http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–5 ! 2015 Informa UK Ltd. DOI: 10.3109/14767058.2015.1019854
ORIGINAL ARTICLE
Synbiotics for decreasing incidence of necrotizing enterocolitis among preterm neonates – a randomized controlled trial L. P. Nandhini1, Niranjan Biswal1, B. Adhisivam1, Jharna Mandal2, Vishnu Bhat. B1, and Betsy Mathai1 J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Nyu Medical Center on 04/29/15 For personal use only.
1
Department of Pediatrics and 2Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India Abstract
Keywords
Objective: To study the effect of synbiotics in reducing incidence and severity of necrotizing enterocolitis (NEC) among preterm neonates. Methods: This randomized controlled trial conducted in a tertiary care teaching hospital, south India, included 220 enterally fed preterm neonates who were randomized to receive either synbiotics or no intervention. The synbiotic contained Lactobacillus, Bifidobacterium and fructo-oligosaccharide. The demographic parameters, risk factors for NEC and outcome including incidence of NEC, its severity, sepsis and mortality were evaluated. Results: Multiple pregnancies, preeclampsia and prolonged rupture of membranes were important maternal characteristics. The average birth weight and gestational age of the preterm neonates was 1.4 kg and 31 weeks, respectively. There was a 50% reduction in the incidence of NEC of all stages in preterm infants who received synbiotics compared to the non-intervention group (7.4% versus 14.5%). Administration of synbiotics did not reduce the severity of NEC, sepsis or mortality. Conclusion: Enteral supplementation of synbiotics along with breastmilk results in a tendancy to decrease the incidence of NEC among preterm neonates.
Necrotizing enterocolitis, neonate, preterm, probiotic, synbiotic, sepsis
Introduction Necrotizing enterocolitis (NEC) is the most common gastrointestinal problem among neonates with high morbidity and mortality [1]. Prematurity is the single most important risk factor for the development of NEC accounting for 90% of all cases with NEC [2]. Probiotics are oral supplements or food products that contain a sufficient number of viable microorganisms to alter the microflora of the host and have the potential for beneficial health effects. Prebiotics are nondigestible food ingredients that benefit the host by selectively stimulating the favorable growth and/or activity of one or more indigenous probiotic bacteria. Synbiotics contain both probiotics and prebiotics [3]. According to a recent Cochrane review, enteral supplementation of probiotics prevents severe NEC and all-cause mortality in preterm infants [4]. Prebiotics and synbiotics appear to be safe, but extremely limited amount of data are available in neonates and hence this study [5].
Material and methods This open label randomized controlled trial was conducted in the Pediatrics Department, JIPMER, Pondicherry, a tertiary Address for correspondence: Dr. Niranjan Biswal, Department of Pediatrics, JIPMER, Pondicherry 605 006, India. Tel: 0413 – 2273139/ 9442528402. Fax: 0413 – 2272067. E-mail: [email protected]
History Received 7 December 2014 Revised 4 January 2015 Accepted 12 February 2015 Published online 10 March 2015
care teaching hospital, after due approval from the Institute Ethical Committee and registered in the Clinical Trials Registry – India (CTRI/2012/09/003008). All intramural enterally fed preterm neonates with gestational age 28–34 weeks and birth weight 4 1000 g were included in the study. Neonates with major congenital anomalies, surgical problems of the gastrointestinal tract, severe birth asphyxia and early onset sepsis were excluded. After obtaining informed consent from the parents, neonates satisfying the inclusion criteria were randomized prior to starting enteral feeds into two groups using computer generated random numbers kept in opaque sealed envelopes. Infants were assessed for eligibility and randomization within 72 h of life. Neonates in group A were administered synbiotics while those in group B received no intervention. No placebo was used due to the logistics involved. However neonates in both the groups received their mothers’ own milk. For an incidence of NEC being 15% in our Institute, keeping the power of the study as 80% and confidence level of 95% and an expected 10% reduction in incidence of NEC by the intervention, sample size in the each group was calculated to be 108. Each symbiotic capsule (PREPRO HS – manufactured by Fourrts (India) Laboratories Pvt Ltd., Chennai, India) contained Lactobacillus acidophilus (700 million CFU), Bifidobacterium longum (400 million CFU), Lactobacillus rhamnosus (400 million CFU), Lactobacillus plantaris
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(300 million CFU), Lactobacillus casei (300 million CFU), Lactobacillus bulgaricus (300 million CFU), Bifidobacterium infantis (300 million CFU) and Bifidobacterium breve (300 million CFU) and 100 mg of fructo-oligosaccharide (prebiotic). Neonates in group A were administered synbiotics along with expressed breast milk twice a day orally or through nasogastric tube for a total duration of seven days. Aseptic precautions were taken while mixing synbiotics with expressed breast milk and all neonates were fed with their own mother’s milk. Feeds were started and advanced according to standard NICU protocol. Synbiotics were administered only after the neonates were started on enteral feeds. Nutritional supplements (Vitamin D, Calcium, and Vitamin E) were added in standard doses once full enteral feeding was achieved (100 ml/kg/day). Neonates were monitored for clinical features of NEC and sepsis including vomiting, abdominal distention, abdominal wall erythema, tenderness and bleeding manifestations. Neonates in both groups were managed as per standard NICU protocol. The demographic parameters and risk factors for NEC were evaluated. The primary outcomes were the incidence and severity of NEC. The secondary outcomes included incidence of sepsis, mortality, duration of hospital stay, number of days to full enteral feeds and colony counts in stool culture. The following definitions and procedures were used in the study. The gestational age was assessed using the New Ballard score [6]. Neonates with birth weight less that 10th percentile for gestational age according to Lubchencho’s intra uterine growth charts were termed small for gestational age [7]. Prolonged rupture of amniotic membrane was considered as risk factor if membranes had ruptured418 h prior to delivery. Mothers who had received 2 doses of betamethasone 12 h apart or 4 doses of dexamethasone 6 h apart were considered to have received a complete course of steroids. Maternal chorioamnionitis was defined as maternal fever with any of the following: foul smelling liquor, uterine tenderness or elevated leukocyte count. Clinical sepsis was defined if a neonate had clinical features like apnea, lethargy, poor feeding, temperature instability, respiratory distress or seizures and one or more of the following laboratory parameters: (1) positive septic screen (micro ESR 15 mm at 1 h or CRP 46 mg/L), (2) leukocyte count: 55000/mm3, (3) Immature/ mature neutrophil ratio 0.2 [8]. Sepsis was considered proven when microorganisms were isolated from blood, urine or CSF. NEC was staged according to the modified Bell’s staging by a neonatologist not involved in the study [9]. Neonates with stage II and III NEC were classified as severe NEC [10] Synbiotics were discontinued when babies developed features of NEC or sepsis. Babies who developed NEC and sepsis were treated according to the standard NICU protocol. Stool culture for estimating the colony counts of Bifidobacteria and Lactobacillus was done on day 7 of the study for 22 neonates. Stool sample was collected using a sterile swab and transported to the laboratory in Cary Blair transport media. One gram of the stool sample was diluted in 9 ml of sterile distilled water and this suspension was serially diluted till 108 dilutions. Using a standard one-microliter loop, the suspension was plated on the surface of MRS agar and Bifidobacterium agar. The plates were incubated for 48 h
J Matern Fetal Neonatal Med, Early Online: 1–5
under anaerobic conditions at 37 C. The colony counts were calculated at the end of 48 h [11]. Statistical analysis of data was done using SPSS (Statistical Packages for Social Sciences) version 13 (SPSS Inc., Chicago, IL). Intention to treat analysis was done. Chi square test and Fisher’s exact test were used for analyzing discrete variables. A p value 50.05 was considered statistically significant.
Results The CONSORT diagram for the study is depicted in Figure 1. Two neonates in group A did not receive the intervention as one of them developed intestinal obstruction soon after starting feeds and the other neonate’s general condition worsened. The clinical profile of neonates in the two groups is shown in Table 1. The average birth weight and gestational age of the preterm neonates was 1.4 kg and 31 weeks, respectively. The baseline characteristics in both the groups were comparable. Though the incidence of preeclampsia was slightly more in the mothers belonging to the synbiotic group, it was not statistically significant. The comparison of outcome variables between the two groups is depicted in Table 2. There was a 50% reduction in the incidence of NEC of all stages in preterm infants who received synbiotics compared to the nonintervention group (7.4% versus 14.5%). However, it was not statistically significant. The severity of NEC, sepsis and mortality were similar in both the groups. An improvement in the time to achieve full enteral feeds was noted in the synbiotic group. There were no adverse events due to the administration of synbiotics. The stool cultures done at the end of 7 days of administration of synbiotics showed increased colonization by Lactobacilli as compared to the non-intervention group.
Discussion NEC is an acute inflammatory disease characterized by variable bowel wall necrosis and perforation occurs in onethird of the affected neonates [12]. It is primarily a disease of preterm infants and a majority of cases occur in very low birth weight (VLBW) infants (birth weight51500 g) [13]. Although neonatal survival has improved due to the advancement in intensive care, the incidence and long-term outcome due to NEC have remain unchanged. Restoration of gut flora towards a full-term/healthy signature pattern may confer protection against NEC. With an aim to achieve this, several preventive strategies including probiotics have been tried with varied but encouraging results. Indigestible prebiotic sugars known to promote the growth of probiotic bacteria in the colon have also been tried with no clear benefit. In the literature, there is a serious lack of well-designed clinical trials of probiotics and prebiotics in NEC, and hence this study combining both probiotics and prebiotics gains importance. In our study, 7.4% of neonates in the synbiotic group developed NEC compared to 14.5% of neonates in the nonintervention group. Though a 50% reduction in the incidence of NEC of all stages was noted among neonates who received synbiotics compared to the non-intervention group, this difference was not found to be statistically significant (p ¼ 0.18). Several other systematic reviews and metaanalysis have found similar results with probiotics
Synbiotics for decreasing NEC
DOI: 10.3109/14767058.2015.1019854
Enrollment
3
Assessed for eligibility (n=280)
Excluded (n= 60) ♦ Not meeting inclusion criteria (n=55 ) ♦ Declined to participate (n=5 )
Randomized (n=220)
Group A
Group B
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Allocaon Allocated to NO intervention (n=110) ♦ Received NO intervention (n=110)
Allocated to intervention (synbiotics) (n=110) ♦ Received allocated intervention (n=108) ♦ Did not receive allocated intervention (developed intestinal obstruction -1, worsened general condition before initiating feeds -1) (n=2)
Clinical course NEC = 7.4% (n =8) Severe NEC = 0% (n= 0) Mortality = 9.2% (n= 10)
NEC = 14.5% (n= 16) Severe NEC = 2.7% (n= 3) Mortality = 8.1% (n=9)
Analysis Analysed (n=108) ♦ Excluded from analysis (n=0)
Analysed (n=110) ♦ Excluded from analysis (n=0)
Figure 1. CONSORT flow diagram.
Table 1. Baseline characteristics of the participants.
Characteristics
Group B Group A (Synbiotics) (No intervention) (n ¼ 110) p value (n ¼ 108)
Maternal characteristics Preeclampsia 40 (37 %) Antenatal steroids 27 (25%) Multiple pregnancy 38 (35%) Gestational diabetes 3 (2.7%) Antepartum hemorrhage 3 (2.7%) Chorioamnionitis 1 (0.92 %) Caesarean section 9 (8.3%) Prolonged rupture of 17 (15%) membranes 418 hours Neonatal characteristics Birth weight (grams) 1430 ± 209 Gestational age (weeks) 31.6 ± 1.4 Small for gestational age 13 (12%) Age at enrolment (Days) 2.0 Use of surfactant 5 (4.6%) Requirement of mechanical 5 (4.6%) ventilation Requirement of CPAP 38 (35%)
28 30 44 3 7 0 11 16
(25 %) (27%) (40%) (2.7%) (6.3%) (0%) (10%) (14.5%)
0.07 0.94 0.48 1.00 0.33 0.49 0.81 0.85
1444 ± 217 31.4 ± 1.4 11 (10%) 1.69 6 (5.4%) 3 (2.7%)
0.63 1.00 0.67 0.07 1.00 0.49
31 (28%)
1.00
[4,5,14–16]. The incidence of severe NEC was 2.7% in the non-intervention group and no such severe case was noted in the synbiotic group. This could be explained by the fact that our study did not include preterm babies below 1000 g where one would expect more incidence of severe NEC. The recent Cochrane review also concluded that the administration of
prophylactic probiotics significantly reduced the incidence of severe NEC [4]. In our RCT, the incidence of sepsis and mortality was not significantly reduced by the intervention. However, a majority of the studies in literature have found probiotics to reduce overall mortality while few have shown to reduce sepsis also [4,17–19]. It is noteworthy that an improvement in the time to achieve full enteral feeds was noted in the synbiotic group. Pooled data of studies have also shown similar reduction in time to reach full enteral feeds [4]. This particular benefit of the intervention is likely to have a positive impact on the weight gain of the neonate and an early NICU discharge. Potential mechanisms by which probiotics and synbiotics may protect neonates from developing NEC or sepsis, or both, include an increased barrier to migration bacteria and their products across the mucosa, competitive exclusion of potential pathogens, modification of host response to microbial products, augmentation of immunoglobulin A (IGA) mucosal responses, enhancement of enteral nutrition that inhibits the growth of pathogens, and up-regulation of immune responses [4]. A new approach in microflora management procedures is the use of synbiotics, in which probiotics and prebiotics are used in combination. The live microbial additions (probiotics) may be used in conjunction with specific substrates (prebiotics) for growth (e.g. a fructo-oligosaccharide in conjunction with a bifidobacterial strain or lactitol in conjunction with a Lactobacillus organism). This combination could improve
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J Matern Fetal Neonatal Med, Early Online: 1–5
Table 2. Comparison of outcome variables.
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Variables Incidence of NEC of all stages Incidence of NEC stage II and III Incidence of clinical sepsis Culture proven sepsis among clinical sepsis Mortality due to sepsis/NEC Duration of antibiotic therapy (no. of days) Duration of hospital stay (no. of days) Number of days to full enteral feeds Requirement of parenteral nutrition Requirement of inotropes Requirement of phototherapy Requirement of platelet transfusion Requirement FFP transfusion Colony counts in stool culture Bifidobacteria 4 108 CFU Lactobacillus 4 108 CFU
Group A (Synbiotics) (n ¼ 108)
Group B (No intervention) (n ¼ 110)
8 (7.4%) 0 30 (14%) 4 (13.3%) 10 (9.2%) 6.11 ± 4.3 8.29 ± 4.5 2.17 ± 2.05 0 (0%) 8 (7.4%) 29 (26%) 15 (7%) 11 (5%) (n ¼ 11) 8 10
16 (14.5%) 3 (2.7%) 38 (17%) 4 (10.5%) 9 (8.1%) 7.06 ± 4.7 8.40 ± 5.1 2.96 ± 2.92 1 (0.9 %) 4 (3.6%) 23 (20.9 %) 11(5%) 12(6%) (n ¼ 11) 3 4
the survival of the probiotic organism, because its specific substrate is readily available for its fermentation [20]. The synbiotic used in our RCT contained Lactobacillus, Bifidobacterium (probiotics) and fructo-oligosaccharide (prebiotic). Generally, probiotic preparations containing either Lactobacillus alone or in combination with Bifidobacterium have been found to be effective [4]. The stool cultures done at the end of 7 days of administration of synbiotic showed increased colonization by Lactobacilli, which is a proof of the gut colonization with the desirable flora. Both the synbiotic and breastmilk could have contributed for this colonization. However, as there is statistically significant increased colonization in the synbiotic group, it is possible that the use of synbiotics augments Lactobacillus colonization. According to a RCT from Turkey, introduction of synbiotics (Bifidobacterium lactis + Chicory inulin) with the first enteral feeding reduced feeding intolerance and morbidity in VLBW infants [21]. Though, a beneficial effect of synbiotics is highlighted in this study, the role of breastmilk in preventing NEC cannot be undermined. According to the recent Cochrane review, the use of probiotics was described as safe and well tolerated [4]. We also did not observe any adverse events due to administration of synbiotics and the intervention appears to be safe. However, there is always a theoretical risk of septicemia due to probiotics, especially in immunocompromised neonates. Contraindications for probiotic use include extremely low birth weight, clinical instability, abnormal abdominal examination, the presence of congenital abnormalities, post-NEC, stage III asphyxia, and umbilical catheters [22]. A rare case report of infant death due to contaminated probiotic powder has been documented [23]. According to a recent study, early microbial colonization has a critical role in the development of NEC and urinary marker (alanine:histidine ratio 44) was associated with microbial community composition and had a predictive value of 78% for prediction of NEC [24]. Probiotics, prebiotics and synbiotics can alter the early microflora in the neonatal gut. As the properties of different probiotic species vary and can be strain-specific, the effects of one probiotic strain should not be generalized to others without
p value 0.18 0.24 0.30 0.72 0.85 0.14 0.42 0.005 1.00 0.37 0.55 0.40 1.00 0.08 0.02
confirmation in separate studies. Careful consideration should be given to these issues before patients are advised to use probiotic supplements in clinical practice [25].
Declaration of interest The authors report no conflicts of interest.
References 1. Kasivajjula H, Maheshwari A. Pathophysiology and current management of necrotizing enterocolitis. Indian J Pediatr 2014;81: 489–97. 2. Sharma R, Hudak ML, Tepas JJ, et al. Impact of gestational age on the clinical presentation and surgical outcome of necrotizing enterocolitis. J Perinatol 2006;26:342–7. 3. Thomas DW, Greer FR; American Academy of Pediatrics Committee on Nutrition; American Academy of Pediatrics Section on Gastroenterology, Hepatology, and Nutrition. Probiotics and prebiotics in pediatrics. Pediatrics 2010;126: 1217–31. 4. AlFaleh K, Anabrees J. Probiotics for prevention of necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev 2014;(4):CD005496. 5. Panigrahi P. Probiotics and prebiotics in neonatal necrotizing enterocolitis: new opportunities for translational research. Pathophysiology 2014;21:35–46. 6. Ballard JL, Khoury JC, Wedig K, et al. New Ballard Score, expanded to include extremely premature infants. J Pediatr 1991; 119:417–23. 7. Lubchenco LO, Hansman C, Boyd E. Intrauterine growth in length and head circumference as estimated from live births at gestational ages from 26 to 42 weeks. Pediatrics 1966;37:403–8. 8. Doellner H, Arntzen KJ, Haereid PE, et al. Interleukin-6 concentrations in neonates evaluated for sepsis. J. Pediatr 1998;132:295–9. 9. Walsh MC, Kliegman RM. Necrotizing enterocolitis: treatment based on staging criteria. Pediatr Clin North Am 1986; 33:179–201. 10. Samanta M, Sarkar M, Ghosh P, et al. Prophylactic probiotics for prevention of necrotizing enterocolitis in very low birth weight newborns. J Trop Pediatr 2009;55:128–31. 11. Mello RMP, Morais MB, Tahan S, et al. Lactobacilli and Bifidobacteria in the feces of schoolchildren of two different socioeconomic groups: children from a favela and children from a private school. J Pediatr (Rio J) 2009; 85:307–14. 12. Kafetzis DA, Skevaki C, Costalos C. Neonatal necrotizing enterocolitis: an overview. Curr Opin Infect Dis 2003;16:349–55. 13. Kosloske AM. Epidemiology of necrotizing enterocolitis. Acta Paediatrica 1994;396:2–7.
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DOI: 10.3109/14767058.2015.1019854
14. Lin HC, Su BH, Chen AC, et al. Oral probiotics reduce the incidence and severity of necrotizing enterocolitis in very low birth weight infants. Pediatrics 2005;115:1–4. 15. Claud EC. Probiotics and neonatal necrotizing enterocolitis. Anaerobe 2011;17:180–5. 16. Wang Q, Dong J, Zhu Y, Probiotic supplement reduces risk of necrotizing enterocolitis and mortality in preterm very low-birthweight infants: an updated meta-analysis of 20 randomized, controlled trials. J. Pediatr Surg 2012;47:241–8. 17. Al-Hosni M, Duenas M, Hawk M, et al. Probiotics-supplemented feeding in extremely low-birth-weight infants. J Perinatol 2012;32: 253–9. 18. Ferna´ndez-Carrocera LA, Solis-Herrera A, Cabanillas-Ayo´n M, et al. Double-blind, randomised clinical assay to evaluate the efficacy of probiotics in preterm newborns weighing less than 1500 g in the prevention of necrotising enterocolitis. Arch Dis Child. Fetal Neonatal Ed 2013;98:F5–9. 19. Demirel G, Erdeve O, Celik IH, Dilmen U. Saccharomyces boulardii for prevention of necrotizing enterocolitis in preterm infants: a randomized, controlled study. Acta Paediatr 2013;102:e560–5.
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20. Collins MD, Gibson GR. Probiotics, prebiotics, and synbiotics: approaches for modulating the microbial ecology of the gut. Am J Clin Nutr 1999;69:1052S–7S. 21. Aydin B, Dilli D, Erol S, et al. The effects of synbiotic use on morbidity and mortality in premature infants: a prospective randomized controlled trial. Arch Dis Child 2012;97:A462. 22. Parker R. Probiotic guideline for necrotizing enterocolitis prevention in very low-birth-weight neonates. Adv Neonatal Care 2014; 14:88–95. 23. CDC Advisory. Infant death caused by contaminated probiotic powder. Available from: http://www.philly.com/philly/blogs/ healthy_kids/CDC-advisory-infant-death-caused-by-probioticpowder.html [last accessed 4 Jan 2015]. 24. Morrow AL, Lagomarcino AJ, Schibler KR, et al. Early microbial and metabolomic signatures predict later onset of necrotizing enterocolitis in preterm infants. Microbiome 2013; 1:13. 25. Boyle RJ, Roy MR-B, Tang MLK. Probiotic use in clinical practice: what are the risks? Am J Clin Nutr 2006;83: 1256–64.
ORIGINAL ARTICLE
Synbiotics for decreasing incidence of necrotizing enterocolitis among preterm neonates – a randomized controlled trial L. P. Nandhini1, Niranjan Biswal1, B. Adhisivam1, Jharna Mandal2, Vishnu Bhat. B1, and Betsy Mathai1 J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Nyu Medical Center on 04/29/15 For personal use only.
1
Department of Pediatrics and 2Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India Abstract
Keywords
Objective: To study the effect of synbiotics in reducing incidence and severity of necrotizing enterocolitis (NEC) among preterm neonates. Methods: This randomized controlled trial conducted in a tertiary care teaching hospital, south India, included 220 enterally fed preterm neonates who were randomized to receive either synbiotics or no intervention. The synbiotic contained Lactobacillus, Bifidobacterium and fructo-oligosaccharide. The demographic parameters, risk factors for NEC and outcome including incidence of NEC, its severity, sepsis and mortality were evaluated. Results: Multiple pregnancies, preeclampsia and prolonged rupture of membranes were important maternal characteristics. The average birth weight and gestational age of the preterm neonates was 1.4 kg and 31 weeks, respectively. There was a 50% reduction in the incidence of NEC of all stages in preterm infants who received synbiotics compared to the non-intervention group (7.4% versus 14.5%). Administration of synbiotics did not reduce the severity of NEC, sepsis or mortality. Conclusion: Enteral supplementation of synbiotics along with breastmilk results in a tendancy to decrease the incidence of NEC among preterm neonates.
Necrotizing enterocolitis, neonate, preterm, probiotic, synbiotic, sepsis
Introduction Necrotizing enterocolitis (NEC) is the most common gastrointestinal problem among neonates with high morbidity and mortality [1]. Prematurity is the single most important risk factor for the development of NEC accounting for 90% of all cases with NEC [2]. Probiotics are oral supplements or food products that contain a sufficient number of viable microorganisms to alter the microflora of the host and have the potential for beneficial health effects. Prebiotics are nondigestible food ingredients that benefit the host by selectively stimulating the favorable growth and/or activity of one or more indigenous probiotic bacteria. Synbiotics contain both probiotics and prebiotics [3]. According to a recent Cochrane review, enteral supplementation of probiotics prevents severe NEC and all-cause mortality in preterm infants [4]. Prebiotics and synbiotics appear to be safe, but extremely limited amount of data are available in neonates and hence this study [5].
Material and methods This open label randomized controlled trial was conducted in the Pediatrics Department, JIPMER, Pondicherry, a tertiary Address for correspondence: Dr. Niranjan Biswal, Department of Pediatrics, JIPMER, Pondicherry 605 006, India. Tel: 0413 – 2273139/ 9442528402. Fax: 0413 – 2272067. E-mail: [email protected]
History Received 7 December 2014 Revised 4 January 2015 Accepted 12 February 2015 Published online 10 March 2015
care teaching hospital, after due approval from the Institute Ethical Committee and registered in the Clinical Trials Registry – India (CTRI/2012/09/003008). All intramural enterally fed preterm neonates with gestational age 28–34 weeks and birth weight 4 1000 g were included in the study. Neonates with major congenital anomalies, surgical problems of the gastrointestinal tract, severe birth asphyxia and early onset sepsis were excluded. After obtaining informed consent from the parents, neonates satisfying the inclusion criteria were randomized prior to starting enteral feeds into two groups using computer generated random numbers kept in opaque sealed envelopes. Infants were assessed for eligibility and randomization within 72 h of life. Neonates in group A were administered synbiotics while those in group B received no intervention. No placebo was used due to the logistics involved. However neonates in both the groups received their mothers’ own milk. For an incidence of NEC being 15% in our Institute, keeping the power of the study as 80% and confidence level of 95% and an expected 10% reduction in incidence of NEC by the intervention, sample size in the each group was calculated to be 108. Each symbiotic capsule (PREPRO HS – manufactured by Fourrts (India) Laboratories Pvt Ltd., Chennai, India) contained Lactobacillus acidophilus (700 million CFU), Bifidobacterium longum (400 million CFU), Lactobacillus rhamnosus (400 million CFU), Lactobacillus plantaris
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Nyu Medical Center on 04/29/15 For personal use only.
2
L. P. Nandhini et al.
(300 million CFU), Lactobacillus casei (300 million CFU), Lactobacillus bulgaricus (300 million CFU), Bifidobacterium infantis (300 million CFU) and Bifidobacterium breve (300 million CFU) and 100 mg of fructo-oligosaccharide (prebiotic). Neonates in group A were administered synbiotics along with expressed breast milk twice a day orally or through nasogastric tube for a total duration of seven days. Aseptic precautions were taken while mixing synbiotics with expressed breast milk and all neonates were fed with their own mother’s milk. Feeds were started and advanced according to standard NICU protocol. Synbiotics were administered only after the neonates were started on enteral feeds. Nutritional supplements (Vitamin D, Calcium, and Vitamin E) were added in standard doses once full enteral feeding was achieved (100 ml/kg/day). Neonates were monitored for clinical features of NEC and sepsis including vomiting, abdominal distention, abdominal wall erythema, tenderness and bleeding manifestations. Neonates in both groups were managed as per standard NICU protocol. The demographic parameters and risk factors for NEC were evaluated. The primary outcomes were the incidence and severity of NEC. The secondary outcomes included incidence of sepsis, mortality, duration of hospital stay, number of days to full enteral feeds and colony counts in stool culture. The following definitions and procedures were used in the study. The gestational age was assessed using the New Ballard score [6]. Neonates with birth weight less that 10th percentile for gestational age according to Lubchencho’s intra uterine growth charts were termed small for gestational age [7]. Prolonged rupture of amniotic membrane was considered as risk factor if membranes had ruptured418 h prior to delivery. Mothers who had received 2 doses of betamethasone 12 h apart or 4 doses of dexamethasone 6 h apart were considered to have received a complete course of steroids. Maternal chorioamnionitis was defined as maternal fever with any of the following: foul smelling liquor, uterine tenderness or elevated leukocyte count. Clinical sepsis was defined if a neonate had clinical features like apnea, lethargy, poor feeding, temperature instability, respiratory distress or seizures and one or more of the following laboratory parameters: (1) positive septic screen (micro ESR 15 mm at 1 h or CRP 46 mg/L), (2) leukocyte count: 55000/mm3, (3) Immature/ mature neutrophil ratio 0.2 [8]. Sepsis was considered proven when microorganisms were isolated from blood, urine or CSF. NEC was staged according to the modified Bell’s staging by a neonatologist not involved in the study [9]. Neonates with stage II and III NEC were classified as severe NEC [10] Synbiotics were discontinued when babies developed features of NEC or sepsis. Babies who developed NEC and sepsis were treated according to the standard NICU protocol. Stool culture for estimating the colony counts of Bifidobacteria and Lactobacillus was done on day 7 of the study for 22 neonates. Stool sample was collected using a sterile swab and transported to the laboratory in Cary Blair transport media. One gram of the stool sample was diluted in 9 ml of sterile distilled water and this suspension was serially diluted till 108 dilutions. Using a standard one-microliter loop, the suspension was plated on the surface of MRS agar and Bifidobacterium agar. The plates were incubated for 48 h
J Matern Fetal Neonatal Med, Early Online: 1–5
under anaerobic conditions at 37 C. The colony counts were calculated at the end of 48 h [11]. Statistical analysis of data was done using SPSS (Statistical Packages for Social Sciences) version 13 (SPSS Inc., Chicago, IL). Intention to treat analysis was done. Chi square test and Fisher’s exact test were used for analyzing discrete variables. A p value 50.05 was considered statistically significant.
Results The CONSORT diagram for the study is depicted in Figure 1. Two neonates in group A did not receive the intervention as one of them developed intestinal obstruction soon after starting feeds and the other neonate’s general condition worsened. The clinical profile of neonates in the two groups is shown in Table 1. The average birth weight and gestational age of the preterm neonates was 1.4 kg and 31 weeks, respectively. The baseline characteristics in both the groups were comparable. Though the incidence of preeclampsia was slightly more in the mothers belonging to the synbiotic group, it was not statistically significant. The comparison of outcome variables between the two groups is depicted in Table 2. There was a 50% reduction in the incidence of NEC of all stages in preterm infants who received synbiotics compared to the nonintervention group (7.4% versus 14.5%). However, it was not statistically significant. The severity of NEC, sepsis and mortality were similar in both the groups. An improvement in the time to achieve full enteral feeds was noted in the synbiotic group. There were no adverse events due to the administration of synbiotics. The stool cultures done at the end of 7 days of administration of synbiotics showed increased colonization by Lactobacilli as compared to the non-intervention group.
Discussion NEC is an acute inflammatory disease characterized by variable bowel wall necrosis and perforation occurs in onethird of the affected neonates [12]. It is primarily a disease of preterm infants and a majority of cases occur in very low birth weight (VLBW) infants (birth weight51500 g) [13]. Although neonatal survival has improved due to the advancement in intensive care, the incidence and long-term outcome due to NEC have remain unchanged. Restoration of gut flora towards a full-term/healthy signature pattern may confer protection against NEC. With an aim to achieve this, several preventive strategies including probiotics have been tried with varied but encouraging results. Indigestible prebiotic sugars known to promote the growth of probiotic bacteria in the colon have also been tried with no clear benefit. In the literature, there is a serious lack of well-designed clinical trials of probiotics and prebiotics in NEC, and hence this study combining both probiotics and prebiotics gains importance. In our study, 7.4% of neonates in the synbiotic group developed NEC compared to 14.5% of neonates in the nonintervention group. Though a 50% reduction in the incidence of NEC of all stages was noted among neonates who received synbiotics compared to the non-intervention group, this difference was not found to be statistically significant (p ¼ 0.18). Several other systematic reviews and metaanalysis have found similar results with probiotics
Synbiotics for decreasing NEC
DOI: 10.3109/14767058.2015.1019854
Enrollment
3
Assessed for eligibility (n=280)
Excluded (n= 60) ♦ Not meeting inclusion criteria (n=55 ) ♦ Declined to participate (n=5 )
Randomized (n=220)
Group A
Group B
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Allocaon Allocated to NO intervention (n=110) ♦ Received NO intervention (n=110)
Allocated to intervention (synbiotics) (n=110) ♦ Received allocated intervention (n=108) ♦ Did not receive allocated intervention (developed intestinal obstruction -1, worsened general condition before initiating feeds -1) (n=2)
Clinical course NEC = 7.4% (n =8) Severe NEC = 0% (n= 0) Mortality = 9.2% (n= 10)
NEC = 14.5% (n= 16) Severe NEC = 2.7% (n= 3) Mortality = 8.1% (n=9)
Analysis Analysed (n=108) ♦ Excluded from analysis (n=0)
Analysed (n=110) ♦ Excluded from analysis (n=0)
Figure 1. CONSORT flow diagram.
Table 1. Baseline characteristics of the participants.
Characteristics
Group B Group A (Synbiotics) (No intervention) (n ¼ 110) p value (n ¼ 108)
Maternal characteristics Preeclampsia 40 (37 %) Antenatal steroids 27 (25%) Multiple pregnancy 38 (35%) Gestational diabetes 3 (2.7%) Antepartum hemorrhage 3 (2.7%) Chorioamnionitis 1 (0.92 %) Caesarean section 9 (8.3%) Prolonged rupture of 17 (15%) membranes 418 hours Neonatal characteristics Birth weight (grams) 1430 ± 209 Gestational age (weeks) 31.6 ± 1.4 Small for gestational age 13 (12%) Age at enrolment (Days) 2.0 Use of surfactant 5 (4.6%) Requirement of mechanical 5 (4.6%) ventilation Requirement of CPAP 38 (35%)
28 30 44 3 7 0 11 16
(25 %) (27%) (40%) (2.7%) (6.3%) (0%) (10%) (14.5%)
0.07 0.94 0.48 1.00 0.33 0.49 0.81 0.85
1444 ± 217 31.4 ± 1.4 11 (10%) 1.69 6 (5.4%) 3 (2.7%)
0.63 1.00 0.67 0.07 1.00 0.49
31 (28%)
1.00
[4,5,14–16]. The incidence of severe NEC was 2.7% in the non-intervention group and no such severe case was noted in the synbiotic group. This could be explained by the fact that our study did not include preterm babies below 1000 g where one would expect more incidence of severe NEC. The recent Cochrane review also concluded that the administration of
prophylactic probiotics significantly reduced the incidence of severe NEC [4]. In our RCT, the incidence of sepsis and mortality was not significantly reduced by the intervention. However, a majority of the studies in literature have found probiotics to reduce overall mortality while few have shown to reduce sepsis also [4,17–19]. It is noteworthy that an improvement in the time to achieve full enteral feeds was noted in the synbiotic group. Pooled data of studies have also shown similar reduction in time to reach full enteral feeds [4]. This particular benefit of the intervention is likely to have a positive impact on the weight gain of the neonate and an early NICU discharge. Potential mechanisms by which probiotics and synbiotics may protect neonates from developing NEC or sepsis, or both, include an increased barrier to migration bacteria and their products across the mucosa, competitive exclusion of potential pathogens, modification of host response to microbial products, augmentation of immunoglobulin A (IGA) mucosal responses, enhancement of enteral nutrition that inhibits the growth of pathogens, and up-regulation of immune responses [4]. A new approach in microflora management procedures is the use of synbiotics, in which probiotics and prebiotics are used in combination. The live microbial additions (probiotics) may be used in conjunction with specific substrates (prebiotics) for growth (e.g. a fructo-oligosaccharide in conjunction with a bifidobacterial strain or lactitol in conjunction with a Lactobacillus organism). This combination could improve
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Table 2. Comparison of outcome variables.
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Variables Incidence of NEC of all stages Incidence of NEC stage II and III Incidence of clinical sepsis Culture proven sepsis among clinical sepsis Mortality due to sepsis/NEC Duration of antibiotic therapy (no. of days) Duration of hospital stay (no. of days) Number of days to full enteral feeds Requirement of parenteral nutrition Requirement of inotropes Requirement of phototherapy Requirement of platelet transfusion Requirement FFP transfusion Colony counts in stool culture Bifidobacteria 4 108 CFU Lactobacillus 4 108 CFU
Group A (Synbiotics) (n ¼ 108)
Group B (No intervention) (n ¼ 110)
8 (7.4%) 0 30 (14%) 4 (13.3%) 10 (9.2%) 6.11 ± 4.3 8.29 ± 4.5 2.17 ± 2.05 0 (0%) 8 (7.4%) 29 (26%) 15 (7%) 11 (5%) (n ¼ 11) 8 10
16 (14.5%) 3 (2.7%) 38 (17%) 4 (10.5%) 9 (8.1%) 7.06 ± 4.7 8.40 ± 5.1 2.96 ± 2.92 1 (0.9 %) 4 (3.6%) 23 (20.9 %) 11(5%) 12(6%) (n ¼ 11) 3 4
the survival of the probiotic organism, because its specific substrate is readily available for its fermentation [20]. The synbiotic used in our RCT contained Lactobacillus, Bifidobacterium (probiotics) and fructo-oligosaccharide (prebiotic). Generally, probiotic preparations containing either Lactobacillus alone or in combination with Bifidobacterium have been found to be effective [4]. The stool cultures done at the end of 7 days of administration of synbiotic showed increased colonization by Lactobacilli, which is a proof of the gut colonization with the desirable flora. Both the synbiotic and breastmilk could have contributed for this colonization. However, as there is statistically significant increased colonization in the synbiotic group, it is possible that the use of synbiotics augments Lactobacillus colonization. According to a RCT from Turkey, introduction of synbiotics (Bifidobacterium lactis + Chicory inulin) with the first enteral feeding reduced feeding intolerance and morbidity in VLBW infants [21]. Though, a beneficial effect of synbiotics is highlighted in this study, the role of breastmilk in preventing NEC cannot be undermined. According to the recent Cochrane review, the use of probiotics was described as safe and well tolerated [4]. We also did not observe any adverse events due to administration of synbiotics and the intervention appears to be safe. However, there is always a theoretical risk of septicemia due to probiotics, especially in immunocompromised neonates. Contraindications for probiotic use include extremely low birth weight, clinical instability, abnormal abdominal examination, the presence of congenital abnormalities, post-NEC, stage III asphyxia, and umbilical catheters [22]. A rare case report of infant death due to contaminated probiotic powder has been documented [23]. According to a recent study, early microbial colonization has a critical role in the development of NEC and urinary marker (alanine:histidine ratio 44) was associated with microbial community composition and had a predictive value of 78% for prediction of NEC [24]. Probiotics, prebiotics and synbiotics can alter the early microflora in the neonatal gut. As the properties of different probiotic species vary and can be strain-specific, the effects of one probiotic strain should not be generalized to others without
p value 0.18 0.24 0.30 0.72 0.85 0.14 0.42 0.005 1.00 0.37 0.55 0.40 1.00 0.08 0.02
confirmation in separate studies. Careful consideration should be given to these issues before patients are advised to use probiotic supplements in clinical practice [25].
Declaration of interest The authors report no conflicts of interest.
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