行政院國家科學委員會專題研究計畫 成果報告
極低體重早產兒口服原生素對壞死性腸炎的影響
計畫類別： 個別型計畫 計畫編號： NSC94-2314-B-039-007- 執行期間： 94 年 08 月 01 日至 95 年 07 月 31 日 執行單位： 中國醫藥大學附設醫院小兒科 計畫主持人： 林鴻志 共同主持人： 鍾美勇，謝武勳，穆淑琪，陳德人，陳昭惠，許瓊心 計畫參與人員： 廖仁敏 報告類型： 精簡報告 處理方式： 本計畫可公開查詢中 華 民 國 95 年 10 月 31 日
行政院國家科學委員會補助專題研究計畫
■ 成 果 報 告
□期中進度報告
「極低體重早產兒口服原生素對壞死性腸炎的影響」
Impact of oral Probiotics on Necrotizing Enterocolitis for Preterm Extremely Low
Birth Weight Infants
計畫類別﹕■ 個別型計畫
□ 整合型計畫
計畫編號﹕
NSC 94－2314－B－039－007－
執行期間﹕
94 年 08 月 01 日 至 95 年 07 月 31 日
計畫主持人﹕林鴻志
共同主持人﹕謝武勳､陳昭惠､鍾美勇､許瓊心､穆淑琪､陳德人
計畫參與人員﹕ 廖仁敏､杜濠仲､洪雪敏
成果報告類型
(依經費核定清單規定繳交)﹕■精簡報告 □完整報告
本成果報告包括以下應繳交之附件﹕
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
□出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
處理方式﹕除產學合作研究計畫､提升產業技術及人才培育研究計畫､
列管計畫及下列情形者外﹐得立即公開查詢
□涉及專利或其他智慧財產權﹐□一年□二年後可公開查詢
執行單位﹕中國醫藥大學附設醫院 兒科部
中 華 民 國 95
年
10
月
1 日
中文摘要:
研究宗旨﹕我們研究在極低體重早產兒(VLBW) 以益生菌減少壞死性腸炎(NEC)發生率的有
效性。
患 者 和 方 法 ﹕ 我 們 假 設 益 生 菌 Infloran （ 含 Lactobacillus acidophilus 和
Bifidobacterium infantis）可以減少極低體重早產兒 (出生重量＜1500 gm)的 NEC 或死 亡率。從 2005 年 4 月 1 日﹐在八個新生兒加護病房進行一個前瞻､遮蓋､隨機化的研究。 存活且有腸道餵食的 VLBW 才納入試驗。實驗組給母乳或混合哺乳與 Infloran125/毫克/公 斤/劑﹐每日兩次-共六週﹔控制組只餵食母奶或混合哺乳。母奶來自嬰兒母親或母奶銀行﹐ 所有母奶都由不參與嬰兒照護的小組準備﹐且遵從來自密封的醫囑﹔臨床照護者嬰兒不知 嬰兒組別。主要結果是測量死亡或 NEC (≧stage 2)的發生率 。 結果﹕共有三百二十個嬰兒納入研究﹐163 人在實驗組和 157 人在控制組。人口特徵和臨 床變項在兩組是相似。死亡或 NEC 的發生(≧階段 2) 在實驗組顯著較低(3/163 對 10/ 157)。與控制群組比較實驗組 NEC 的發生(≧ 階段 2) 也是顯著的低 (3/163 對 7/157)。 沒有血液培養長出乳酸桿菌屬或比菲德氏菌也沒有發現其它副作用。 結論﹕以 Infloran 為益生菌哺餵極低體重早產兒 6 個星期可以減少 NEC 的發生。 關鍵字﹕益生菌､壞死性腸炎､極低體重早產兒
ABSTRACT
Objective: We investigated the efficacy of probiotics in reducing the incidence of necrotizing
enterocolitis (NEC) for very low birth weight (VLBW) infants.
Patients and Methods: A prospective, masked, multi-center randomized control trial was
conducted at eight neonatal centers to evaluate the beneficial effects of probiotics for NEC among VLBW (<1500 g) infants. VLBW infants who started to feed enterally were eligible and were randomized into 2 groups after parental informed consents were obtained. Infants in the study group were fed with Infloran (Lactobacillus acidophilus and Bifidobacterium bifidus) with breast milk or mixed feeding (breast and formula) twice daily for 6 weeks. Infants in the control group were fed with breast milk or mixed feeding. The clinicians caring for the infants were blinded to the group assignment. The primary outcome measurement was death or NEC ( stage 2).
Results: Three hundred and twenty infants were enrolled: 161 in the study group and 157 in the
control group. The demographic and clinical variables were similar in both groups. The incidence of death or NEC ( stage 2) was significantly lower in the study group (3 of 163 vs 10 of 157). The incidence of NEC ( stage 2) was also significantly lower in the study when compared with the control group (3 of 163 vs 7 of 157). None of the positive blood culture grew Lactobacillus or Bifidobacterium species. No other adverse effect was noted.
Conclusion. Infloran as probiotics fed enterally to preterm VLBW infants for 6 weeks reduces the incidence of NEC.
Keywords: probiotics, necrotizing enterocolitis, very low birth weight infants.
報告內容﹕
Introduction
Necrotising enterocolitis NEC is one of the most common gastrointestinal emergencies in preterm very low birth weight (VLBW) infants with highly variable incidence affecting 7% to 14% of these infants; [1] Recent reports suggest increasing occurrence of NEC, estimating up to 9000 cases of NEC in the United States every year, with death occurring in 15 % to 30% of affected individuals. [2]
As the survival rate of VLBW infants increases, NEC remains a critical eminent problem.
The pathogenesis of NEC is unknown, but is widely considered as a multi-factorial disease; prematurity, enteral feeding, intestinal hypoxia-ischemia, and bacterial colonization are considered major risk factors. [3] Most likely, NEC is the clinical culmination of multiple different risk factors interacting with each other to produce bowel injury through a final, common inflammatory pathway. Researches showed that platelet aggregation factor (PAF) interleukin-1.6.12.18 (IL-1, IL-6, IL-12, IL-18) and tumor necrosis factor- (TNF -) [4-8] and anti-inflammatory cytokines such as interleukin 4.10 (IL-4.10) [9.10] by intestinal epithelial cells is highly relevant to be involved in the final common pathway of NEC. [4-11]
Recent studies indicated that commensal bacteria block ubiquination and degradation of inhibitory factor that results in very low level of inflammatory response continuously at mucosa.[12.13] Unfortunately pathogenic floras attach to the epithelium cell of preterm infants much easier than to the full term infants and the fetal enterocyte have exaggerated IL-8 response.[14] It has bee thought that inappropriate, accentuated inflammatory response to colonizing pathogenic flora at premature gut plays a major role, the inflammatory cascade further promotes spread of bacteria or toxin, and end up with ischemia, necrosis and even perforation. [15-17]
We and Bin-Nun had proved that probitics reduces the incidence and severity of necrotizing enterocolitis in preterm VLBW infants. [18.19] Nevertheless, there is no solid data in the
literature as to the optima strains, timing, dosage and duration of probiotics administering to preterm VLBW infants at preset; and these need further investigate. Bifidobacteria and lactobacilli are commonly found in breast-fed infants; [20] and most of the NEC in VLBW infants occurred before 6-8 weeks of age; [21]; we therefore hypothesized that oral probiotics contains Bifidobacteria and lactobacilli fed to preterm VLBW infants for 6 weeks would reduce the incidence and severity of NEC.
PATIENTS AND METHODS
From April 1, 2005 to Oct 31, 2006, a prospective masked randomized control trial was conducted at the level III Neonatal Intensive Care Unit (NICU) of eight medical centers in Taiwan. Study protocol was approved by Institutional Review Board (IRB) of each hospital. Preterm VLBW infants (birth weight < 1500 gm) who were fed enterally were eligible for the trial. They were randomized into the study or control group by a random number table sequence after informed parental consents were obtained. The allocations were in sequentially numbered and were sent by computer center located at the children hospital of China Medical University when the participate hospital registered. Preterm VLBW infants who had severe asphyxia (stage III), fetal chromosome anomalies, fetal cyanotic congenital heart disease, congenital intestine atresia, gastroschisis, omphalocele, exclusive formula feeding and nothing per oral was more than 3 weeks were excluded.
The clinicians who provided care to the infants were blinded to the grouping. Investigators were not involved in the care of these infants. Study group was fed with infloran (Lactobacillus acidophilus and Bifidobacterium bifidus,Swiss Serum and Vaccine Institute Berne, Switzerland) 125mg/kg/dose (CFU 109) twice daily with breast milk or mixed feeding (breast and formula) for 6 weeks; the control group was fed with breast milk or mixed feeding. Infloran was stored in refrigerator at a tempreture between 2 –80 C and mixed with breast milk or formula before feeding. Breast milk was either from infant’s own mother’s milk or from breast milk bank. Both breast milk of the study and control group were prepared by breast milk team who were not involved in the care of the infant and follow the order from a sealed envelop. Feeding was started
when the infant,s vital signs were stable, had active bowel movement, without abdominal distension, without bile or blood from the oral-gastric tube, and did not have an umbilical artery or umbilical venous catheter in place for at least 24 hours.
A strict feeding protocol was followed for all study infants. Depending on the birth weight and gestational age, a certain amount of breast milk was initiated after the infant tolerated one trial of distilled water. On the first day, 1 mL/kg - distilled water was given twice, followed by breast milk. The amount of feeding was advanced slowly if tolerated, with no more than 20 mL/kg/day of increment per feed. An oral intake of 100 mL/kg/d was defined as complete enteral feeding. Feeding was stopped if there was any sign of feeding intolerance, defined as the presence of gastric aspirate in the amount that was more than half of the previous feeding, twice, with abdominal distension. Infants who weighed under 1,000gm received total parenteral nutrition (TPN) until half of the calories were supplied by oral route. The same attending physician was in charge of the care of the infants during their hospital stay. The residents who rotated through the NICU provided the care following established protocols in the unit. Definition of prenatal steroid, small for gestational age, prolonged rupture of amniotic membrane, chorioamnionitis, asphyxia, respiratory distress syndrome, patent ductus arteriosus, intraventricular hemorrhage (IVH), sepsis; indication of surfactant and indomethacin were described as in our previous study. [18] There were no modifications in any management protocols, clinical practices, equipment, infrastructure, or any other elements in each unit during the study period. All the definitions and protocols got consensus among the eight centers after several discussions before the study was begun.
NEC was classified by modified Bell’s classification. [22]The final determination of the diagnosis of NEC (≧ stage2) was made by two independent attending physicians who did not know the group assignment of the infant. Demographic and clinical variables that were potential risk factors for NEC were prospectively abstracted from the medical records. These factors were as our previous study except added antibiotics use per weeks and weight gain per week; these factors were prospectively abstracted from the medical records.18
Primary outcome measurement was the incidence and severity of NEC (≧ stage 2); or death. Secondary outcome were culture proved sepsis, chronic lung disease, periventricular leukomalacia (PVL), weight gain per week, duration of TPN, lengthy of stay.
SAMPLE SIZE CALCULATION AND STATISTICS
The recent data showed that the combined incidence of NEC (≧stage 2) or death was about 26 % in Taiwan. [23] Setting the α error <0.05 and β error <0.2, and an absolute reduction of the incidence of NEC or death by 50%, the number needed to verify our hypothesis was 264 by two-tail.
Chi-square test was used to examine the association between categorical risk factors and mortality when its assumption was held (less than 20% of cells had expected values of less than five), whilst a two-tailed Fisher*s exact test was used when assumption of Chi-squared test was not held. Student*s t-test was used to explore the differences in continuous risk factors.
RESULTS
There were 413 VLBW infants admitted to the eight NICU during the 1.5-year study period. Of these infants, 95 were either expired (n =69) or had met the exclusi on creteria before enrolled to the study (n = 9) or the family members declined consent for study (n = 17). A total of 320 infants were enrolled in the trial: 163 in the study arm and 157 in the control arm. Fifty-six infants in the study group and 61 infants in the control group were fed with mixed feeding. The maternal clinical and infant’s demographic and clinical characteristics did not differ between the 2 groups (Table 1). The infants’ clinical characteristics also did not differ between the 2 groups (Table 2). Table 3 shows the outcomes of the study by logistic regression analysis. The incidence of death or NEC was significantly lower in the probiotics group when compared with the control group ( 3 of 163 [1.8 %] vs 10 of 157 [6.4 %], respectively; P .009). The incidence of NEC was also lower in the probiotics when compared with the control group (3 of 163 [1.8%] vs 7 of 157 [4.5 %], respectively; P .04).
There were 4 cases of severe NEC (stage 3) in the control group and one in the probiotics group (P .03 by bivariate analysis). The incidence of culture proved sepsis, CLD, PVL were no
significant difference between both groups. None of the positive blood cultures grew Lactobacillus or Bifidobacterium species. No other adverse effect was noted. The administration of infloran to the study group resulted in a significantly earlier colonization of Lactobacillus acidophilus and bifidobacteria (8.1 +/- 3.9 days of life) than in the control group (11.3 +/- 4.7 days of life). On day 7 after infloran, Lactobacillus acidophilus and bifidobacterial dominance (> 90% of the intestinal microflora) could be found in 26% of infants in the study group and only in 2% of the control group (p < 0.001).
DISCUSSION
This multi-center control trial showed that probiotics as Infloran reduces the incidence of NEC or death for preterm VLBW infants. We also found that the study group had a lower incidence of NEC and sepsis. We found that bifidobacteria and lactobacilli were more prominent with less pathogenic microorganisms in stool culture of the study group compared to the control group. According to our data, the number needed to treat to prevent 1 case of NEC is 32, and the number needed to treat to prevent 1 death due to NEC is 29.
Although many variables are associated with development of NEC, only prematurity and low birth weight have been consistently identified in case controlled studies.[24.25]Many other factors that were associated with an increased risk of NEC had been reported; [24.25] because the current multi-center study was designed as a randomized, controlled trial, all the risk factors were distributed randomly and showed no difference between the 2 study groups.
A major component of the proposed pathogenesis of NEC is the interaction of bacteria with the premature gut. [26-33] Sakata H and Gewolb IH found that the Bifidobacteria were undetectable in the intestinal flora during the first 1 to 2 weeks after birth and did not predominate until after the third week of life among VLBW infants even receiving breast milk. [26.27] Lawrence and Shah demonstratedthat delay gut colonization in NICU, make the intestine absorbed of intact bacterial toxin, which damaged the immature ileum and developed NEC.[28.29] Hoy andMillar found that there were quantitative and qualitative changes in the fecal flora before NEC.[30.31] Panigrafi and Dai showed that bacterial translocation leading to NEC.[32.33] All these studies
suggest a strong relationship of delay and low colonization of commensal flora and proliferation of pathogenic flora in the immature gut that predisposing preterm infants to develop NEC. Using animal model Caplan and Butel showed that Bifidobacteria supplement in neonate rat and quail model resulted inintestinal colonization and subsequently reduction in NEC-like lesion.[34.35] Bifidobacteria and lactobacilli could break down ingested sugars to form lactic acid, inhibite pathogenic microorganisms, not undergo translocation, producing protective nutrients (arginine, glutamine, short-chain fatty acids) protective against the translocation of other bacteria. [36] These characteristics make that probiotics contained Lactobacillus and Bifidobacterium become best shot for preventing of NEC.
There were five clinical trials till now that evaluated the impact of probiotics on NEC. In Hoyos,s [37] historical control study, infloran was given to all infants admitted to NICU and showed significant reduction in the incidence of NEC and NEC associated death. Dani performed a multi-center study double blind control trial involving 585 preterm VLBW infants with LGG as probiotics in Italy. [38]There were no significant differences between the probiotic and placebo groups in regards to any of the three outcome variables, including NEC. However, the event rate was low for all three variables and needed a much larger sample size to verify their hypothesis. A recent study by Dr. Bin Nun from Israel and published in Journal of Peds showed similar results as our previous study with significant reduction of death or NEC using Bifidobacteria infantis, Streptococcus thermophillus, and Bifidobacteria bifidus. 19
Although the four control studies were different somewhat in study design and methodology, the four randomized trials can be compared because the Breslow–Day test shows that the relative risk assessments were homogeneity. With method of Mantel– Haenszel, the weighted, pooled estimate of relative risk is 0.34, with a 95% confidence interval of 0.20 to 0.90, suggesting a beneficial effect of probiotic treatment on reducing the incidence of NEC. The weighted risk difference summarising the presented studies, 0.021, indicates that the number needed to treat to prevent one case of NEC is 41 infants.
(Bifidobacteria infantis was changed to Bifidobacteria bifidus); although the results of ours two studies seemed similar, the previous study did not have stage III NEC. It seemed that Bifidobacterium infantis might be more effective to prevent NEC. Evidence disclosed that oral administration of specific strains of Lactobacillus species, stimulated the Bifidobacterium microbiota; [39] Combination of this type of probiotic microbes with breastfeeding have synergistic effect on gut humoral immunity and microbiota modification. Giving that Bifidobacterium infantis, B. longum and B. breve are the most common strain in health breast feeding infants, [40] probiotics which contains these three Bifidobacterium might be more appropriate to prevent NEC.
Breast milk and even donors breast milk could reduce the incidence of NEC, but breast milk along could not eradicate NEC.[41] Evidence showed that those infants who fed breast milk still got NEC was because IL-10 deficiency; [42]probiotics could induce IL10 production, this may explained partially why that breast milk has synergistic effect with the Bifidobacteria and Lactobacilla to inhibit the inflammation response of NEC.
Because there were very few preterm VLBW infants fed with formula milk alone; in reality, it will be extremely difficult to conduct the study adopting formula milk with probiotics.
Although some reports demonstrated that probiotics reduced the incidence of sepsis.[43]This current study didn’tshowed that probiotics reduced the incidence of sepsis in VLBW infants as in our previous study.18 This might result from the the different enrolled time and the short period of probiotics usage.
Many clinicians worry the safety of probiotics treatment in immunodeficient hosts such as neonates. Others and we did not observe complications (such as Lactobacillus or Bifidobacterium sepsis) due to probiotics during study. [18.19.40] The number accumulated from all the clinical trial might be power to state that it is relative safe if we compare the incidence and disaster of NEC ( 7-14%) and the possible sepsis due to probiotics ( 0/715, sum of the clinical trial) for VLBW infants.
experience from clinical studies show that the efficacy varies from none to significant as the treatment expands from single-strain to full flora replacement single-strain probiotic is less effective than multistrain probiotic.[45] we therefore suggest that multiple strains contains Bifidobacterium and Lactobacillus are superior to single strain; probiotics should be used as early as possible and a dosage of around 10 9 is enough to colonize in the intestine. Because most of the NEC in VLBW infants occurred before 6-8 weeks of age, therefore, we suggest that a 6-8 weeks period of probiotics should be adequate to prevent NEC.
The issue of prevalence of atopic disease such as atopic dermamitis[46], or allergic rhinitis [47] in those preterm infants fed with probiotics would be very interesting;if probiotics indeed reduces the incidence of atopic diseases in infancy, these observation would further extend the rationale of treating preterm VLBW infants with probiotics.
We observed 6 infants developed NEC without enteral feeding and before enrolled to the study, 5 of them were weighted less than 1000 g. Probiotics alone could not eliminate NEC. Future study should focus on these victims; immediate oral probiotics soon after birth or probiotic mixed with amniotic fluid that contains many growth factors [48]might be the ideal silver bullet to prevent NEC effectively.
We concluded that oral Infloran as probiotics administration for 6 weeks reduces the incidence of NEC in preterm VLBW infants.
ACKNOWLEDGMENTS
This study was supported by the National Science Council of Taiwan (grant ) and approved by IRB of China Medical University Hospital (DMR94-IRB-14). We appreciate Associate Professor Li Tsai-Chung for help with statistics.
參考文獻﹕
1. Hintz SR, Kendrick DE, Stoll BJ, et al. Neurodevelopmental and growth outcomes of extremely low birth weight infants after necrotizing enterocolitis. Pediatrics 2005;115:696-e703.
2. Luig M, Lui K. Epidemiology of necrotizing enterocolitis— Part II: Risks and susceptibility of premature infants during the surfactant era: a regional study. J Paediatr Child Health 2005; 41: 174-179.
3. Caplan MS, MacKendrick W. Necrotizing enterocolitis: a review of pathogenetic mechanisms and implications for prevention. Pediatr Pathol 1993;13:357-369.
4. Caplan MS, Simon D, Jilling T. The role of PAF, TLR, and the infl ammatory response in neonatal necrotizing enterocolitis. Semin Pediatr Surg 2005; 14: 145-151.
5. Viscardi RM, Lyon NH, Sun CC, Hebel JR, Hasday JD: Inflammatory cytokine mRNAs in surgical specimens of necrotizing enterocolitis and normal newborn intestine. Pediatr Pathol Lab Med 1997;17:547-559.
6. Goepfert AR, Andrews WW, Waldemar C, Ramsey PS, Cliver SP, Goldenberg RL, Hauth JC: Umbilical cord plasma interleukin-6 concentrations in preterm infants and risk of neonatal morbidity. Am J Obstet Gynecol 2004;191:1375-1381.
7. Halpern MD, Holubec H, Dominguez JA, Williams CS, Meza YG, McWilliam DL, Payne CM, McCuskey RS, Besselsen DG, Dvorak B: Up-regulation of IL-18 and IL-12 in the ileum of neonatal rats with necrotizing enterocolitis. Pediatr Res 2002;51:733-739.
8. Halpern MD, Holubec H, Dominguez JA, Meza YG, Williams CS, Ruth MC, McCuskey RS, Dvorak B: Hepatic inflammatory mediators contribute to intestinal damage in necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 2003; 284:G695-G702.
9. Treszl A, Heninger E, Kalman A, Schuler A, Tullassay T, Vasarhelyi B: Lower prevalence of IL-4 receptor alpha-chain gene G variant in very-low-birth-weight infants with necrotizing enterocolitis. J Pediatr Surg 2003; 38:1374-1378.
10. Kling KM, Kirby L, Kwan KY, Kim F, McFadden DW: Interleukin-10 inhibits inducible nitric oxide synthase in an animal model of necrotizing pancreatitis. Int J Surg Investig 1999;1:337-342.
11. Harris MC, D’Angio CT, Gallagher PR, Kaufman D, Evans J, Kilpatrick L. Cytokine elaboration in critically ill infants with bacterial sepsis, necrotizing entercolitis, or sepsis syndrome: correlation with clinical parameters of infl ammation and mortality. J Pediatr 2005; 147: 462-468.
12. Kelly D, Campbell JI, King TP, et al. Commensal anaerobic gut bacteria attenuate infl ammation by regulating nuclearcytoplasmic shuttling of PPAR-gamma and RelA. Nat Immunol 2004; 5: 104-112.
13. Collier-Hyams LS, Sloane V, Batten BC, Neish AS. Cutting edge: bacterial modulation of epithelial signaling via changes in neddylation of cullin-1. J Immunol 2005; 175: 4194-4198. 14. Markel TA, Crisostomo PR, Wairiuko GM, Pitcher J, Tsai BM, Meldrum DR. Cytokines in
necrotizing enterocolitis.Shock. 2006 Apr;25(4):329-337.
15. Claud EC, Walker WA. Hypothesis: inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis. FASEB J. 2001 ;15:1398-1403.
16. Nanthakumar NN, Fusunyan RD, Sanderson I, Walker WA. Inflammation in the developing human intestine: A possible pathophysiologic contribution to necrotizing enterocolitis.Proc Natl Acad Sci U S A. 2000; 23;97:6043-6048.
17. Hsueh W, Caplan MS, Qu XW, Tan XD, De Plaen IG, Gonzalez-Crussi F: Neonatal necrotizing enterocolitis: clinical considerations and pathogenetic concepts. Pediatr Dev Pathol 2002;6:6-23.
18. Lin CH, Su BH, Chen AC, Lin TW, Tsai CH, Yeh TF, et al. Oral probiotics reduce the incidence and severity of necrotizing enterocolitis in very low birth weight infants. Pediatrics 2005;115:1-4.
19. Bin-Nun A, Bromiker R, Wilschanski M, Kaplan M, Rudensky B, Caplan M. Oral probiotics prevent necrotizing enterocolitis in very low birth weight neonates. J Pediatr 2005;
147:192-196.
20. Pietzak M. Bacterial colonization of the neonatal gut. J Pediatr Gastroenterol Nutr 2004;38:389–91.
21. Wilson R, Kanto WP Jr, McCarthy BJ, Burton A, Lewin P, Feldman RA. Age at onset of necrotizing enterocolitis: an epidemiologic analysis. Pediatr Res 1982; 16: 82-85.
22. Bell MJ, Ternberg JL, Feigin RD, Keating JP, Marshall R, Barton L, et al. Neonatal necrotizing enterocolitis: therapeutic decisions based upon clinical staging. Ann Surg 1978;187:1-7.
23. SS Lin. BH Su. TW Lin. HC Lin. CT Peng. A comparison of survival in extremely low birth weight infants between periods 1997-1998 and 1998-2000. Acta Paediatrica Taiwanica. 2002; 43:193-198.
24. Hsueh W, Caplan MS, Qu XW, Tan XD, De Plaen IG, Gonzalez-Crussi F. Neonatal necrotizing enterocolitis: clinical considerations and pathogenetic concepts. Pediatr Dev Pathol 2003; 6: 6-23.
25. Llanos AR, Moss ME, Pinzon MC, Dye T, Sinkin RA, Kendig JW. Epidemiology of neonatal necrotising enterocolitis: a populationbased study. Paediatr Perinat Epidemiol 2002; 16: 342-49.
26. Sakata H, Yoshioka H, Fujita K. Development of the intestinal flora in very low birth weight infants compared to normal full-term newborns. Eur J Pediatr. 1985;144(2):186-90.
27. Gewolb IH, Schwalbe RS, Taciak VL, Harrison TS, Panigrahi P. Stool microflora inextremely low birthweight infants. Arch Dis Child Fetal Neonatal Ed 1999; 80:F167-73.
28. Lawrence G, Bates J, Gaul A. Pathogenesis of neonatal necrotizing enterocolitis. Lancet 1982; 1:137-9.
29. Shah U, Walker WA. Adverse host responses to bacterial toxins inhuman infants.J Nutr. 2000;130(2S Suppl):420S-425S.
30. Hoy C, Millar MR, Mackey P, godwin PCR, Langdale V, Levene MI. Quantitative changes in faecal microflora preceding necrotizing enterocolitis in premature neonates. Arch Dis Child
1990; 65:1057-1059.
31. Millar MR, Mackey P, Levene MI. Enterobacteriaceae and necrotizing enterocolitis. Arch Dis Child 1992; 67: 53-56.
32. Panigrahi P, Bamford P, Horvath K, Morris JG Jr, Gewolb IH. Escherichia coli transcytosis in a Caco-2 cell model: implications in neonatal necrotizing enterocolitis.Pediatr Res. 1996 Sep;40(3):415-21.
33. Dingwei DAI, W Allan Walke. Role of bacterial colonization in neonatal necrotizing enterocolitis and its prevention. Acta Paeditr Sin 1998; 39: 357-366.
34. Caplan MS, Miller-Catchpole R, Kaup S, Russell T, Lickerman M, Amer M, Xiao Y, Thomson R Jr. Bifidobacterial supplementation reduces the incidence of necrotizing enterocolitis in a neonatal rat model. Gastroenterology 1999; 117: 577-583.
35. Butel MJ, Roland N, Hibert A, et al. Clostridial pathogenicity in experimental necrotising enterocolitis in gnotobiotic quails and protective role of bifidobacteria. J Med Microbiol. 1998;47:391-399.
36. Hammerman C, Kaplan M. Germ warfare: probiotics in defense of the premature gut. Clin Perinatol 2004;31: 489-500.
37. Hoyos AB. Reduced incidence of necrotizing enterocolitis associated with enteral administration of Lactobacillus acidophilus and Bifidobacterium infantis to neonates in an intensive care unit. Int J Infect Dis 1999; 3:197-202.
38. Dani C, Biadaioli R, Bertini G. Pobiotics feeding in prevention of urinary tract infection, bacterial sepsis and necrotizing enterocolitis in preterm infants. A prospective double-blind study. Biol Neonate 2002; 82:103-8.
39. Gueimonde M, Sakata S, Kalliomaki M, et al. Effect of maternal consumption of Lactobacillus GG on transfer and establishment of fecal bifidobacterial microbiota in neonates. J Pediatr Gastroenterol Nutr. 2006;42: 166-70.
40. Harmsen H.J., Wildeboer-Veloo A.C., Raangs G.C., Wagendorp A., Klijn N., Bindels J., et al. Analysis of intestinal flora development in breast-fed and formula-fed infants by using
molecular identification and detection methods. J Pediatr Gastroenterol Nutr (2000) 30 : pp 61-67.
41. Donor breast milk versus infant formula for preterm infants: a systematic review and meta-analysis.Arch Dis Child Fetal Neonatal Ed. 2006 Apr 5.
42. Fituch CC, Palkowetz KH, Goldman AS, Schanler RJ. Concentrations of IL-10 in preterm human milk and in milk from mothers of infants with necrotizing enterocolitis Acta Paediatr. 2004 Nov;93(11):1496-500.
43. Strategies for prevention of nosocomial sepsis in the neonatal intensive care unit. Curr Opin Pediatr. 2006 Apr;18(2):101-106.
44. Tannock GW, Munro K, Harmsen HJ, Welling GW, Smart J, Gopal PK. Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20. Appl Environ Microbiol. 2000;66:2578-2588.
45. Borody T.J., Warren E.F., Leis S.M., et al. Bacteriotherapy using fecal flora: toying with human motions. J Clin Gastroenterol (2004) 38 : pp 475-483.
46.Kalliomaki M, Salminen S, Poussa T, Arvilommi H, Isolauri E. Probiotics and prevention of atopic disease: 4-year follow-up of a randomised placebo-controlled trial.
47.The efficacy and safety of heat-killed Lactobacillus paracasei for treatment of perennial allergic rhinitis induced by house-dust mite.Pediatr Allergy Immunol. 2005;16(5):433-8.Lancet. 2003: 31;361(9372):1869-71.
48.Barney CK, Purser N, Christensen RD. A phase 1 trial testing an enteral solution patterned after human amniotic fluid to treat feeding intolerance. Adv Neonatal Care. 2006 ;6(2):89-95.
計畫成果自評﹕
本研究評估極低體重早產兒服用INFLORAN 後 NEC 或死亡的發生率﹐共有三百二十 個嬰兒納入研究﹐163 人在實驗組､157 人在控制組﹐人口特徵和臨床變項在兩組是相似。 死亡或NEC 的發生(≧階段 2) 在實驗組顯著較低 (3/163 對 10/ 157)。與控制群組比較實驗 組NEC 的發生(≧ 階段 2) 也是顯著的低 (3/163 對 7/154)。沒有血液培養長出乳酸桿菌屬 或比菲德氏菌也沒有發現其它副作用。 結論﹕以Infloran 為益生菌哺餵極低體重早產兒 6 個星期可以減少 NEC 的發生。至於 其他次要的指標如慢性肺病的發生率﹐細菌培養證實的敗血病､每星期增加的體重､住院 天數則沒有差別﹐可惜因為研究經費不足必須結案﹐但統計上仍有個案數不足的疑慮。附件一､
Table1. Maternal and Clinical and Infants demographic and clinical characteristics characterisitcs Control group
(N= 157)
Study group
(N= 163) P-value Prolonged rupture of amniotic membrane,
n (%) 45 44 1.00 Preeclampsia, n (%) 21 25 0.73 Prenatal steroid, n (%) 70 57 0.41 Mutipregnancy, n (%) 46 32 0.14 Chorioamnionitis, n (%) 8 7 0.92 Male, n (%) 91 86 0.39
Small for gestational age, n (%) 126 120 0.17
Gestation, wk 28.55±2.58 28.09±2.78 0.13 Birth weigh, g 1117.30±246.47 1029.40±258.00 0.00 Apgar (5 min) <3 30 29 4-6 24 50 0.00 >7 102 83 PH 7.29±0.10 7.26±0.13 0.03 Age at enrollment,*d 6.19±5.70 5.94±3.19 0.65 Antibiotics use during 1st wk 135 145 1.00
Antibiotics use during 2nd wk 60 71 0.50
Antibiotics use during 3rd wk 43 54 0.44
Antibiotics use during 4th wk 34 43 0.55
Antibiotics use during 5th wk 30 41 0.40
Antibiotics use during 6th wk 23 30 0.70
Ues of surfactant, n 74 97 0.03
Umbilical artery catheter, *d 0.63±1.44 0.97±1.75 0.06 Umbilical venous cathter, *d 1.14±2.06 1.56±2.27 0.09 Intermittent mandatory ventilation, *d 13.77±23.61 14.64±21.49 0.74
Pneumothorax, n (%) 0 4 0.14
Use of dopamine, n (%) 66 79 0.51
Dopamine, d 2.48±5.83 2.44±3.85 0.95
Indomethacin, n (%) 63 90 0.01
IVH >3 6 5 0.96
Table2.impact of Probiotics on feeding amount and weight gain
Variables Control group
(N= 157)
Study group
(N= 163) P-value Total parenteral nutrition, d 10.63±16.44 13.95±15.57 0.07 Feeding amount at 42 d 139.09±67.57 148.20±74.90 0.32 Full feeding day 28.80±18.34 26.30±18.44 0.24 Body wt gain at 14d 64.94±65.36 49.97±56.36 0.03 Body wt gain at 28d 115.54±73.81 107.02±69.20 0.30 Body wt gain at 42d 149.56±86.70 127.50±83.49 0.03
Table.3 Outcome variable after oral probiotics
Variables Control group (N= 157) Study group (N= 163) P values Death or NEC 10 3 0.04 Death 3 0 0.05 NEC grade 2 or 3 7/154 3/163 0.04
Sepsis (culture proven) 21 34 0.09
NEC or sepsis 5 3 0.68
CLD 30 40 0.27
