Role of Probiotics in Prevention of Necrotising Enterocolitis in Preterm Babies

DESSERTATION ON

ROLE OF PROBIOTICS IN PREVENTION OF NECROTISING

ENTEROCOLITIS IN PRETERM BABIES

Dissertation Submitted to

THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY

In partial fulfillment of the regulations

for the award of the degree of

M.D IN PAEDIATRIC MEDICINE

BRANCH VII

THANJAVUR MEDICAL COLLEGE

THANJAVUR - 613004.

THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY

CHENNAI – 600 032.

CERTIFICATE

I certify that the dissertation titled “A ROLE OF ORAL PROBIOTIC IN

PREVENTION OF NECROTISING ENTEROCOLITIS IN PRETERM

BABIES”, submitted by Dr. K.RAJAPRIYA, for the Degree of DOCTOR OF

MEDICINE (PAEDIATRICS) (BRANCH VII), to The TamilNadu Dr.M.G.R.

Medical University, Chennai, is the result of original research work undertaken

by her in the Department of Paediatrics, ThanjavurMedical College, Thanjavur.

Prof.Dr.M.SINGARAVELU Prof Dr.S.RAJASEKAR MD,DCH

MD,DCH,DNB,MNAMS(PED)FIAP Professor of paediatrics, Professor and HOD of paediatrics Department of paediatrics, Department of paediatrics Thanjavur medical college. Thanjavur medical college.

Place: Thanjavur. DEAN

DECLARATION

I hereby solemnly declare that the dissertation titled “A ROLE OF ORAL PROBIOTIC IN PREVENTION OF NECROTISING ENTEROCOLITIS IN PRETERM BABIES” has been prepared by me under the guidance of

PROF. DR. M. SINGARAVELU MD., DCH., DNB(paed)., MNAMS(Paed),FIAP PROFESSOR AND HOD, DEPARTMENT Of PAEDIATRICS, THANJAVUR MEDICAL COLLEGE, THANJAVUR. This

is submitted to THE TAMILNADU DR.M.G.RMEDICALUNIVERSITY,

CHENNAI, in partial fulfillment of the requirement for the degree of DOCTOR

OF MEDICINE (PAEDIATRICS) (BRANCH VII).

DATE:

ACKNOWLEDGEMENT

I express my sincere gratitude to Prof Dr. M. SINGARAVELU MD., DCH., DNB(Paed)., MNAMS(Paed),FIAP Professor and Head of the Department, Department of Pediatrics, Thanjavur Medical College, Thanjavur,

a versatile personality and dynamic person for his constant support,

encouragement and guiding me in preparing and conducting the study. I am

very much indebted to him for his generous help and honoured to have his

blessings throughout the study period.

I am extremely grateful to my beloved teacher Prof Dr.

S.RAJASEKARMD., DCH., Professor of Pediatrics, Department of Pediatrics, Thanjavur Medical College, Thanjavur, for his constant

encouragement and timely help. I wish to express my sincere gratitude to my

Professor for his enormous help and guiding me throughout the study period.

I sincerely thank my Assistant Professors for their guidance and

I also express my gratitude to the Dean, Thanjavur Medical College, Thanjavur and the Ethical Committee for allowing me to conduct this study.

I am extremely thankful to my parents and to my beloved husband

Dr.V.Thamilarasu for their moral support during the study period.

I have to specially thank all my teachers in the Department of

Paediatrics for their guidance, encouragement, inspiration and moral

support during my career as a postgraduate.

A special thanks to my dear friends and colleagues Dr. Varun, Dr.radhakrishnan, and Dr. Madhina for their kind support. I also thank all the postgraduates of the Department of Paediatrics, for all their help and

support they gave me.

Finally, I pay my prayers to the Almighty God for His blessings and

wishes and making me pursue the post-graduation in my field of interest.

Date: Postgraduate in

Thanjavur Department of Pediatrics,

Thanjavur Medical College,

CONTENT

S.NO TOPIC PAGE NO

1. Abstract 1

2. Introduction 3

3. Aims and objectives 9

4. Review of literature 11

5. Materials and methods 56

6. Observation and results 59

7. Discussion 71

8. Conclusion 78

9. Summary 82

10. Bibilography 84

11. Proforma 90

ABSTRACT

BACKGROUND AND OBJECTIVES

Necrotizing enterocolitis(NEC) is a common emergency condition of

preterm babies. It has got a potential to cause significant mortality and

morbidity. There are many preventive strategies for NEC. one among

them is administration of oral probiotics. The aim of the study is to

prove that use of oral probiotics can significantly reduce the incidence

of NEC among the preterm babies

METHODS

The study design was a prospective randomized control trial. Only

babies <34weeks of gestation were included in the study. The selected

sample of babies were randomly divided into two groups viz, the test

and the study group. Babies in the test group were fed with Darolac

0.5g/day (Lactobacillus acidophilus, Lactobacillus rhamnosus,

Bifidobacterium longum, and Saccharomyces boluardi) with breast

milk twice daily till they reach full feeds. Babies in the control group

were fed with breast milk alone. The two groups were compared for

the incidence of NEC.

RESULTS

Sample size was limited to 200 babies. They were

randomized as control and test groups with 100 each. It was found

that the incidence of NEC was lower in the test group(3 of 100 vs. 11

of 100). The results were statistically significant. There was 3 case in

stage 2 NEC and 1 case of severe stage 3 NEC in control group

CONCLUSION

Prophylactic probiotic has a beneficial role in prevention of

necrotising enterocolitis in premature low birth weight babies.

INTRODUCTION

Newborn is the period between births to first 28 days of life. Neonatology is

rapidly growing subpeciality in paediatrics. In the past 20 years level of NICU

care improved and helps in reducing preterm mortality.

Preterm is defined as baby delivered before 37 completed gestation weeks.

“Low birth weigh less than 2.5 kg

Very low birth weight less than1.5kg

Extremely low birth weight les than1 kg”

Healthy term babies can be managed by mother under the guidance of health

care professional .But, preterm low birth weight babies are fragile and they need

special neonatal intensive care (NICU).

“

under-five mortality rate has decreased by 53%, from an estimated rate of 91/ 1000 live births in 1990 to 43/ 1000 live births in 2015”.

• 41% of under five death, constitutes by neonatal death.

• Number of preterm birth is increasing and it was estimated 15millon

preterm per year.

• Complications of preterm is important cause for underfive death

• 75% of prematuremature babies could be saved by advanced maternal

COMPLICATIONS OF PRETERM

Respiratory distress syndrome

Bronchopulmonary dysplasia

Apnea of prematurity

Intraventricuar hemorrhage

Periventricular leukomalacia

Retinopathy of prematurity

Infections

Patent ductus arteriosus

Necrotising enterocolitis

• Necrotizing Enterocolitis (NEC) is the commonest life threatening

emergency of the gastrointestinal tract in the neonatal period. The gross

pathological change in intestine is variable stages of transmural necrosis.

“

The cause of NEC remains multifactorial”.

Most common risk factors that have been significantly associated with NEC

are prematurity and enteral feeding.

• Necrotizing enterocolitis is a leading cause of neonatal morbidity and

mortality and it is the most common gastrointestinal emergency in neonates

Necrotizing enterocolitis (NEC) affects about 5% of all very low birth

weight neonates and about 10% of all extremely low birth weight 4. The rate of

NEC-associated acute mortality is about 10% overall. Mortality is more than

25% in preterms with severe necrotising enterocolitis. long-term complications

of those premature babies who survived from severe necrotising enterocolitis

are neuro developmental impairment, short bowel syndrome, and growth

impairment.

• “enteric feeding, abnormal bacterial colonization of the neonatal

gastrointestinal tract, bacterial translocation and activation of the cytokine

cascade, decreased epidermal growth factor, increased platelet activating

factor, and mucosal damage from free radical production”.

• Clinical manifestations of NEC , are non specific signs like apnea,

bradycardia,hypothermia(temperature instability ),lethargy and specific

GIT symptoms such as feed intolerance, vomiting, abdominal distention

and tenderness,blood in stools and abdominal wall erythema.

• Lab values are diagnostic of necrotizing enterocolitis.lab report mayshow

evidence of infection and coagulation abnormalities .

• Radiological signs may include dilated and/ or fixed bowel

loops,pneumotosis intestinalis( air within the intestinal wall),

• Medical management is mainly complete bowel rest and decompression

by nasogastric drainage, antibiotics, and treatment for hematological

abnormality like thrombocytopenia and treatment of electrolyte

imbalances. Hemodynamically unstable preterm may needs

cardiorespiratory supportslike mechanical ventilation and inotrope

support.

• Surgical management is needed in case of advanced necrotising

enterocolitis if presents with pneumoperitoneum .

Prevention is better than cure- so advanced antenatal,neonatal care and

preventive measures needed to prevent necrotising enterocolitis.

There are many preventive stratergies for NEC, my proposed stratergy is role

of probiotics in prevention of NEC.

AIMS AND OBJECTIVES

Prospective study about the role of oral probiotics in prevention of

MATERIALS AND METHODS

A prospective randomized control study was done on the selected sample of 200

preterm neonates inNeonatal ICU in government rajamirasudhar

hospital,department of paediatrics,thanjavur medical college. Preterm babies

(gestational age <34 weeks) who were stable enough to take oral feeds were

eligible for the trial. The selected sample of babies were randomly assigned as

either control or test groups. Informed parental consents were obtained.

The test group received their regular feeds plus daily probiotic supplement

DAROLAC 0.5gram/day (1.25billion cells)(lactobacillus acidophilus,

lactobacillus rhamnosus,bifidobacter longum,sacchromyces boulardi) in two

divided doses. They were administered orally mixing with expressed breast

milk from the initiation of enteral feedings till the baby matures enough to

REVIEW OF LITERATURE

REVIEW OF LITERATURE

HISTORICAL REVIEW

1. First NEC was described in the 19th century11

2. The term “NEC” was coined by Schemid and Quaiser12

3. Recognized as a an important neonatal disorder in the 1960’s13

4. Published in English literature in 196511

5. The first epidemic of NEC was reported in south Africa 197215 and in India

in1973

6. In 1978 Bell et al. proposed a system for staging of patients of NEC,

Bell’s staging was later modified by Walsh and Kliegman. “They included the

systemic, intestinal and radiological signs of NEC and suggested treatment

based on stage and severity”.

Necrotizing enterocolitis (NEC) is the most common life threatening

gastrointestinal disorder affecting very preterm or very low birth weight infants.

The risk of NEC is inversely proportional to gestational age and birth weight of

EPIDEMIOLOGY

Incidence of necrotising enterocolitis varies from centre to centre. NEC occurs in 2-5% of NICU(neonatal intensive care unit)admitted

babies.NEC occurs in 5-10%of VLBW infants.incidence increase with

decreasing gestational age.

Sex,race,climate,season doesn’t play any role in necrotiing enterocolitis

RISK FACTORS FOR NECROTISING ENTEROCOLITIS

• Prematurity.

• Enteral feeding.

• IUGR baby

• Gestational hypertension

• Abruption placenta

• End diastolic flow velocity that is either absent or reversed.

• Use of umbilical catheters .

• Poor Apgar scores .

PREMATURITY

The most important risk factor for NEC is prematurity. Decreasing

gestational age is associated with increased risk of NEC. The mean gestational

age for Necrotizing Entero colitis is 30-32 weeks, and the infants are generally

weight appropriate for gestational age. The overall mortality is 9%-28%

regardless of medical or surgical intervention. The mortality for infants

weighing <1500 grams can be as high has 45%. Approximately 10% of infants

with NEC are full-term. The postnatal age of onset of NEC is inversely related

to the birth weight and the gestational age of the baby. The mean age of onset is

12 days.

Factors which increase the susceptibility of preterm infants to NEC include

1. A compromised immune system and abnormal secretory IgA function.

Abnormal IgA function leads to less efficient binding and clearance of bacterial

endotoxin which stimulates the inflammatory cascade.

2. An immature intestinal mucosa

3. Suppressed mucosal gastro intestinal enzyme and hormonal activity and

Factors Making Premature Infant's Gut Susceptible to necrotiing enterocolitis

Poor GI peristalsis,

deficient mucus layer,

Abnormal composition of lipids in gut (premature gut is more

permeable).

Late and abnormal bacterial colonization,

low anaerobic bacteria.

Subnormal gastric acid production,

low lactase levels

POLYCYTHEMIA:

Leake et al.20 reported an increased incidence of NEC in polycythemic

infants.Hyperviscosity associated with severe polycythemia compromises

cardiac output and regional perfusion of blood and that the presence of

excessive erythrocytes impairs the movement of the cells within the capillary

network and polycythemia impairs oxygenation. This leads to decreased

EXCHANGE TRANSFUSION

There is a strong correlation to exchange transfusion through an umbilical vein

catheter to an increased incidence of NEC. During exchange transfusion wide

variations in portal venous pressure occurs that impairs the intestinal

perfusion.26 During exchange transfusion acute elevation of intestinal venous

pressure occurs and it causes reflex vaso constriction within intestinal resistance

vessels. This leads to decreased flow to intestine that leads to ischaemic

necrosis of bowel and NEC.

NEC WITH CONGENITAL HEART DISEASE

Congenital heart disease is a predisposing factor for NEC. The altered

hemodynamics are blamed for the development of intestinal lesion. The

intestinal ischemia is believed to be caused by the diastolic steal of blood from

descending aorta in PDA.27 PGE2 given to babies with congenital heart

conditions to maintain the duct patency causes apnea and hypotension which

may in turn causes poor gut perfusion and NEC.The impaired or reversed

diastolic umbilical blood flow seen in intra uterine growth retarded babies

ENTERAL FEEDINGS

Enteral feedings have been implicated in the pathogenesis of NEC. Factors that

have been considered include

1.osmolality of formula feeds

2 . the lack of immunoprotective factors in formula milk

3 .timing, volume, and the rate of feeding.

Breast milk has been shown to have

protective factors against necrotising enterocolitis.

Preterm formulas provides adequate calories and nutrient essential for growth

of premature babies. But, preterm formulas do not contain the non-nutrient

components like “secretory IgA, lysozyme, oligosaccharides, PUFA, and

platelet-activating factor (PAF)acetylhydrolase”. These non-nutrient

components of human milk essential for intetinal mucosal integrity, and it gives

ABNORMAL BACTERIAL COLONIZATION

• Prolonged empirical antibiotic therapy

• Imbalance between normal commensal flora and pathogenic bacteria.(low

commensals and high pathogens)

Premature infants are more likely to be colonized with more virulent organisms

and they have delayed acquisition of commensal bacteria, particularly

bifidobacter.

Preterm infants and are commonly exposed to antibiotic therapy,and they have

been associated with an increased risk of NEC .

Preterm infants are often delivered by caesarean and have delayed enteral

feeding, they are less likely to acquire commensal flora perinatally from passage

through the birth canal or from human milk feeding. This may lead to decreased

colonization of beneficia probiotic bacteria ,including Bifidobacteria,

Lactobacillus and Bacteroides . The hospital environment, with its

preponderance of pathogenic organisms , may also negatively affect the

intestinal colonization of beneficial commensal bacteria. Since abnormal

bacterial colonization plays a role in the pathogenesis of necrotising

enterocolitis, probiotics may exert their beneficial effects by restoring the

commensal organisms essential for maintaining gut integrity.

Prebiotics and probiotics may also accomplish this goal, through the promotion

INTRA UTERINE GROWTH RETARTATION

Intra uterine hypoxia leads to hypoperfusion and ischaemic necrosis of

bowel.

ABRUPTIO PLACENTA

Placental insufficiency leads to hypoperfusion and ischaemic necrosis

TRANSFUSION RELATED NEC

Blood transfusion is also one of the reasons for necrotising enterocolitis. Mechanism behind necrotising enterocolitis following blood transfusion is-

1. Adverse reaction to blood transfusion similar to transfusion related acute lung

injury.

2. Due to stored red blood cells(RBCs).stored RBC are less competent to

deliver oxygen to tissues leads to mesenteric vasoconstriction and ischemia and

finally necrosis of bowel wall.

IL-8 recruits inflammatory “cells. The synthesis of TNFα _{is generated in part by}

microbial products and other cytokines. TNFα _{recruits inflammatory cells, but}

is also involved in cytotoxicity and programmed cell death.9

Platelet activating factor (PAF) is one of the inflammatory mediator most

intensely studied.PAF is an endogenous phospholipid inflammatory mediator

that is produced by inflammatory cells, endothelial cells, platelets and bacteria.

There are PAF receptors on most cells. The receptor gene is expressed in many

organs, but evidence exists that the greatest receptor expression is found in the

ileum-the most common site for necrotising enterocolitis. Normal bacterial

colonization is established once enteral feeding is achieved.In the anaerobic

environment of the colon, bacteria rapidly ferment carbohydrates to gases and

short chain fatty acids. In the premature infant who has a relative lactase

deficiency, lactose ingested in the form of milk may be fermented into short

chain fatty acids and subsequently absorbed. Levels of short chain fatty acids in

the distal ileum may also increase by way of reflux across the ileo-cecal” valve

or due to local bacterial overgrowth.

NEC pathogenesis is considered multifactorial. The loss of

epithelial barrier; allows pathogens translocation from the intestinal lumen to

the mucosa. Innate immunity; is regulated by the epithelial barrier in

infection,antibiotic use,enteral nutritionhypoxia, microcirculatory dysfunction

will induce epithelial injury.

Hyperactivation of TLR 4 affects healing process in gut and favours

pathological bacterial translocation across” epithelial barrier.

The activation of TLR4 “inhibits enterocyte migration and leads

to enterocyte apoptosis via nuclear factor kappa light chain enhancer of

activated B cells (NFB) pathway activation, whereas the inhibition of TLR4

signaling in the intestinal epithelium prevents NEC development and attenuates

the degree” of enterocyte apoptosis .

CLINICAL FEATURES:

The clinical presentation of necrotizing enterocolitis (NEC)4 includes

vomiting,

diarrhea,

feeding intolerance and high gastric residuals .

abdominal distention

frank or occult blood in the stools.

when disease progress, abdominal tenderness, abdominal wall edema, erythema,

or palpable bowel loops indicating a fixed and dilated loop of bowel may

Systemic signs- apnea,

bradycardia,

lethargy,

labile body temperature,

hypoglycemia,

shock

systemic signs are indicators of physiologic instability

MODIFIED BELL STAGING

(NEC)

Stage

Systemic signs Abdominal signs Radiographic signs Treatment IA Suspected Temperature instability, apnea, bradycardia, lethargy Gastric retention, abdominal distention, vomiting, heme-positive stool Normal or intestinal dilation, mild ileus NPO, antibiotics x 3 days

IB Suspected Temperature instability,apnea bradycardia, lethargy Grossly bloody stool Normal or intestinal dilation, mild ileus NPO, antibiotics 3 days IIA Definite, mildly ill Temperature instability,apnea bradycardia, lethargy

Same as above, plus absent bowel sounds with or without abdominal tenderness Intestinal dilation, ileus, pneumatosis intestinalis NPO, antibiotics x 7 to 10 days

IIB Definite, moderatel y ill

Same as above, plus mild metabolic acidosis and thrombocytopen ia

Same as above, plus absent bowel sounds, definite tenderness, with or without abdominal cellulitis or right lower quadrant mass

Same as IIA, plus ascites

NPO, antibiotics x 14 days

IIIA Advanced, severely ill, intact bowel

Same as IIB, plus

hypotension, bradycardia, severe apnea,

Same as above, plus signs of peritonitis, marked tenderness, and abdominal

Same as IIA, plus ascites

combined respiratory and metabolic acidosis,DIC, and neutropenia

distention paracentesis

IIIB Advanced, severely ill,

perforated bowel

Same as IIIA Same as IIIA Same as above,

plus

pneumoperitone um

Same as IIA, plus surgery

DIC: disseminated intravascular coagulation

NPO: “nil per oral”

The severity of the disease; was categorized in stages by

Bell et al7 in 1978 and “later modified by Walsh and Kliegman8 in 1986.

Briefly, abdominal distention in stage I (mild), Pneumatosis intestinalis in stage

II (moderate), and pneumoperitoneum in stage III (severe) are the diagnostic

parameters. In 25% of cases, Necrotizing Enterocolitis is suspected but not

confirmed (stage I). The symptoms resolve gradually in these infants. In

25-40% of cases, the progression of Necrotizing Enterocolitis is fulminant with

INVESTIGATION

Necrotising enterocolitis is diagnosed clinically, no lab values support its

diagnosis

RADIOLOGICAL STUDY

The abdominal X-ray will often reveal an abnormal gas pattern

consistent with ileus. Both anteriorposterior or left lateral decubitus views

should be included. These films may reveal bowel wall edema, a fixed position

loop on serial studies, the appearance of a mass, pneumatosis intestinalis (the

radiological hallmark used to confirm the diagnosis), portal or hepatic venous

air, pneumobilia, or pneumoperitoneum. Isolated intestinal perforation may

X-ray showing dilated bowel loop

The non-specific radiographic findigs of necrotizing enterocolitis is dilated

bowel loops. Generalized bowel distension is the earliest radiographic sign of

NEC. Dilated bowel loops of NEC may be localized, especially in the right

Pneumatosis intestinalis

It is the diagnostic feature of NEC. This gas in the bowel wall is hydrogen, a

product of bacterial metabolism. A linear or cresent gas shadow in bowel wall is

characteristic of NEC.

X- Ray showing pneumoperitoneum

air in peritoneal cavity following perforation of bowel.

portal gas shadow

Pneumotosis intestinalis extends into portal venous circulation . it can be

seen in plain xray or in ultrasound abdomen.portal gas shadow associated with

severe disease

TREATMENT

Stage 1- NPO, IV antibiotics - ampicillin,and gentamycin for 3 days

Stage 2a- NPO,IVantibiotics- ampicillin, gentamycin and clindamycin for 7 to

10 days

Stage 2b- NPO, IV antibiotics- ampicillin,gentamycin and clindmycin for 14

days

Stage 3- NPO, IV antibiotics – ampicillin,gentamycin and clindamycinfor 14

SURGICAL INTERVENTION

Intestinal perforation usually occurs within 12 to 48 hours after onset of

necrotising enterocolitis.

Main surgical treatment is resection with enterostomy or

resection with primary anastomosis in selected cases.

Peritoneal drainage can be considered in ELBW

infants(<1000g),hemodynamically unstable babies

Up to 50% of neonates with NEC develop advanced disease that requires

operative treatment 3.

The indications for surgery 3

• pneumoperitoneum, indicating perforation of the intestine,

• clinical deterioration despite maximal medical treatment, • abdominal mass with intestinal obstruction.

Relative indications

• fixed dilated intestinal loop,

• presence of portal gas,

• thrombocytopenia.

PREVENTIVE STRATEGIES

2. Alteration of the immunologic status of the intestine

Oral immuno-globulins may have potential benefit, immunoglobulin A (IgA)

and immunoglobulin G (IgG) supplementation in feedings reduced the incidence of

NEC.

3.Breast milk

Human breast mik contains many immunoprotective factors. Incidence of

NEC is lower in premature infants fed only with breast milk. Human “milk has

been reported to reduce the incidence of Necrotizing Enterocolitis by upto ten

fold compared to infant formula. The protective factors of breast milk are IL10,

modulate intestinal microflora composition to the advantage of the host. The

activity of acetyl hydrolase (PAF-AH), an enzyme that degrades PAF, is lower

in” neonateswith probiotics.

4. Feeding

5. Trophic Feeding (Minimal Enteral Nutrition)

MEN is safe alternative to complete fasting before initiation of

progressive feeds. Minimal enteral nutrition does not increase the

incidence of necrotising enterocolitis.

6. Probiotics

Probiotics are defined as “live microorganisms which when administered in

adequate amounts confer a health benefit on the host”. “Compared with healthy,

full-term infants, the intestinal microbiota in preterm infants features a low

number of species, with typically only 3 bacterial species found at 10 days of

enterococci such as E. faecalis, and staphylococci such as S. epidermidis, S.

aureus, and S. haemolyticus, are the most frequently retrieved . All of these

facultative anaerobes persist at high levels in the fecal flora of preterm infants

and there is significantly delayed colonization with anaerobes, especially

Bifidobacteria, compared with that seen in healthy, full-term infants . It has

been suggested that the enteral administration of probiotics to preterm newborns

could prevent infections, prevent NEC, and reduce the use of antibiotics.”

7. Prebiotics

The prebiotics are “nondigestible food components that has beneficial effects in

the host by selectively stimulating the growth and/or activity of one or a limited

number of bacteria in the colon Oligosaccharides in human breast milk are

considered to be prebiotics, as they facilitate the growth of bifidobacteria and

lactobacilli in the colon of breast-fed neonates .”

8. Synbiotics

The “synbiotic contains both probiotics and prebiotics in same product” . In a

“recently published RCT, 90 preterm infants received a dietary supplement

containing 2 lactobacillus species plus fructooligosaccharides, a supplement

containing several species of Lactobacilli and Bifidobacteria plus

fructooligosaccharides, or placebo twice daily for 28 days or until discharge if

earlier. The study found that preterm infants who received the supplement

fructooligosaccharides were more likely to become colonized” with

bifidobacteria.

9. Fluid Restriction

Excess fluid intake has been implicated in the pathogenesis of NEC . Cochrane

review which included 3 studies concluded that restricted water intake

In animal “models of experimental enterocolitis, glutamine

supplementation reduces mucosal damage and lowers the risk of invasive

infection and death. Glutamine is abundant in human milk but present only in

much lower levels in cow milk formula and absent in standard parenteral

nutrition solutions. A relative deficiency of arginine leading to inadequate NO

levels, vasoconstriction, ischemic-reperfusion injury, and ultimately the

development” of NEC.

Amin et al. in “a prospective trial on 152 neonates showed that

the incidence of NEC was significantly lower in group receiving supplemental

arginine with feeds till 28 days compared with group not receiving supplemental

16. Acidification of Gastric Contents

Carrion and “Egan have documented that acidifying the feedings of preterm

neonates to a pH low enough to inhibit gastric bacterial proliferation

significantly lowers the risk of NEC. Evidence exists that the use of

histamine-receptor type 2 (H2) blockers to suppress gastric acidity is associated with a

higher risk of NEC (and nosocomial infection) in VLBW” infant.

BACTERIAL COLONIZATION OF GROWING GUT:

The fetus “lives in a sterile intrauterine environment

are protected by layer of chorioamniotic membranes. At birth, the

gastrointestinal tract of neonate is sterile. The gut becomes colonized quickly

after birth with various organism environmental and maternal vaginal flora .

The pattern and rates of neonatal colonization are influenced by gestational age,

mode of delivery, maternal bacterial flora, antenatal and postnatal antibiotic use,

environment hygiene, and type of feeding. There are significant differences in

the intestinal colonization pattern between preterm and term infants. Healthy

full term neonates delivered vaginally are colonized by anaerobic bacteria

predominantly Bacteroides by one week of age. However, infants delivered by

cesarean section exhibit delayed colonization by anaerobes and are colonized

be colonized predominantly by Bacteroides spp., E. coli, and Klebsiella. The

healthy commensal organisms like” Bifidobacterium andLactobacilli “tend to

appear only in the third week of life in preterm infants. Stools of breast fed

infants have oBifidobacterium and Lactobacillus species,which will compete

with Bacteroides, Clostridia, and Enterobacteriaceae found as intestinal flora in

formula fed infants . Exposure to maternal antibiotics as well as postnatal

antibiotic therapy, total parenteral nutrition, or nursing in the incubator can

Miller et al- in 1993,studied 20 preterm infants with mean gestational age 33

weeks.infants were randomized to receive either milk or milk along with

lactobacillus GG 108 CFU(colony forming units) twice daily for 2weeks or till

discharge of the baby. They found “lactobacillus GG was well tolerated and it

Kitajima et al in 1997 in japan randomized trial in 91 infants. 45 in study group

were given probiotic bifidobacterium breve and control group 46 were given

only distilled water till 28 days of life. Study showed colonization of bowel with

bifidobacter breve was 73% in “study group compared with control group

infants.”

Angela B hoyos in 1999 has done a trial of probiotics in pretem infants and

showed reduction of necrotising enterocolitis. In her study total 1237 newborn

received probiotic lactobacillusGG daily were compared with historical controls

.the incidence of NEC in study group is reduced to one- third compared to

historical controls.(18vs47) with p value<0.0005.

In a multicentre, double-blind study from Italy, done by Dani et al. in 2002,

Studied in 585 infants with a gestational age <33 weeks or birth weight <1,500

g were randomized to receive Lactobacillus GG in a dose of 6 × 109

colony-forming units once a day until discharge, starting with the first feed . Outcome

measure included the incidence of Urinary Tract Infection, Necrotizing

Enterocolitis and bacterial sepsis. Incidence of necrotising enterocolitis in tudy

group was 1.4% and in control group was 2.8%.but satistically not significant.

The number of babies with any of the three outcome(NEC, bacterial sepsis,UTI)

were low, but there were no significant differences between the probiotic and

Ramesh Agarwal et al. in 2003, studied “the ability of lactobacillus GG to

colonise the neonatal gut and modify its microbial ecology, a prospective,

randomized study was performed in 71 preterm infants less than 2 kilograms

birth weight.” 109lactobacillus GG was given orally twice a day for 21 days.

The babies with a weight of 1500-2000 grams were treated for 8 days. Stools

were analysed on day 7 and 8 and day 14 and 21 for qualitative aerobic and

anaerobic cultures. It was found that colonization occurred more in larger babies

11 of 23 compared to 5 of 24 in infants with weight less 1500 grams. It was

found that although lactobacillus GG is relatively poor colonizer in infants, “it

does appear to affect neonatal intestinal colonization patterns and” there by

preventing Necrotizing Enterocolitis in preterm infants. Limitation of the study

–those baby discharged earlier probiotic therapy not continued.

Bin-nun et al-israle in 2005 ,studied 145 infants ,who are less than 1.5 kg. 72were given probiotics and 73in placebo group.probiotic used in this study is

(B.infantis 0.3x109,S.thermophilus 0.3x109,B.bifidis 0.35x109 )given till 36

weeks of getational age. “The combined incidence of NEC and mortality was

compared” and was found that significantly more in the control group (17/73)

were affected as compared with the study group (6/72), (p=0.025;pr-0.358;95%

CI=0.150-0.856).incidence was 4% in study group and 16.4%in control group

of generating and blinding of randomization has not been described and the

completeness of follow up has not mentioned.

Lin et al in 005 in Taiwan has done a prospective randomised control study in

37 preterm low birth weight babies<1.5kg. 180 in study group and 187 in

control group. Probiotics given to study group was lactobacillus acidophilus

1x106 and Bifidobacterium.infantis1x106 . incidence of severe NEC is

significantly lower in study group.

He also found that “neonates with a birth weight of 1000-1500 grams were

of < 1000 grams and the limitations of this study was dropouts were not

mentioned.”

A meta-analysis was performed by Girish Deshpande et al. in 2010 to update

the 2007 systematic review of randomized controlled trials of probiotic

supplementation for preventing NEC in preterm VLBW neonates. “A total of

11 (N=2176) including 4 new (N=783) trials were included in the meta-analysis.

The risk for NEC and death was significantly lower. Risk for sepsis did not

differ significantly. No significant adverse effects were reported. Trial showed

30% reduction in the incidence of NEC.”

Conchrane review in 2014. In this meta analysis 24 trails were included.

It showed significant reduction in severe NEC( stage2 and 3)with(RR 0.43 at

CI 95% 0.33-0.56).

Significant reduction in mortality (RR-O.65 at 95%CI 0.52-0.81)

Reduction in nosocomial infection is not significant(RR-0.95 at 95% CI

0.8-1.03)

PROBIOTICS

The term "probiotic" was first used in 1965, by Lilly and Stillwell for describing

substances secreted by one organism which stimulate the growth of another.

probiotics: viable microorganism that has beneficial effects to host

Prebiotic: food substances that are nondigestable ingredient that selectively

stimulates the growth and activity of indigenous bacteria

Post biotic –non viable bacterial products or metabolic byproducts from

probiotic which has beneficial effects to host

Symbiotic – combination of probiotic and prebiotic

The “term 'probiotics' was derived from the Greek word, meaning "for life".

FAO (Food and Agriculture Organization) and WHO defined probiotic as

"livemicro-organisms," which, when administered in adequate amounts confers

a health benefit on the host. Various bacterial genera most commonly used in

probiotic preparations are Lactobacillus,

Bifidobacterium,Escherichia,Enterococcus,Bacillus, Streptococcus . Some

fungal strains belonging to Saccaromyces have also been used as” probiotic

most clinical attention to date. The Lactobacillus strain used traditionally for

fermentation by dairy industry was unable to implant the gut,

so, Lactobacillus rhamnosus strain GG was discovered in 1985, by

developing a list of ideal qualities for probiotics. Lactobacillus

rhamnosus strain GG has proven beneficial affects on intestinal immunity. It

increases the number of IgA and other immunoglobulins secreting cells in the

intestinal mucosa, stimulates local release of interferons and facilitates antigen

transport to underlying lymphoid cells. .

For “adequate amount of health benefits, a dose of five

billion colony forming units a day (5x10 9CFU/day has been recommended, for

bile, hydrochloric acid and pancreatic juice, have anti-carcinogenic activity and

stimulate immune-system, have reduced intestinal permeability, produce lactic

acid, able to survive both acidic conditions of the stomach and alkaline

conditions of the duodenum. Foods for human consumption that contain mainly

lactic acid bacteria include fermented milks, cheeses, fruit juices, wine, and”

sausages.

PROPERTIES OF PROBIOTICS

1.Viable

2. resistant to acids

3 able to persist in gut even if probiotic strain cannot colonize the gut

4.should be human origin

5.should be non pathogenic

6.resistance to processing

7.adhere to gut epithelium to cancel the flushing effects of peristalisis

ORGANISMS USED AS PROBIOTICS

1.LACTOBACILLUS- L.acidophilus

L.casei

L.fermentum

L.lactis

L.gasseri

L.johnsonii

L.paracasei

L.reuteri

L.salivarius

L.bulgaricus

2 BIFIDOBACTERIUM- B.bifidum

B.breve

B.lactis

B.longum

B.infantis

B.adolescentis

3 . SACCHAROMYCES- S.boulardi

4.STREPTOCOCCUS- S.thermophilus

S.salivarius

5 .OTHERS – Bacillus cereus

E.coli

The “microbiota of a newborn develops rapidly after the birth and it is initially

dependent mainly on the mother’s microbiota, mode of delivery, birth

environment,postnatal care and rarely genetic factors. The maternal vaginal and

intestinal flora constitutes the source of bacteria, which colonizes the intestine

of the newborn, the dominating strains being facultative anaerobes such as the

enterobacteria, coliforms, and lactobacilli. After weaning the composition of the

microflora gradually alters to resemble that of the adult. The bacterial strains

having beneficial properties are mainly bifidobacteria, lactobacilli and

streptococcus.”

The fecal flora of 46 preterm infants and 52 born at full

term was studied at 10 days of age; 46 born at full term and 37 preterm infants

were also studied at 30 days of age. “Gas liquid chromatography was used to

identify the anaerobes lactobacilli, but not bifidobacteria, were found in high

counts in the stools of most of infants born at full term by 30” days of age.

The mode of delivery had a significant influence on

early colonization. A selective deficiency of lactobacilli compared with

coliform organism was found in preterm infants. Their study indicated “that

lactobacilli may be an important part of normal stool flora in early infancy, and

colonization. A high proportion of preterm infants receiving intensive care

suffer episodes of systemic infection with antibiotic resistant bacteria and fungi.

These infections further increase the risk of necrotising enterocolitis.

MECHANISM OF ACTION OF PROBIOTICS

1. act as a barrier and prevents migration of bacteria into GI mucosa

2. Compete with potential pathogens,

3. modifys the host response to microbial products

4. augmentation of IgA mucosal responses.

5. enhances enteral nutrition that wil inhibits the growth of pathogens and

for the care of these babies were carried out in both groups. On admission to

NICU a septic work up which included complete blood count, C-reactive

protein and blood cultures were done to all babies.

INCLUSION CRITERIA

1. Preterm neonates <34 weeks

2. Hemodynamically stable

3. Parents giving consent to participate in study

EXCLUSION CRITERIA

1. Gestational age > 34 weeks

2. Hemodynamically unstable

3. Birth asphyxia

4. IUGR

5. Congenital anomalies

6. Parental refusal

RESULTS

Analysis of cases and results

There were 200 preterm neonates < 34 weeks of gestation admitted to NICU

of Rajamirasudhar government hospital attached to Thanjavur medical college,

during the time period between December 2014 and august 2015. They were

assigned randomly to the study or control group. The study group was fed with

probiotic and the control group was fed with breast milk without the addition of

probiotics.

The study and control groups were compared according to their age at

admission, sex, birth weight, gestational age, mode of delivery, antenatal risk

factors and age of initiation of feed.

Statistical Analysis

The statistical analysis was done using statistical package for social sciences version 16.

The data was tested for normality using the Shapiro Wilk test.

The comparability of the two groups was assessed using Chi-square test for categorical variables and T test for independent samples for quantitative variables.

The outcome, namely the duration of hospital stay was analysed using the T test for independent samples.

The association of incidence of NEC with the intervention was analysed using Fisher’s exact T test.

1.comparison of preterm with respect to sex

Females

Males

Marginal Row Totals

Experimental

group

49 (48) [0.02]

51 (52) [0.02]

100

Control group

47 (48) [0.02]

53 (52) [0.02]

100

Marginal

Column Totals

96

104

200 (Grand Total)

“The Chi-square statistic is 0.0801. The P value is 0.777124. This

result is

not

significant at p < 0.05.”

When sex distribution of the two groups were compared, it was

observed that there was no difference between the two groups

44 46 48 50 52 54

Females Males

Control group

2.Comparison of experimental group and control

group with respect to weight

T test for difference between means of the two groups

Experimental group mean weight : 1.462 kg SD : 0.272 kg

Control Group mean weight : 1.476 kg SD: 0.283 kg

T value : -0.4076

P value : 0.684

The experimental group and control group were not significantly different with

3.Comparison of experimental group and control

group with respect to gestational age

Experimental group mean gestational age: 31.99weeks SD

1.778

Control group mean gestational age: 32.15weeks

SD:

1.641

T value : -0.6631

P value : 0.5081

The two samples were not significantly different with respect to

gestational age

0 5 10 15 20 25 30 35

Weeks of Age Standard Deviation 31.99

1.778 32.15

1.641

Comparison of the study group in terms of Gestational Age

4.Comparison of experimental group and control

group with respect to weight ( AGA or SGA)

Appropriate for

GA

Small for GA

Marginal Row Totals

Experimental

group

82 (84.5) [0.07] 18 (15.5) [0.4] 100

Control group

87 (84.5) [0.07] 13 (15.5) [0.4] 100

Marginal

Column Totals

169

31

200 (Grand Total)

“The Chi-square statistic is 0.9544. The P value is 0.328607. This

result is

not

significant at p < 0.05.”

82 18

87 13

0 20 40 60 80 100

Appropriate for Gestational age Small for Gestational age

Control group

5.Comparison of experimental group and control

group with respect to Mode of delivery

ND

LSCS

Marginal Row Totals

experimental

group

73 (78) [0.32] 27 (22) [1.14] 100

control group

83 (78) [0.32] 17 (22) [1.14] 100

Marginal

Column Totals

156

44

200 (Grand Total)

“The Chi-square statistic is 2.9138. The P value is

0.087827. This result is

not

significant at p < 0.05.”

73 27

83 17

0 20 40 60 80 100

Normal Delivery LSCS

control group

6.Comparison of age at initiation of feeds between

experimental group and control group

T value:

1.64

P value: 0.102

The two groups did not vary with respect to age at initiation of

feeds

7.

Comparison of duration of hospital stay between

study and control group

8.

INCIDENCE OF NECROTISING

ENTEROCOLITIS

The incidence of NEC was compared thetwo groups using the fisher’s

exact test. The P value was 0.0489. There was a statistically

significant difference between the two groups. The experimental

group was less likely to develop NEC when compared to the control

group

Results

NEC

No NEC

Marginal Row

Totals

Study Group

3

97

100

Control Group

11

89

100

Marginal Column Totals

14

186

200 (Grand Total)

NEC NO NEC

3

97

11

89

Comparison of incidence of NEC

9.STAGING

The three neonates who developed NEC in the experimental group belonged to

stage 1 according to bell-staging. In the control group, 7 neonates belonged to

stage 1, 3 neonates belonged to stage 2 and 1 neonate to stage 3. Fisher’s exact

test did not show a statistically significant difference between the two groups

according to staging of the disease. P = 0.462

Experimental

Group

Control

group

Stage 1 3 7

Stage 2 0 3

Stage 3 0 1

0 2 4 6 8 10 12

STAGE 1 STAGE 2 STAGE 3

3

0 0

11

3

1

Comparison of NEC staging

9.

comparison of sepsis between two groups

27 neonates in the experimental group were positive for CRP. 15 of those

neonates had positive blood cultutre. . Out of those who tested negative for

CRP, none of them had a positive blood culture

Among the neonates in the control group, 42 were positive for CRP. 17 of these

neonates had a positive blood culture. . Out of those neonates who tested

negative for CRP, 6 of them had positive blood culture. A total of 48 neonates

had evidence of sepsis.

The chi square value for the above parameters was 9.408 with a ‘p’ value of

0.00216. incidence of sepis is less likely in study group.

Sepsis No sepsis

Marginal Row

Totals

Experimental Group

27 (37.5) [2.94] 73 (62.5) [1.76] 100

Control Group 48 (37.5) [2.94] 52 (62.5) [1.76] 100

Marginal Column

Totals

75 125 200 (Grand Total

DISCUSSION

NEC(necrotising enterocolitis) is most common devasting intra abdominal emergency in preterm neonates with mortality approaching 30%.cause for NEC

is multifactorial, common fators are prematurity,enteral feeding and abnormal

bacterial colonization.

The proposed stratergy for prevention of NEC is oral probiotics.

Choice of probiotics

Probiotics are live microbial supplements that colonise the gut and provide

benefit to the host.

Many different species of bacteria and fungi have been used as probiotics in

various studies

Probiotic organisms generally consist of strains of Lactobacillus,

CHOICE OFPROBIOTICS IN VARIOUS STUDIES

S NO STUDY PROBIOTICS

1 HOYOS et al in 1995 Bifidobacteriuminfantis,

2 DANI et al in 2002 Lactobacillus rhamnosusGG

3 COSTALOS et al in 2003 Saccharomyces boulardi

4 LIN et al 2005 Lactobacillus acidophilus

5 BIN NUN et al 2005 Bifidobaerium infantis

Streptococcu thermophilus

Bifidobacterium bifidis

6 MANZONI et al 2006 Lactobacillus rhamnosus

7 LIN et al 2008 Lactobacillus acidophilus

Bifidobacterium bifidis

8 SAMANTA et al 2009 Bifidobacterium infantis

Bifidobacterium bifidis

Bifidobacterium lactis

Lactobacillus acidophilus

9 AWAD et al 2010 Lactobacillus acidophilus

10 MIHATSH et al 2010 Bifidobacterium lactis

11 ROMEO et al 2011 Lactobacillus reuteri

12 FERNANDEZ et al2013 Lactobacillus acidophilu

Lactobacillus GG

Lactobacillus pantarum

Streptococcus thermophillus

Bifidobacterium infantis

13 BRAGAet al 2011 Lactobacillus casei

Bifidobacterium brevi

14 SARIET at al 2011 Lactobacillus sporogens

15 Present study Lactobacillus rhamnosus

Lactobacillus acidophilus

Sacchromyces boulardi

Similar observations were seen in study by Lin et al. They reported a lower

incidence of NEC in the probiotic group (1.1% Vs 5.3%; p=0.04).

Bin-Nun et al. found a significantly lower incidence of all cases of NEC in the

probiotic group (4% Vs 16.6%; p=0.031).

Dani et al. found a lower incidence of NEC (1.4 Vs 2.7%) in the probiotic

group, but his study not reach statistical significance.

Costalos et al. reported a non significant trend lowered less NEC of any

severity in the probiotic group (9.8% Vs 16%; p=0.5).

Manzoni et al. also reported a non-significant trend towards less severe NEC

in the probiotic group (2.6% Vs 4.9%; p=0.51).

• Our study showed that the test group has a lower incidence of

necrotizing enterocolitis and lower incidence of sepsis in study

group.both are statistically significant. Severity of disease(stage 2 and

NEC and Bell Staging

• In our study, out the 14 babies developed NEC, 3 babies in study

group and 11 infants in observational group.

Incidence of necrotiing enterocolitis was statistically significant.

In contol group, 3 babies in stage II NEC and 1 baby in stage III NEC ,

which was statistically not significant (p>0.05).

The study by Bin Nun et al. reported that “three deaths in the control group

were due to NEC, whereas there were no NECrelated deaths among the test

neonates (p = 0.87).

The study done by Hoyos MD et al. showed NEC-associated mortality is more

in the non probiotic group (35/1282 Vs 14/1237; p-value<0.005) which was

statistically significant.”

Studies done by Lin et al. and Manzoni et al. reported a significantly lower

mortally rate in the probiotic group but did not differentiate between death

attributed to NEC byother cases.

NEC and Sepsis

• In our present study, the incidence of sepsis in test group is 27% and

in the

control group is 48%. It is found that the incidence of sepsis is less in the test

Hung–Chin Lin et al. in 2005 reported a lower

incidence of sepsis in the probiotic group (22/180 Vs 36/183; p=0.03).

“The mechanism for the efficacy of probiotics in reducing the incidence of

sepsis in VLBW infants is probably similar to NEC and possibly a result of

increased

colonization of desirable microflora supplemented through” probiotics.

Dani et al. and Bin Nun et al. did not show any reduced

incidence of sepsis in the probiotic group. Their studies reported that the

pathogens were most often related to catheter related infections in both groups.

Probiotics alone could not over come the invasive procedures inducing

infections.

CONCLUSION

Necrotizing Enterocolitis is a world wide problem in very low birth weight

infants (VLBW), causing significant mortality and morbidity.

The present study found that probiotic supplementation has reduced both

incidence and seventy of NEC in preterm neonates < 34 weeks of gestation.

Probiotic supplementation has also reduced the incidence of sepsis in the

preterm neonates. Both incidence of NEC,incidence of sepsis were decreased in

study group and it is statistically significant but there were no significant

differences between test

and control groups in severity of disease and mean duration of hospital stay.

However more research is required involving more sample size to support the

use of probiotics in preterm neonates.

RECOMMENDATIONS

Probiotics offers many potential benefits for premature infant.

probiotic treatment provides a promising strategy to prevent NEC in premature

neonates. “probiotics have three advantages, compared to other strategies

proposed for the prevention” of NEC

1.probiotics represent a simple, non invasive attempt to recreate

a natural normal florarather than a disruption of nature.

2. it is effective in preventing major source of morbidity in low

birth weight infant.

3 .is safety record renders it an attractive alternative to many of

the more aggressive therapeutic options.

By demonstrating a probiotic mediated reduction in both the incidence and

of NEC in premature infants, our results lend further support to the

consideration of inclusion of probiotics in the prevention of necrotizing

LIMITATION OF THE STUDY

1.Adverse effects of probiotics was not analysed.

2. Necrotising enterocolitis is multifactorial .other factors contributing for NEC

SUMMARY

• Two hundred preterm neonates less than 34 weeks were studied

during march 2015 to august 2015 in raja mirasudhar hospital –

thanjavur medical college

• They were randomly assigned in study and control group

• Study group received probiotics till they reach full feed. Control group

only breast milk

• Clinical and demographic variables between two groups were

compared and it was statistically not significant.

• Incidence of NEC is 3% in study group and 11% control group (p

value<0.05 )

• Severity of NEC is high in control group 3 in stage and 1 in stage 3,

but not statistically significant (p value>0.05)

• Incidence of sepis 27% in study group and 48% control group.( p

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