijnWEvan ElburgRMvan KemaIPGoudoeverJBMethods Mol Biol20117639510410.1007/978-1-61779-191-8_621874446The effect of enteral supplementation of a prebiotic mixture of non-human milk galacto-, fructo- and acidic oligosaccharides on intestinal permeability in preterm infantsWesterbeekEAMvan den BergALafeberHNFetterWPFvan ElburgRMBr J Nutr201110526827410.1017/S000711451000340520863418Probiotic Bacteria in dietetic products for infants: a commentary by the ESPGHAN Committee on NutritionAgostoniCAxelssonIBraeggerCGouletOKoletzkoBMichaelsenKFRigoJShamirRSzajewskaHTurckDWeaverLJ Pediatr Gastroenterol Nutr20043836537410.1097/00005176-200404000-0000115085012Severe sepsis after probiotic treatment with Escherichia coli nissle 1917GuentherKStraubeEPfisterWGuentherAHueblerAPediatr Infect Dis J20102918818920118748Two cases of Lactobacillus bacteremia during probiotic treatment of short gut syndromeKunzANNoelJMFairchokMPJ Pediatr Gastroenterol Nutr20043845745810.1097/00005176-200404000-0001715085028Lactobacillus sepsis associated with probiotic therapyLandMHRouster-StevensKWoodsCRCannonMLCnotaJShettyAKPediatrics200511517818115629999Pediatric applications of inulin and oligofructoseVeeremanGJ Nutr20071372585S2589S17951508