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Efficacy of saccharomyces boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: A randomised controlled trial
EHD-03851; No of Pages 4 Early Human Development xxx (2013) xxx–xxx
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Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Efficacy of saccharomyces boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: A randomised controlled trial Ozge Serce ⁎, Derya Benzer, Tugba Gursoy, Guner Karatekin, Fahri Ovali Zeynep Kamil Maternity and Children's Research and Training Hospital, Neonatology Unit, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 31 May 2013 Received in revised form 18 August 2013 Accepted 20 August 2013 Available online xxxx Keywords: Necrotizing enterocolitis Probiotics Saccharomyces boulardii Sepsis Very low birth weight
a b s t r a c t Background: Probiotics have strain specific effects and the effects of fungi in preventing diseases in preterm infants have been investigated poorly. Saccharomyces boulardii is a yeast which acts both as a probiotic and a polyamine producer. Aim: The objective of this study was to investigate the efficacy of S. boulardii in preventing necrotizing enterocolitis (NEC) or sepsis in very low birth weight infants. Study design and subjects: A prospective, double blind, placebo controlled trial was conducted in preterm infants (≤32 GWs, ≤1500 g birth weight). They were randomized either to receive feeding supplementation with S. boulardii 50 mg/kg every 12 h or placebo, starting with the first feed until discharged. Outcome measures: Necrotizing enterocolitis (NEC) or sepsis and NEC or death. Results: Birth weight and gestational age of the study (n = 104) and the control (n = 104) groups were 1126 ± 232 vs 1162 ± 216 g and 28.8 ± 2.2 vs 28.7 ± 2.1 weeks, respectively. Neither the incidence of stage ≥ 2 NEC or death nor stage ≥2 NEC or late onset culture proven sepsis was significantly lower in the study group when compared with the control group (9.6% vs 7.7%, p = 0.62; 28.8% vs 23%, p = 0.34). Time to reach 100 mL/kg/day of enteral feeding (11.9 ± 7 vs 12.6 ± 7 days, p = 0.37) was not different between the groups. Conclusions: Saccharomyces boulardii did not decrease the incidence of NEC or sepsis. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Necrotizing enterocolitis (NEC) is the most serious acquired disease of the gastrointestinal tract characterized by bowel wall necrosis in very low birth weight infants (VLBW) [1]. The incidence varies among centers [2]. Recent studies report up to 9000 cases of NEC in the United States every year, with a case fatality rate of 15% to 30% [3]. The pathogenesis of NEC remains incompletely understood but it is considered to be due to a complex interaction of factors such as; prematurity, enteral feeding, intestinal hypoxia–ischemia, and bacterial colonization causing mucosal injury through a final, common, inflammatory pathway [2,4,5]. VLBW infants at risk of NEC have abnormal fecal colonization, demonstrate a paucity of normal enteric bacterial species, and have delayed onset of bacterial colonization [5]. Nosocomial infection is also a frequent complication in VLBW infants. Twenty five percent of these infants have at least one or more
⁎ Corresponding author at: Zeynep Kamil Maternity and Children's Research and Training Hospital, Neonatalogy Unit, Zeynep Kamil Mahallesi, Op. Dr. Burhanettin Ustunel Caddesi No:10 Uskudar 34668 Istanbul, Turkey. Tel.: +90 532 327 03 75; fax: +90 216 391 06 90. E-mail addresses: [email protected] (O. Serce), [email protected] (D. Benzer), [email protected] (T. Gursoy), [email protected] (G. Karatekin), [email protected] (F. Ovali).
positive blood cultures over the course of their hospitalization [6]. Late onset sepsis is associated with an increased risk of death, neonatal morbidity and prolonged hospitalization [7]. Probiotic bacteria are live microbial supplements that colonize the gastrointestinal tract and potentially provide benefit to the host. Probiotics may prevent NEC by promoting colonization of the gut with beneficial organisms, preventing colonization by pathogens, improving the maturity and function of gut mucosal barrier, and modulating the immune system [1]. Many clinical trials have evaluated the safety and benefits of probiotic supplementation in VLBW neonates. It is important to note that there are many different mechanisms producing the benefits of probiotics and there are also strain-specific effects [8]. Researchers have generally selected strains belonging to bacterial species naturally present in the intestinal flora. Saccharomyces boulardii (SB) is a yeast which acts both as a probiotic and polyamine producer [9]. Polyamines which are essential for cell growth and differentiation enhance intestinal maturation [9]. SB is used for the treatment of a number of gastrointestinal disorders related to abnormal bacterial growth [9]. Therefore, we hypothesized that SB may be an attractive candidate for the prevention of NEC which is characterized by bacterial colonization causing mucosal injury, and sepsis by overwhelming bacterial translocation with affects on intestinal maturation. SB can be a better strategy than other bacterial strains due to the ability to produce polyamines. We therefore designed a randomized, double-blind placebo controlled
0378-3782/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.earlhumdev.2013.08.013
Please cite this article as: Serce O, et al, Efficacy of saccharomyces boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: A randomised controlled trial, Early Hum Dev (2013), http://dx.doi.org/10.1016/j.earlhumdev.2013.08.013
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O. Serce et al. / Early Human Development xxx (2013) xxx–xxx
clinical trial to evaluate the influence of SB supplementation on the incidence and severity of NEC and sepsis in VLBW infants. 2. Patients and method A prospective, masked, randomized, controlled trial was conducted from October 1, 2010 to November 30, 2011, in Zeynep Kamil Maternity and Children's Research and Training Hospital, Neonatal Intensive Care Unit (NICU) after the approval from ethical committee. VLBW infants (gestational age ≤32 weeks; birth weight ≤1500 g) who survived to feed enterally were eligible for the trial. Infants who had severe asphyxia (stage III), major congenital anomalies, those who had been fasted for more than 3 weeks, died in the first postnatal 14 days and infants who used antifungal therapy were excluded. The event rate was determined by unpublished data based on 2009–2010 of our NICU. Our recent event rate for NEC or sepsis was 31% and for death or NEC was 35% in our NICU (for NEC alone 17%, for sepsis alone 19%, and for death alone 18%). With the α-error set at 0.05 and the β-error set at 0.2, and an absolute reduction in the incidence of either NEC/sepsis or NEC/death of 50%, the number of infants needed to verify our hypothesis was 104 for NEC or sepsis and 92 for NEC or death for each group. The infants enrolled in the study were assigned randomly to the study or control group prospectively after informed parental consent was obtained. Randomization was performed by using sequential numbers generated at the computer center. The allocations were contained in opaque, sequentially numbered sealed envelopes. The study group received Saccharomyces boulardii (Reflor®, Biocodex, France, 50 mg/kg equal to 0.5 × 109 cell/kg per dose twice daily); whereas the control group received placebo (distilled water; 1 mL per dose twice daily) which were added to breast milk or formula [Aptamil Prematil Formula (Milupa AG, Friedrichsdorf, Germany)] starting with the first feed until discharged. The median duration of SB supplementation and follow up period was 44 days. The dosage of SB used in this study was the same as the one used in Costalo's study [9]. SB was kept in a dry place at room temperature, away from light and humidity, in tightly closed container, and mixed with breast milk or formula before feeding. One milliliter suspension was prepared with mixture of the powder of SB with distilled water. This supplementation did not change the appearance of the milk or the formula. Fresh suspension of supplements was prepared by personnel in the breast milk team who were not involved in the care of the infant and who followed the instructions from the sealed envelope. Thus, the only personnel who knew of the infants' group assignments were the investigator and those in the breast milk team who were not involved in the care of the infants. Depending on the birth weight and gestational age, 10–20 mL/kg of breast milk or formula was initiated on the first postnatal day if there were not any contraindications (such as severe IUGR, severe asphyxia, severe preeclampsia, reversed end diastolic flow in umbilical artery) and if the infant tolerated the feeding. The amount of feeding was increased based on birth weight (10 mL/kg increment for b750 g; 20 mL/kg for 750–1250 g; 30 mL/kg for 1250–1500 g birth weight). Aptamil Eoprotin human milk fortifier (Milupa AG, Friedrichsdorf, Germany) was used for fortification of the human milk when the oral intake of 100 mL/kg per day was reached. Feeding was stopped if there was any sign of feeding intolerance which was defined as the presence of gastric aspirate in an amount that was more than one half of the previous feeding with abdominal distension. Infants received total parenteral nutrition until 100 mL/kg per day of enteral feeding was achieved. The gestational age was estimated based on ultrasound examination before the end of 20 GWs (73%) or from the last menstrual period. Infants with a birth weight below 2 SD according to Turkish intrauterine growth curves were classified as small for gestational age (SGA) [10]. “Modified Bell Classification” was used to define NEC [11], and “Papille Classification” for IVH [12]. The culture proven late onset sepsis was defined in neonates who have at least two clinical symptoms and two
laboratory signs mentioned below with positive blood culture after 72 h of life; a) Clinical signs: 1) Core temperature ≥38.5 °C or ≤36 °C; 2) Bradycardia (mean heart rate b10th percentile for age in the absence of external vagal stimulus, beta-blockers or congenital heart disease or tachycardia (mean heart rate 2 SD above normal for age in the absence of external stimulus, chronic drugs and painful stimuli) and/or rhythm instability; 3) Reduced urinary output (b 1 mL/kg/h), hypotension (mean arterial pressure b5th percentile for age); 4) impaired peripheral perfusion; 5) Petechial rash, sclerema; 6) Apnea or tachypnea (mean respiratory rate over 2 SD above normal for age) or increased oxygen requirements or requirement for ventilation support); 7) Feeding intolerance, poor sucking, abdominal distention; 8) Irritability, lethargy and hypotonia b) Laboratory Signs: 1) WBC count: b4000 × 109 cells/L or N 20000 × 109 cells/L; 2) Immature to total neutrophil ratio 0.2; 3) Platelet count: b100 000 × 109 cells/L; 4) C reactive protein N15 mg/L or procalcitonin ≥ 1 ng/mL; 5) Blood glucose N 180 mg/dL or b45 mg/dL; 6) Metabolic acidosis: Base excess b− 10 mEq/L or serum lactate N2mMol/L. Survival was defined as the infant being alive at discharge. Demographic and clinical variables that were potential risk factors for NEC and sepsis were recorded. The primary outcomes were stage ≥2 NEC or late onset culture proven sepsis and stage ≥2 NEC or death. Secondary endpoints were the time to reach 100 mL/kg/day of enteral feeding, weight gain per week, oxygen dependency at 36 weeks, mortality until hospital discharge, and duration of hospitalization. Adverse effects, including culture proven sepsis attributable to SB, anaphylaxis and diarrhea, were recorded. Statistical analyses were performed by SPSS statistics programme v.13.0 (SPSS, Chicago, IL, USA). Demographic data were stated as ratio, mean (±standard deviation), and median (25%–75%). Dependent and independent variables were compared by chi-square and Fisher exact test. Independent samples t-test and Mann–Whitney U test were used to compare continuous variables. Results are stated as relative risk ratio (RR) and 95% confidence interval, CI). All statistical tests were 2-sided, and a P value of ≤ 0.05 was considered to indicate statistical significance. 3. Results Infants assessed for eligibility were 292; 48 of them were excluded because of major congenital malformation (n = 9), severe asphyxia (n = 4), death occurred in 24 h (n = 10), and lack of parental consent (n = 25). A total of 244 infants were enrolled in the study. Three infants were excluded due to fasting more than 3 weeks. Eleven infants were lost to follow up due to family withdraw, and a total of 19 infants died during the first 2 week period after enrollment. Finally, 104 infants per group completed the study protocol. Demographic characteristics of the infants and clinical characteristics of the mothers did not differ between the groups (Table 1). The infants' clinical variables also did not differ between the groups, except for earlier time to start minimal enteral feeding (MEF) in the placebo group (Table 2). Table 3 shows the outcomes of the study. The incidence of stage ≥2 NEC was 7 (6.7%) both in the control and study group, and it did not reach statistical significance (p = 1). Six of fifteen babies who developed stage ≥2 NEC died (40%), and the number of NEC-related deaths was same in both groups. Neither the incidence of stage ≥2 NEC or death nor the incidence of stage ≥2 NEC or late onset culture proven sepsis differed between the groups (Table 3). Twenty five (24.3%) neonates in the control group and 19 (18.3%) neonates in the study group developed culture proven sepsis. Of these
Please cite this article as: Serce O, et al, Efficacy of saccharomyces boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: A randomised controlled trial, Early Hum Dev (2013), http://dx.doi.org/10.1016/j.earlhumdev.2013.08.013
O. Serce et al. / Early Human Development xxx (2013) xxx–xxx Table 1 Demographic and clinical characteristics of the mothers and infants.
Birth weight (g), mean ± SD Gestational age (weeks), mean ± SD 5th min. Apgar, median (25%–75%) Male infants, n (%) Cesarean section, n (%) PROM N18 h, n (%) Preeclampsia, n (%) Antenatal steroid treatment, n (%) SGA, n (%)
Table 3 Outcomes of the study.
Control group (n = 104)
Study group (n = 104)
p
1162 ± 216 28.7 ± 2.1 7(6–8) 56 (53.8) 92 (88.5) 30 (28.8) 16 (15.4) 73 (70.2) 9 (8.7)
1126 ± 232 28.8 ± 2.2 8(6–8) 51 (49) 84 (80.8) 26 (25) 22 (21.2) 71 (68.3) 14 (13.5)
0.3 0.72 0.9 0.48 0.12 0.53 0.28 0.76 0.26
PROM, prolonged rupture of membrane; SGA, small for gestational age.
microorganisms that grew, 72% were gram positive and 28% were gram negative in control group and 73% were gram positive and 27% were gram negative in study group. We did not demonstrate any fungal sepsis in our cohort. There was no statistically significant difference in the time to reach 100 mL/kg/day of enteral feeding, weight gain per week, deaths not attributable to NEC, and duration of hospitalization between the groups. No side effect attributed to SB was observed. 4. Discussion Probiotics have become a hope for NEC prophylaxis and treatment and many trials have been performed. Cochrane meta-analysis indicates that enteral probiotic supplementation such as Lactobacillus and Bifidobacterium species which are members of the normal flora of gut significantly reduces the incidence of stage ≥2 NEC and mortality in preterm infants [13]. Probiotics act through many different mechanisms which are strain-specific. The use of probiotic bacteria to inhibit pathogenic bacteria has been studied extensively over the years, while little attention has been given to yeasts in a similar role. However, yeasts have been reported to antagonise both other yeasts and bacteria in several ways. In an animal study SB was reported to attenuate NEC induced intestinal tissue damage through mechanisms inhibiting intestinal proinflammatory mediator release [14]. In another study, while SB-supplemented formula was shown to have a beneficial effect on gut flora in healthy preterm infants, bringing it closer to that of breastfed infants, no effect on NEC could be shown [9]. We also could not demonstrate any statistically significant difference in NEC or sepsis rate between the groups. This controversy with other studies that used probiotic bacteria and demonstrated reduction in the incidence of NEC or mortality may be explained by various mechanisms. First of all, there are substantial differences in the cell wall composition of bacteria and yeast which are responsible for the modulation of the mucosal immune response by probiotics. This can account for the different responses obtained by these probiotic microorganisms [15]. Secondly, probiotics inhibit the production of proinflammatory cytokines and the predominant cytokine profile under influence depends on the types of probiotics used [16]. SB was predominantly used in adults and older children and was found to be effective in inflammatory bowel disease and gastroenteritis Table 2 Postnatal clinical characteristics of the infants.
Respiratory distress syndrome, n (%) Patent ductus arteriosus, n (%) Intraventricular haemorrhage, n (%) Start on MEF (day), mean ± SD Increment of MEF (mL/kg/day), mean ± SD % of breast milk used, mean ± SD
Control group (n = 104)
Study group (n = 104)
P
56 (53.8) 31 (29.8) 23 (22.1) 1.8 ± 1.1 17 ± 5 65 ± 31
48 (46.2) 20 (19.2) 21 (20.2) 2±1 19 ± 6 64 ± 31
0.26 0.07 0.73 0.03 0.10 0.74
MEF, minimal enteral feeding; PMA, postmenstrual age.
3
Control group Study group P (n = 104) (n = 104) Time to reach 100 mL/kg/day of oral feeding (day), mean ± SD Weight gain (g/week), mean ± SD Oxygen dependency at 36. PMA, n (%) Late onset culture (+) sepsis, n (%) NEC ≥ stage 2, n (%) Stage ≥ 2 NEC or late onset culture (+) sepsis, n (%) Stage ≥ 2 NEC or death, n (%) Deaths attributable to stage ≥ 2 NEC n (%) Deaths (all), n (%) Duration of hospitalization (day), median (25%–75%)
RR (95% CI)
12 ± 7
11 ± 7
0.37
129 ± 65
113 ± 61
0.31
11 (10.6)
12 (11.5)
0.82
25 (24.3)
19 (18.3)
0.29 0.69 (0.35–1.36)
7 (6.7) 30 (28.8)
7 (6.7) 24 (23)
1 1 (0.58–1.72) 0.34 0.86 (0.64–1.15)
10 (9.6) 3 (2.8)
8 (7.7) 3 (2.8)
0.62 0.89 (0.57–1.37) 1 1 (0.44–2.25)
4 (3.8) 43 (29–60)
5 (4.8) 39 (28–60)
0.74 0.89 (0.49–1.63) 0.62
PMA, postmenstrual age; NEC, necrotizing enterocolitis.
[15,17–19]. NEC is the most common inflammatory gut emergency diagnosed in neonatal intensive care units. The diminished innate immunity of premature neonates may be responsible for this conflict observed between adults and neonates [20]. Many preterm infants require intensive care procedures and receive antibiotics early in life, which may be one of the major factors determining intestinal microbiota development. [21]. They are more susceptible to infection as they have an immature immune system, which may in part be due to the abnormal development of their gastrointestinal microflora [22]. They also have delayed colonization with healthy commensals, such as Lactobacillus and Bifidobacterium species, which may lead to altered function of the gut microbial community, particularly the immune functions [22]. So it has been hypothesized that very preterm infants may benefit from colonization with exogenously administered probiotics. However, in most of the studies and meta-analysis the beneficial effects on sepsis could not be supported [4,13,23]. SB, similar to the other studies reported before, did not decrease the incidence of late onset culture proven sepsis in our study. Feeding difficulties that lead to prolonged deprivation of enteral feeds and dependence on total parenteral nutrition are a major issue in preterm neonates. Probiotics improve gastric emptying and gut barrier function [24,25]. Given the importance of optimal enteral nutrition in early postnatal life, the benefits of probiotics have significant implications in improving the overall prognosis of this high-risk population. The study results about the benefit of probiotics for the time to reach full enteral feeding are conflicting due to the significant heterogeneity in feeding protocols as the definition of full enteral feeding varies significantly [13]. Since the definition of full enteral feeding may be different for each infant, we decided to analyse the time to reach 100 mL/kg/day of enteral feeding as a standard measure. Although probiotics were reported to decrease the time to reach full enteral feeding in some studies [13], we did not demonstrate any positive effect of SB on the time to reach 100 mL/kg/day of enteral feeding like in the Costalos' study [9]. This may be due to the different probiotics used in those studies. Clinical findings of gastrointestinal intolerance (higher osmotic load causing abdominal distension, diarrhea or vomiting) or sepsis due to probiotics were the main possible adverse effects of probiotics [8]. However; we did not observe any side effects during the study. So far, probiotics that contain bacterial strains were found to decrease the incidence and severity of NEC and were suggested to be used for the prophylaxis. However, in this study and in the study of Costalos, the reduction in NEC incidence could not be demonstrated with SB. Although this result might be due to several differences observed between bacterial and yeast strains, as mentioned before, it
Please cite this article as: Serce O, et al, Efficacy of saccharomyces boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: A randomised controlled trial, Early Hum Dev (2013), http://dx.doi.org/10.1016/j.earlhumdev.2013.08.013
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is not that clear and more studies and investigations may be warranted. Future research can be done with mixed probiotics that contain yeast and bacteria which have different mechanisms of action and synergistic effect. Besides, higher viability might be expected by mixing both types of probiotics [26] as several studies showed that yeasts could positively interact with bacterial probiotics by enhancing their survival and stimulating their growth [27,28]. However, until such studies are performed, we cannot recommend SB to be used alone in the prevention of NEC or sepsis in preterm babies. Funding and competing interests The drugs were supplied by Biocodex. Ethics approval The trial was approved by the central ethical committee in Ankara, Turkey. Registration This trial was registered by IRCT with ID as IRCT2012071410279N1. References [1] Deshpande G, Rao S, Patole S, Bulsara M. Updated meta-analysis of probiotics for preventing necrotizing enterocolitis in preterm neonates. Pediatrics 2010;125:921–30. [2] Neu J. Necrotizing enterocolitis: the search for a unifying pathogenic theory leading to prevention. Pediatr Clin North Am 1996;43:409–32. [3] Lin PW, Stoll BJ. Necrotizing enterocolitis. Lancet 2006;368:1271–83. [4] Lin HC, Hsu CH, Chen HL, Chung MY, Hsu JF, Lien RI. Oral probiotics prevent necrotizing enterocolitis in very low birth weight preterm infants: a multicenter, randomized, controlled trial. Pediatrics 2008;122:693–700. [5] Gewolb IH, Schwalbe RS, Taciak VL, Harrison TS, Panigrahi P. Stool microflora in extremely low birthweight infants. Arch Dis Child Fetal Neonatal Ed 1999;80:F167–73. [6] Stoll BJ, Gordon T, Korones SB, Shankaran S, Tyson JE, Bauer CR, et al. Late-onset sepsis in very low birth weight neonates: a report from the National Institute of Child Health and Human Development Neonatal Research Network. J Pediatr 1996;129:63–71. [7] Stoll BJ, Hansen N, Fanaroff AA, Wright LL, Carlo WA, Ehrenkranz RA, et al. Late-onset sepsis in very low birth weight neonates: the experience of the NICHD Neonatal Research Network. Pediatrics 2002;110:285–91. [8] Deshpande GC, Rao SC, Keil AD, Patole SK. Evidence-based guidelines for use of probiotics in preterm neonates. BMC Med 2011;9:92.
[9] Costalos C, Skouteri V, Gounaris A, Sevastiadou S, Triandafilidou A, Ekonomidou C, et al. Enteral feeding of premature infants with Saccharomyces boulardii. Early Hum Dev 2003;74:89–96. [10] Ovalı F. Intrauterine growth curves for Turkish infants born between 25 and 42 weeks of gestation. J Trop Pediatr 2002;49:381–3. [11] Walsh MC, Kliegman RM, Fanaroff AA. Necrotiing enterocolitis: a practitioner's perpective. Pediatr Rev 1988;9:219–26. [12] Papile LA, Burstein J, Burstein R, Koffler H. Incidence and evolution of subependymal and intraventricular hemorrhage: a study of infants with birth weight less than 1500 grams. J Pediatr 1978;92:529–34. [13] Alfaleh K, Anabrees J, Bassler D, Al-Kharfi T. Probiotics for prevention of necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev 2011;3:CD005496. [14] Akisu M, Baka M, Yalaz M, Huseyinov A, Kultursay N. Supplementation with Saccharomyces boulardii ameliorates hypoxia/reoxygenation-induced necrotizing enterocolitis in young mice. Eur J Pediatr Surg Oct. 2003;13:319–23. [15] Czerucka D, Piche T, Rampal P. Review article: yeast as probiotics --Saccharomyces boulardii. Aliment Pharmacol Ther 2007;15(26):767–78. [16] Ng SC, Hart AL, Kamm MA, Stagg AJ, Knight SC. Mechanisms of action of probiotics: recent advances. Inflamm Bowel Dis 2009;15:300–10. [17] Sazawal S, Hiremath G, Dhingra U, Malik P, Deb S, Black RE. Efficacy of probiotics in prevention of acute diarrhoea: a meta-analysis of masked, randomised, placebo controlled trials. Lancet Infect Dis 2006;6:374–82. [18] Vanderhoof JA, Whitney DB, Antonson DL, Hanner TL, Lupo JV, Young RJ. Lactobacillus GG in the prevention of antibiotic-associated diarrhea in children. J Pediatr 1999;135:564–8. [19] McFarland LV. Systematic review and meta-analysis of Saccharomyces boulardii in adult patients. World J Gastroenterol May 14 2010;16:2202–22. [20] Levy O. Innate immunity of the newborn: basic mechanisms and clinical correlates. Nat Rev Immunol 2007;7:379–90. [21] Indrio F, Neu J. The intestinal microbiome of infants and the use of probiotics. Curr Opin Pediatr 2011;23:145–50. [22] Schwiertz A, Gruhl B, Löbnitz M, Michel P, Radke M, Blaut M. Development of the intestinal bacterial composition in hospitalized preterm infants in comparison with breast-fed, full-term infants. Pediatr Res 2003;54:393–9. [23] Mihatsch WA, Vossbeck S, Eikmanns B, Hoegel J, Pohlandt F. Effect of Bifidobacterium lactis on the incidence of nosocomial infections in very-low-birth weight infants: a randomized controlled trial. Neonatology 2010;98:156e63. [24] Stratiki Z, Costalos C, Sevastiadou S, Kastanidou O, Skouroliakou M, Giakoumatou A, et al. The effect of a Bifidobacter supplemented bovine milk on intestinal permeability of preterm infants. Early Hum Dev 2007;83:575–9. [25] Indrio F, Riezzo G, Raimondi F, Bisceglia M, Cavallo L, Francavilla R. The effects of probiotics on feeding tolerance, bowel habits, and gastrointestinal motility in preterm newborns. J Pediatr 2008;152:801–6. [26] Bisson JF, Hidalgo S, Rozan P, Messaoudi M. Preventive effects of different probiotic formulations on travelers' diarrhea model in wistar rats: preventive effects of probiotics on TD. Dig Dis Sci 2010;55:911–9. [27] Katakura Y, Sano R, Hashimoto T, Ninomiya K, Shioya S. Lactic acid bacteria display on the cell surface cytosolic proteins that recognize yeast mannan. Appl Microbiol Biotechnol 2010;86:319–26. [28] Gobbetti M, Corsetti A, Rossi J. The sourdough microflora. Interactions between lactic acid bacteria and yeasts: metabolism of carbohydrates. Appl Microbiol Biotechnol 1994;41:456–60.
Please cite this article as: Serce O, et al, Efficacy of saccharomyces boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: A randomised controlled trial, Early Hum Dev (2013), http://dx.doi.org/10.1016/j.earlhumdev.2013.08.013
Contents lists available at ScienceDirect
Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Efficacy of saccharomyces boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: A randomised controlled trial Ozge Serce ⁎, Derya Benzer, Tugba Gursoy, Guner Karatekin, Fahri Ovali Zeynep Kamil Maternity and Children's Research and Training Hospital, Neonatology Unit, Istanbul, Turkey
a r t i c l e
i n f o
Article history: Received 31 May 2013 Received in revised form 18 August 2013 Accepted 20 August 2013 Available online xxxx Keywords: Necrotizing enterocolitis Probiotics Saccharomyces boulardii Sepsis Very low birth weight
a b s t r a c t Background: Probiotics have strain specific effects and the effects of fungi in preventing diseases in preterm infants have been investigated poorly. Saccharomyces boulardii is a yeast which acts both as a probiotic and a polyamine producer. Aim: The objective of this study was to investigate the efficacy of S. boulardii in preventing necrotizing enterocolitis (NEC) or sepsis in very low birth weight infants. Study design and subjects: A prospective, double blind, placebo controlled trial was conducted in preterm infants (≤32 GWs, ≤1500 g birth weight). They were randomized either to receive feeding supplementation with S. boulardii 50 mg/kg every 12 h or placebo, starting with the first feed until discharged. Outcome measures: Necrotizing enterocolitis (NEC) or sepsis and NEC or death. Results: Birth weight and gestational age of the study (n = 104) and the control (n = 104) groups were 1126 ± 232 vs 1162 ± 216 g and 28.8 ± 2.2 vs 28.7 ± 2.1 weeks, respectively. Neither the incidence of stage ≥ 2 NEC or death nor stage ≥2 NEC or late onset culture proven sepsis was significantly lower in the study group when compared with the control group (9.6% vs 7.7%, p = 0.62; 28.8% vs 23%, p = 0.34). Time to reach 100 mL/kg/day of enteral feeding (11.9 ± 7 vs 12.6 ± 7 days, p = 0.37) was not different between the groups. Conclusions: Saccharomyces boulardii did not decrease the incidence of NEC or sepsis. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Necrotizing enterocolitis (NEC) is the most serious acquired disease of the gastrointestinal tract characterized by bowel wall necrosis in very low birth weight infants (VLBW) [1]. The incidence varies among centers [2]. Recent studies report up to 9000 cases of NEC in the United States every year, with a case fatality rate of 15% to 30% [3]. The pathogenesis of NEC remains incompletely understood but it is considered to be due to a complex interaction of factors such as; prematurity, enteral feeding, intestinal hypoxia–ischemia, and bacterial colonization causing mucosal injury through a final, common, inflammatory pathway [2,4,5]. VLBW infants at risk of NEC have abnormal fecal colonization, demonstrate a paucity of normal enteric bacterial species, and have delayed onset of bacterial colonization [5]. Nosocomial infection is also a frequent complication in VLBW infants. Twenty five percent of these infants have at least one or more
⁎ Corresponding author at: Zeynep Kamil Maternity and Children's Research and Training Hospital, Neonatalogy Unit, Zeynep Kamil Mahallesi, Op. Dr. Burhanettin Ustunel Caddesi No:10 Uskudar 34668 Istanbul, Turkey. Tel.: +90 532 327 03 75; fax: +90 216 391 06 90. E-mail addresses: [email protected] (O. Serce), [email protected] (D. Benzer), [email protected] (T. Gursoy), [email protected] (G. Karatekin), [email protected] (F. Ovali).
positive blood cultures over the course of their hospitalization [6]. Late onset sepsis is associated with an increased risk of death, neonatal morbidity and prolonged hospitalization [7]. Probiotic bacteria are live microbial supplements that colonize the gastrointestinal tract and potentially provide benefit to the host. Probiotics may prevent NEC by promoting colonization of the gut with beneficial organisms, preventing colonization by pathogens, improving the maturity and function of gut mucosal barrier, and modulating the immune system [1]. Many clinical trials have evaluated the safety and benefits of probiotic supplementation in VLBW neonates. It is important to note that there are many different mechanisms producing the benefits of probiotics and there are also strain-specific effects [8]. Researchers have generally selected strains belonging to bacterial species naturally present in the intestinal flora. Saccharomyces boulardii (SB) is a yeast which acts both as a probiotic and polyamine producer [9]. Polyamines which are essential for cell growth and differentiation enhance intestinal maturation [9]. SB is used for the treatment of a number of gastrointestinal disorders related to abnormal bacterial growth [9]. Therefore, we hypothesized that SB may be an attractive candidate for the prevention of NEC which is characterized by bacterial colonization causing mucosal injury, and sepsis by overwhelming bacterial translocation with affects on intestinal maturation. SB can be a better strategy than other bacterial strains due to the ability to produce polyamines. We therefore designed a randomized, double-blind placebo controlled
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Please cite this article as: Serce O, et al, Efficacy of saccharomyces boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: A randomised controlled trial, Early Hum Dev (2013), http://dx.doi.org/10.1016/j.earlhumdev.2013.08.013
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O. Serce et al. / Early Human Development xxx (2013) xxx–xxx
clinical trial to evaluate the influence of SB supplementation on the incidence and severity of NEC and sepsis in VLBW infants. 2. Patients and method A prospective, masked, randomized, controlled trial was conducted from October 1, 2010 to November 30, 2011, in Zeynep Kamil Maternity and Children's Research and Training Hospital, Neonatal Intensive Care Unit (NICU) after the approval from ethical committee. VLBW infants (gestational age ≤32 weeks; birth weight ≤1500 g) who survived to feed enterally were eligible for the trial. Infants who had severe asphyxia (stage III), major congenital anomalies, those who had been fasted for more than 3 weeks, died in the first postnatal 14 days and infants who used antifungal therapy were excluded. The event rate was determined by unpublished data based on 2009–2010 of our NICU. Our recent event rate for NEC or sepsis was 31% and for death or NEC was 35% in our NICU (for NEC alone 17%, for sepsis alone 19%, and for death alone 18%). With the α-error set at 0.05 and the β-error set at 0.2, and an absolute reduction in the incidence of either NEC/sepsis or NEC/death of 50%, the number of infants needed to verify our hypothesis was 104 for NEC or sepsis and 92 for NEC or death for each group. The infants enrolled in the study were assigned randomly to the study or control group prospectively after informed parental consent was obtained. Randomization was performed by using sequential numbers generated at the computer center. The allocations were contained in opaque, sequentially numbered sealed envelopes. The study group received Saccharomyces boulardii (Reflor®, Biocodex, France, 50 mg/kg equal to 0.5 × 109 cell/kg per dose twice daily); whereas the control group received placebo (distilled water; 1 mL per dose twice daily) which were added to breast milk or formula [Aptamil Prematil Formula (Milupa AG, Friedrichsdorf, Germany)] starting with the first feed until discharged. The median duration of SB supplementation and follow up period was 44 days. The dosage of SB used in this study was the same as the one used in Costalo's study [9]. SB was kept in a dry place at room temperature, away from light and humidity, in tightly closed container, and mixed with breast milk or formula before feeding. One milliliter suspension was prepared with mixture of the powder of SB with distilled water. This supplementation did not change the appearance of the milk or the formula. Fresh suspension of supplements was prepared by personnel in the breast milk team who were not involved in the care of the infant and who followed the instructions from the sealed envelope. Thus, the only personnel who knew of the infants' group assignments were the investigator and those in the breast milk team who were not involved in the care of the infants. Depending on the birth weight and gestational age, 10–20 mL/kg of breast milk or formula was initiated on the first postnatal day if there were not any contraindications (such as severe IUGR, severe asphyxia, severe preeclampsia, reversed end diastolic flow in umbilical artery) and if the infant tolerated the feeding. The amount of feeding was increased based on birth weight (10 mL/kg increment for b750 g; 20 mL/kg for 750–1250 g; 30 mL/kg for 1250–1500 g birth weight). Aptamil Eoprotin human milk fortifier (Milupa AG, Friedrichsdorf, Germany) was used for fortification of the human milk when the oral intake of 100 mL/kg per day was reached. Feeding was stopped if there was any sign of feeding intolerance which was defined as the presence of gastric aspirate in an amount that was more than one half of the previous feeding with abdominal distension. Infants received total parenteral nutrition until 100 mL/kg per day of enteral feeding was achieved. The gestational age was estimated based on ultrasound examination before the end of 20 GWs (73%) or from the last menstrual period. Infants with a birth weight below 2 SD according to Turkish intrauterine growth curves were classified as small for gestational age (SGA) [10]. “Modified Bell Classification” was used to define NEC [11], and “Papille Classification” for IVH [12]. The culture proven late onset sepsis was defined in neonates who have at least two clinical symptoms and two
laboratory signs mentioned below with positive blood culture after 72 h of life; a) Clinical signs: 1) Core temperature ≥38.5 °C or ≤36 °C; 2) Bradycardia (mean heart rate b10th percentile for age in the absence of external vagal stimulus, beta-blockers or congenital heart disease or tachycardia (mean heart rate 2 SD above normal for age in the absence of external stimulus, chronic drugs and painful stimuli) and/or rhythm instability; 3) Reduced urinary output (b 1 mL/kg/h), hypotension (mean arterial pressure b5th percentile for age); 4) impaired peripheral perfusion; 5) Petechial rash, sclerema; 6) Apnea or tachypnea (mean respiratory rate over 2 SD above normal for age) or increased oxygen requirements or requirement for ventilation support); 7) Feeding intolerance, poor sucking, abdominal distention; 8) Irritability, lethargy and hypotonia b) Laboratory Signs: 1) WBC count: b4000 × 109 cells/L or N 20000 × 109 cells/L; 2) Immature to total neutrophil ratio 0.2; 3) Platelet count: b100 000 × 109 cells/L; 4) C reactive protein N15 mg/L or procalcitonin ≥ 1 ng/mL; 5) Blood glucose N 180 mg/dL or b45 mg/dL; 6) Metabolic acidosis: Base excess b− 10 mEq/L or serum lactate N2mMol/L. Survival was defined as the infant being alive at discharge. Demographic and clinical variables that were potential risk factors for NEC and sepsis were recorded. The primary outcomes were stage ≥2 NEC or late onset culture proven sepsis and stage ≥2 NEC or death. Secondary endpoints were the time to reach 100 mL/kg/day of enteral feeding, weight gain per week, oxygen dependency at 36 weeks, mortality until hospital discharge, and duration of hospitalization. Adverse effects, including culture proven sepsis attributable to SB, anaphylaxis and diarrhea, were recorded. Statistical analyses were performed by SPSS statistics programme v.13.0 (SPSS, Chicago, IL, USA). Demographic data were stated as ratio, mean (±standard deviation), and median (25%–75%). Dependent and independent variables were compared by chi-square and Fisher exact test. Independent samples t-test and Mann–Whitney U test were used to compare continuous variables. Results are stated as relative risk ratio (RR) and 95% confidence interval, CI). All statistical tests were 2-sided, and a P value of ≤ 0.05 was considered to indicate statistical significance. 3. Results Infants assessed for eligibility were 292; 48 of them were excluded because of major congenital malformation (n = 9), severe asphyxia (n = 4), death occurred in 24 h (n = 10), and lack of parental consent (n = 25). A total of 244 infants were enrolled in the study. Three infants were excluded due to fasting more than 3 weeks. Eleven infants were lost to follow up due to family withdraw, and a total of 19 infants died during the first 2 week period after enrollment. Finally, 104 infants per group completed the study protocol. Demographic characteristics of the infants and clinical characteristics of the mothers did not differ between the groups (Table 1). The infants' clinical variables also did not differ between the groups, except for earlier time to start minimal enteral feeding (MEF) in the placebo group (Table 2). Table 3 shows the outcomes of the study. The incidence of stage ≥2 NEC was 7 (6.7%) both in the control and study group, and it did not reach statistical significance (p = 1). Six of fifteen babies who developed stage ≥2 NEC died (40%), and the number of NEC-related deaths was same in both groups. Neither the incidence of stage ≥2 NEC or death nor the incidence of stage ≥2 NEC or late onset culture proven sepsis differed between the groups (Table 3). Twenty five (24.3%) neonates in the control group and 19 (18.3%) neonates in the study group developed culture proven sepsis. Of these
Please cite this article as: Serce O, et al, Efficacy of saccharomyces boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: A randomised controlled trial, Early Hum Dev (2013), http://dx.doi.org/10.1016/j.earlhumdev.2013.08.013
O. Serce et al. / Early Human Development xxx (2013) xxx–xxx Table 1 Demographic and clinical characteristics of the mothers and infants.
Birth weight (g), mean ± SD Gestational age (weeks), mean ± SD 5th min. Apgar, median (25%–75%) Male infants, n (%) Cesarean section, n (%) PROM N18 h, n (%) Preeclampsia, n (%) Antenatal steroid treatment, n (%) SGA, n (%)
Table 3 Outcomes of the study.
Control group (n = 104)
Study group (n = 104)
p
1162 ± 216 28.7 ± 2.1 7(6–8) 56 (53.8) 92 (88.5) 30 (28.8) 16 (15.4) 73 (70.2) 9 (8.7)
1126 ± 232 28.8 ± 2.2 8(6–8) 51 (49) 84 (80.8) 26 (25) 22 (21.2) 71 (68.3) 14 (13.5)
0.3 0.72 0.9 0.48 0.12 0.53 0.28 0.76 0.26
PROM, prolonged rupture of membrane; SGA, small for gestational age.
microorganisms that grew, 72% were gram positive and 28% were gram negative in control group and 73% were gram positive and 27% were gram negative in study group. We did not demonstrate any fungal sepsis in our cohort. There was no statistically significant difference in the time to reach 100 mL/kg/day of enteral feeding, weight gain per week, deaths not attributable to NEC, and duration of hospitalization between the groups. No side effect attributed to SB was observed. 4. Discussion Probiotics have become a hope for NEC prophylaxis and treatment and many trials have been performed. Cochrane meta-analysis indicates that enteral probiotic supplementation such as Lactobacillus and Bifidobacterium species which are members of the normal flora of gut significantly reduces the incidence of stage ≥2 NEC and mortality in preterm infants [13]. Probiotics act through many different mechanisms which are strain-specific. The use of probiotic bacteria to inhibit pathogenic bacteria has been studied extensively over the years, while little attention has been given to yeasts in a similar role. However, yeasts have been reported to antagonise both other yeasts and bacteria in several ways. In an animal study SB was reported to attenuate NEC induced intestinal tissue damage through mechanisms inhibiting intestinal proinflammatory mediator release [14]. In another study, while SB-supplemented formula was shown to have a beneficial effect on gut flora in healthy preterm infants, bringing it closer to that of breastfed infants, no effect on NEC could be shown [9]. We also could not demonstrate any statistically significant difference in NEC or sepsis rate between the groups. This controversy with other studies that used probiotic bacteria and demonstrated reduction in the incidence of NEC or mortality may be explained by various mechanisms. First of all, there are substantial differences in the cell wall composition of bacteria and yeast which are responsible for the modulation of the mucosal immune response by probiotics. This can account for the different responses obtained by these probiotic microorganisms [15]. Secondly, probiotics inhibit the production of proinflammatory cytokines and the predominant cytokine profile under influence depends on the types of probiotics used [16]. SB was predominantly used in adults and older children and was found to be effective in inflammatory bowel disease and gastroenteritis Table 2 Postnatal clinical characteristics of the infants.
Respiratory distress syndrome, n (%) Patent ductus arteriosus, n (%) Intraventricular haemorrhage, n (%) Start on MEF (day), mean ± SD Increment of MEF (mL/kg/day), mean ± SD % of breast milk used, mean ± SD
Control group (n = 104)
Study group (n = 104)
P
56 (53.8) 31 (29.8) 23 (22.1) 1.8 ± 1.1 17 ± 5 65 ± 31
48 (46.2) 20 (19.2) 21 (20.2) 2±1 19 ± 6 64 ± 31
0.26 0.07 0.73 0.03 0.10 0.74
MEF, minimal enteral feeding; PMA, postmenstrual age.
3
Control group Study group P (n = 104) (n = 104) Time to reach 100 mL/kg/day of oral feeding (day), mean ± SD Weight gain (g/week), mean ± SD Oxygen dependency at 36. PMA, n (%) Late onset culture (+) sepsis, n (%) NEC ≥ stage 2, n (%) Stage ≥ 2 NEC or late onset culture (+) sepsis, n (%) Stage ≥ 2 NEC or death, n (%) Deaths attributable to stage ≥ 2 NEC n (%) Deaths (all), n (%) Duration of hospitalization (day), median (25%–75%)
RR (95% CI)
12 ± 7
11 ± 7
0.37
129 ± 65
113 ± 61
0.31
11 (10.6)
12 (11.5)
0.82
25 (24.3)
19 (18.3)
0.29 0.69 (0.35–1.36)
7 (6.7) 30 (28.8)
7 (6.7) 24 (23)
1 1 (0.58–1.72) 0.34 0.86 (0.64–1.15)
10 (9.6) 3 (2.8)
8 (7.7) 3 (2.8)
0.62 0.89 (0.57–1.37) 1 1 (0.44–2.25)
4 (3.8) 43 (29–60)
5 (4.8) 39 (28–60)
0.74 0.89 (0.49–1.63) 0.62
PMA, postmenstrual age; NEC, necrotizing enterocolitis.
[15,17–19]. NEC is the most common inflammatory gut emergency diagnosed in neonatal intensive care units. The diminished innate immunity of premature neonates may be responsible for this conflict observed between adults and neonates [20]. Many preterm infants require intensive care procedures and receive antibiotics early in life, which may be one of the major factors determining intestinal microbiota development. [21]. They are more susceptible to infection as they have an immature immune system, which may in part be due to the abnormal development of their gastrointestinal microflora [22]. They also have delayed colonization with healthy commensals, such as Lactobacillus and Bifidobacterium species, which may lead to altered function of the gut microbial community, particularly the immune functions [22]. So it has been hypothesized that very preterm infants may benefit from colonization with exogenously administered probiotics. However, in most of the studies and meta-analysis the beneficial effects on sepsis could not be supported [4,13,23]. SB, similar to the other studies reported before, did not decrease the incidence of late onset culture proven sepsis in our study. Feeding difficulties that lead to prolonged deprivation of enteral feeds and dependence on total parenteral nutrition are a major issue in preterm neonates. Probiotics improve gastric emptying and gut barrier function [24,25]. Given the importance of optimal enteral nutrition in early postnatal life, the benefits of probiotics have significant implications in improving the overall prognosis of this high-risk population. The study results about the benefit of probiotics for the time to reach full enteral feeding are conflicting due to the significant heterogeneity in feeding protocols as the definition of full enteral feeding varies significantly [13]. Since the definition of full enteral feeding may be different for each infant, we decided to analyse the time to reach 100 mL/kg/day of enteral feeding as a standard measure. Although probiotics were reported to decrease the time to reach full enteral feeding in some studies [13], we did not demonstrate any positive effect of SB on the time to reach 100 mL/kg/day of enteral feeding like in the Costalos' study [9]. This may be due to the different probiotics used in those studies. Clinical findings of gastrointestinal intolerance (higher osmotic load causing abdominal distension, diarrhea or vomiting) or sepsis due to probiotics were the main possible adverse effects of probiotics [8]. However; we did not observe any side effects during the study. So far, probiotics that contain bacterial strains were found to decrease the incidence and severity of NEC and were suggested to be used for the prophylaxis. However, in this study and in the study of Costalos, the reduction in NEC incidence could not be demonstrated with SB. Although this result might be due to several differences observed between bacterial and yeast strains, as mentioned before, it
Please cite this article as: Serce O, et al, Efficacy of saccharomyces boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: A randomised controlled trial, Early Hum Dev (2013), http://dx.doi.org/10.1016/j.earlhumdev.2013.08.013
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is not that clear and more studies and investigations may be warranted. Future research can be done with mixed probiotics that contain yeast and bacteria which have different mechanisms of action and synergistic effect. Besides, higher viability might be expected by mixing both types of probiotics [26] as several studies showed that yeasts could positively interact with bacterial probiotics by enhancing their survival and stimulating their growth [27,28]. However, until such studies are performed, we cannot recommend SB to be used alone in the prevention of NEC or sepsis in preterm babies. Funding and competing interests The drugs were supplied by Biocodex. Ethics approval The trial was approved by the central ethical committee in Ankara, Turkey. Registration This trial was registered by IRCT with ID as IRCT2012071410279N1. References [1] Deshpande G, Rao S, Patole S, Bulsara M. Updated meta-analysis of probiotics for preventing necrotizing enterocolitis in preterm neonates. Pediatrics 2010;125:921–30. [2] Neu J. Necrotizing enterocolitis: the search for a unifying pathogenic theory leading to prevention. Pediatr Clin North Am 1996;43:409–32. [3] Lin PW, Stoll BJ. Necrotizing enterocolitis. Lancet 2006;368:1271–83. [4] Lin HC, Hsu CH, Chen HL, Chung MY, Hsu JF, Lien RI. Oral probiotics prevent necrotizing enterocolitis in very low birth weight preterm infants: a multicenter, randomized, controlled trial. Pediatrics 2008;122:693–700. [5] Gewolb IH, Schwalbe RS, Taciak VL, Harrison TS, Panigrahi P. Stool microflora in extremely low birthweight infants. Arch Dis Child Fetal Neonatal Ed 1999;80:F167–73. [6] Stoll BJ, Gordon T, Korones SB, Shankaran S, Tyson JE, Bauer CR, et al. Late-onset sepsis in very low birth weight neonates: a report from the National Institute of Child Health and Human Development Neonatal Research Network. J Pediatr 1996;129:63–71. [7] Stoll BJ, Hansen N, Fanaroff AA, Wright LL, Carlo WA, Ehrenkranz RA, et al. Late-onset sepsis in very low birth weight neonates: the experience of the NICHD Neonatal Research Network. Pediatrics 2002;110:285–91. [8] Deshpande GC, Rao SC, Keil AD, Patole SK. Evidence-based guidelines for use of probiotics in preterm neonates. BMC Med 2011;9:92.
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Please cite this article as: Serce O, et al, Efficacy of saccharomyces boulardii on necrotizing enterocolitis or sepsis in very low birth weight infants: A randomised controlled trial, Early Hum Dev (2013), http://dx.doi.org/10.1016/j.earlhumdev.2013.08.013