Trial document




drksid header

  DRKS00009290

Trial Description

start of 1:1-Block title

Title

Intestinal microbiota composition and diversity in very low and extremely low birthweigt infants related to strategies of NEC prophylaxis - a pilot
study

end of 1:1-Block title
start of 1:1-Block acronym

Trial Acronym

Neonatal Microbiomics and NEC prophylaxis

end of 1:1-Block acronym
start of 1:1-Block url

URL of the Trial

[---]*

end of 1:1-Block url
start of 1:1-Block public summary

Brief Summary in Lay Language

At the Medical University Graz, Department of Pediatrics, Division of Neonatology, we use a special prophylaxis regimen to prevent necrotizing enterocolitis (NEC), achieving low incidence rates of NEC if compared internationally. NEC still constitutes a major factor in neonatal morbidity and mortality, mainly appearing in preterm born infants. Prophylaxis regimen used consists of antibiotic, antimycotic and probiotic treatment combined with a special feeding regimen. We aim to invstigate prophylaxis´ impact on intestinal microbiota composition within the first weeks of life. Stool samples will be teken every 2 days from diapers thorughout the first two weeks of life. Samples will be analyzed in regard of microbiota composition and diversity. As control groups, infants cared for at the general hospital of Klagenfurt am Wörthersee, and hospital of Leoben, not receiving the prophylaxis regimen as do infants cared for in Graz, will be included. Treatment in regard of NEC prophylaxis differs between the two centers in Klagenfurt and Leoben and the center in Graz. We aim to invetigate how the prophylaxis regimen used in Graz influences and alters intestinal microbiota composition and diversity over the first two weeks of life.

end of 1:1-Block public summary
start of 1:1-Block scientific synopsis

Brief Summary in Scientific Language

Hypothesis:
Differences in therapeutic and preventive approach to VLBW infants, in use of probiotics, antibiotics, antifungal agents and feeding strategies all might influence diversity and composition of gut microbiota. Thus intestinal microbial diversity and abundance, as well as development of the intestinal microbiome within the first weeks of life in preterm infants included in our study should differ between the two groups. Due to the different NEC prophylaxis regimen used, differences regarding intestinal microbiota might be found that additionally might have long-term, up to now unknown, impacts.
Intestinal microbiota in neonates
The genesis of the microbiome starts by vertical transmission of maternal microbiota even before delivery. Recent studies suggest the presence of a microbiome even within the placenta and fetal meconium. Delivery mode has been found to significantly influence intestinal colonization of infants. Intestinal colonization patterns established within the first week of life are thought to have effects on the composition of the individual ́s future intestinal microbiota. In term infants, the intestinal microbiome undergoes rapid maturation throughout the first year of life and is considered to be securely established by three years of age. However in preterm infants maturation is delayed. The individual intestinal microbiome of adulthood is likely governed by an interplay between initial colonizing microbiota, genes, gut development, diet, and environment. Nutrition, breastfeeding versus formula milk, plays a major role and alters early colonization patterns of the neonatal intestinal microbiome. Breast milk contains several beneficial components, such as lactoferrin, or secretory IgA, that have antimicrobial and anti-inflammatory effects and stimulate growth of Bifidobacterium. Human milk also contains live bacteria. The intestinal microbiome interacts in a symbiotic relationship with its host. It contributes to nutrient utilization by increasing digestive capacity and the ability to harvest nutrients from food. It supports vitamin biosynthesis and production of other metabolites. In addition the intestinal microbiome can limit nutrient resources available to pathogens, specifically by out-competing them for metabolic resources and physical space. Development of barrier function, integrity and immune function is aided by the intestinal microbiome.

Intestinal microbiota in preterm infants:
The intestinal microbiome of the preterm infant shows reduced microbial diversity with a concurrent increase in colonization with pathogenic organisms. It is less stable compared to that of term born infants, and shows delayed transition to an adult colonization pattern. In preterm infants, facultative anaerobes such as Enterococcus, Enterobacter, and Lactobacillus, as well as numbers of Staphylococcus dominate while numbers of anaerobes like Bifidobacterium, Bacteroides and Atopobium are decreased. Healthy breastfed term born infants were found to be colonized by Bifidobacterium species by day 7 of life while preterm infants were not. Gestational age may contribute to intestinal colonization pattern. Data suggest that the development of the intestinal microbiome may also be influenced by genetics. Evolution of the preterm infants ́ intestinal microbiome is marked by periods of abrupt population changes. Preterm infants exhibit an altered microbial diversity, and are both qualitatively and quantitatively immune compromised caused by their underdeveloped immune system. An insufficient epithelial gut barrier function predisposes them to invasion by pathogens. These can trigger exaggerated inflammatory responses by the infants ́ still developing immune system that may lead to disease processes such as NEC. Preterm infants ́ immune dysfunction coupled with low diversity of intestinal microbiota and an overall predominance of pathogenic bacteria within the infants ́ gut represents a prime example of dysbiosis.
Intestinal microbiota and NEC
Among risk factors, prematurity and accompanying intestinal colonization represent the only consistently identified criteria. Therefore, if prematurity is inevitable, colonization of the infants gut resembles the major modifiable risk factor. Up to date, findings on intestinal colonization and the microbiome aspect of NEC are inconsistent. While some studies found a unique community structure of the intestinal microbiome in preterm infants prior to and at the time of disease onset, others could not. The intestinal microbiome of NEC-infants appears to first diverge from that of healthy controls as early as three weeks prior to disease. Two weeks prior to disease onset, cases intestinal microbiota show an increased proportion of Proteobacteria while the proportion of Bacterioidetes is decreased. Differences in intestinal microbiota abundance and diversity continue until onset of NEC, at which infants have been characterized by an overall lower diversity index compared to the average preterm infant. Whether onset of NEC is early in life (i.e. ≤ 22 days of age) or late (i.e. > 22 days of age), seems to discriminate abundance of intestinal microbiota. Thus, it can be assumed that the specific infectious agent associated with NEC might vary by infants ́ age at onset of disease.

NEC:
NEC resembles the most common life-threatening emergency in the gastrointestinal tract in the newborn period. It was first described in 1965, and occurs mainly in preterm infants, rarely affecting term born infants. The prevalence of NEC accounts 7-11% in VLBW infants, with a prevalence rate of 1-5% of infants in NICUs. Both incidence and case fatality rates increase with decreasing birth weight and gestational age, putting very low and extremely low (<1000g) birth weight infants at highest risk. Although NEC is a multifactorial disease primarily associated with intestinal immaturity, the concept of “risk factors” for NEC is controversial. The greatest risk factor for NEC is prematurity. Pathogenesis: The triad of intestinal ischemia, enteral nutrition and intestinal pathogenic organisms has been linked to NEC. The disorder probably results from an interaction between loss of mucosal integrity due to a variety of factors and the host’s response to that injury, leading to necrosis of the affected area. Various bacterial and viral agents have been recovered from cultures. However, in most situations, no distinct pathogen is identified. NEC rarely occurs before the initiation of enteral feeding and is much less common in infants fed human milk. Aggressive enteral feeding may predispose to the development of NEC. Clinical manifestations: The onset of NEC is usually in the 2nd or 3rd week of life. Age of onset is inversely related to gestational age. There is a variety of signs and symptoms to be found in affected infants that may have an insidious or sudden catastrophic onset. The first signs of impending disease may be nonspecific, including lethargy and temperature instability, or related to gastrointestinal pathology, such as abdominal distention and gastric retention. Bloody stools are seen in 25% of patients. The spectrum of illness is broad, ranging from mild disease to severe illness with bowel perforation, peritonitis, an even shock and death. Progression may be rapid. Diagnosis: Plain abdominal x-rays are essential to make a diagnosis of NEC. The finding of pneumatosis intestinalis (air in the bowel wall) is pathognomonic and confirms the clinical suspicion. To stage the disease, Bell ́s Staging Criteria score, consisting of systemic, intestinal and radiologic signs, is used. Treatment: Rapid initiation of therapy, consisting of supportive care and preventing further injury, is required for suspected as well as proven cases of NEC. Cessation of feeding, nasogastric decompression, and administration of intravenous fluids, as well as systemic broad-spectrum antibiotics are applied. Surgical treatment might be necessary in case of massive hemorrhagic necrosis or intestinal perforation. Prognosis: Medical management fails in about 20-40% of patients presenting with pneumatosis intestinalis. Of these infants, 10-30% die. Premature infants with NEC who require surgical intervention or who have concomitant bacteremia are at increased risk for adverse growth and neurodevelopmental outcome. Economics: The total annual estimated cost of caring for affected infants in the United States amounts between $500 million and $1 billion. Prophylaxis: Newborns exclusively breast-fed have a reduced risk of NEC. There have been concerns about early and aggressive increase in feeding volumes in raising the risk of NEC in VLBW infants, although a safe feeding regimen remains to be unknown. Prophylactic enteral antibiotics can reduce the risk of NEC, although concerns about the development of resistant bacteria persist. Probiotic preparations may also decrease the incidence of NEC. Enteral supplementation of probiotics reduces the risk of severe NEC (Bell ́s stage II or higher) and mortality in preterm infants.

Potential clinical impact of the proposed project:
We aim to elucidate how different prophylaxis strategies lead to differences in development of gut microbiota and influence morbidity, growth and development of VLBW infants. Our findings might contribute to help unrevealing infectious genesis of NEC and lead to an improvement in NEC prophylaxis. Currently no general recommendations on NEC prophylaxis exist. Our data might support establishment of recommendations.

Study design:
Prospective controlled multicenter cohort pilot study including preterm infants with a birth weight of <1500g treated at three NICUs with different regimens of NEC prophylaxis and feeding strategies.

Study protocol:
Stool samples of 60 VLBW preterm infants, treated at the NICUs at the department of pediatrics, Medical University Graz (n=20), Department of Pediatrics, hospital of Leoben (n=20) and the Department of Pediatrics, General Hospital of Klagenfurt (n=20), will be collected every two days within the first weeks of life. 7 samples will be taken of each infant. Samples will be partitioned into 2 containers, so that one can remain stored for future analysis. Prior to sample collection 6 samples from infants <1500g birth weight will be sent to the Core Facility Molecular Biology to establish the DNA isolation method.
At the Division of Neonatology, Department of Pediatrics, Medical University of Graz, a unique regimen of NEC prophylaxis in preterm infants <1500g birth weight is used. The regimen includes oral antibiotic and antifungal therapy and probiotic drugs as well as a standardized feeding regimen.The incidence of NEC in preterm infants treated by this regimen has been shown to be significantly lower, reflecting 0.7% when treatment was initiated at the first day of life, compared to international incidence rates (5.1%). To compare our infants, we cooperate with the Division of Neonatology, Department of Pediatrics, General Hospital of Klagenfurt, and Division of Neonatology, Department of Pediatrics, General Hospital of Leoben, where different prophylactic approaches are performed. Infants will be recruited at both centers to compare microbiota-analysis data between these two groups. Prior to inclusion, infants ́ parents are informed about intention and purpose of the study, procedure of stool sampling, advantages and disadvantages for their infant, and that the infant does not get harmed or treated differently for the purpose of the study. Parental information is provided by a written consent form, as well as by an attending pediatrician. Parents must give written informed consent before inclusion of the infant. 14 stool samples of spontaneously produced feces should be collected throughout 2 weeks, starting at the day of the infants ́ first defecation, i.e. meconium loss. No additional interventions to stimulate defecation, deviating from standard care, are performed in purpose of the study. Collection of stool samples will be performed using sterile stool sample containers. All samples taken will be addressed a running number. Storage of the samples taken will be performed using a deep-freezing storage device at -80°C as soon as possible. Delivery of samples from Klagenfurt will be performed by a professional delivery service using dry ice cooling and a temperature protocol. After collection is completed, microbiome analysis will be performed.
Inclusion criteria comprise birth weight <1500g, treatment at the NICU, department of pediatrics, Medical University Graz, or NICU, department of pediatrics, General hospital of Klagenfurt, or general hospital of Leoben between October 2015 and March 2017.
Exclusion criteria comprise infants nonviable at birth, death within the first 2 weeks of life, infants suffering from genetic diseases, genetic syndromes or congenital anomalies, and meconium transit disorder.
Additionally the following data will be collected throughout hospitalization:
- Maternal and pregnancy associated data: mothers age, number of pregnancies past, number
of infants born, multiple pregnancy, existence of mother-child-passport, pregnancy
associated complications, application of steroids to induce lung maturation, tocolysis.
- Perinatal data: complications during birth, birth-mode, APGAR-score, umbilical cord pH.
- Neonatal data: sex, birthweight, small-for-dateness (SFD, birthweight <10. percentile),
duration of hospitalization, duration of NICU stay, duration of oxygen supplementation, appearance of neonatal morbidities (i.e. NEC, early/late onset sepsis, infant respiratory distress syndrome, bronchopulmonary dysplasia, retinopathy of prematurity, intra- /periventricular hemorrhage, periventricular leucomalazia, delayed meconium passage, spontaneous intestinal perforation), application of coffein and surfactant, as well as inotropes and cardiovascular supportive therapy.
- Data on specific medication: dosage, active pharmaceutical ingredient and time of application of antibiotics, antifungal agents and probiotics, as well as parenteral or enteral nutrition.
All clinical data, as described above, will be recorded using an excel sheet and additionally a hard copy placed at each center. To provide high quality throughout all steps of the study protocol, all procedures as described above will be put down into Standard Operating Procedures (SOP).
Analysis of intestinal microbiota
Microbial DNA will be extracted from stool samples using a combination of mechanical and enzyme- based lysis. From extracted DNA, 16S rRNA genes will be amplified with suitable primers (specific for bacteria, fungi and archaea) and prepared for MiSeq Sequencing [60], which will be performed at the “Zentrum für Medizinische Forschung” (ZMF), core facility for molecular biology, Medical University Graz. Analysis will be performed using public analysis-pipelines, like mothur and Quiime, which allow analysis of high-throughput community sequencing data. After qualitative filtering, remaining sequences will be assigned to phylogenetic taxa and grouped into operational taxonomic units (OTU) using specific databases. Furthermore, chimeras and sequences containing sequencing artifacts will be removed. Based on R-scripts, abundance of single OTU ́s is analyzed and significant changes of abundance are calculated using ANOVA. Further, Adonis Testing and Ordinations will be applied to illustrate correlations. Full analysis will be performed in close cooperation with the core facility for bioinformatics and biostatistics, ZMF, Medical University Graz. In the frame of the Microbiome Initiative Graz (MIGrobeZ; including groups around C. Högenauer, G. Gorkiewicz and C. Moissl-Eichinger) the necessary expertise in order to analyse and interpret the data is available. All tools, pipelines and expert knowledge of the Microbiome Initiative will be made available for the applicant, who is also member of the Microbiome Initiative.

Planned number of included infants:
We plan to include 20 infants <1500g birth weight (VLBW) from each of the participating study centers. This resembles a number of 60 infants included in our study. Starting at the day of the infants ́ first spontaneous stool, collection of 7 samples, acquired every two days throughout the first weeks of life, is performed in every infant. Finally all 420 samples will be analyzed using 16S-rRNA- analysis techniques.

end of 1:1-Block scientific synopsis
start of 1:1-Block forwarded Data

Do you plan to share individual participant data with other researchers?

No

end of 1:1-Block forwarded Data
start of 1:1-Block forwarded Data Content

Description IPD sharing plan:

[---]*

end of 1:1-Block forwarded Data Content
start of 1:1-Block organizational data

Organizational Data

  •   DRKS00009290
  •   2015/10/27
  •   [---]*
  •   yes
  •   Approved
  •   27-366 ex 14/15, Ethikkommission der Medizinischen Universität Graz, Auenbruggerplatz 2, 8036 Graz, Österreich ecs.medunigraz.at
end of 1:1-Block organizational data
start of 1:n-Block secondary IDs

Secondary IDs

  • [---]*
end of 1:n-Block secondary IDs
start of 1:N-Block indications

Health Condition or Problem studied

  •   Necrotizing Enterocolitis
  •   P77 -  Necrotizing enterocolitis of fetus and newborn
end of 1:N-Block indications
start of 1:N-Block interventions

Interventions/Observational Groups

  •   Preterm infants <1500g, treated at the Neonatology ward, Department of Pediatrics, Medical University Graz. NEC prophylaxis used at the Neonatology ward, Department of Pediatrics, Medical University Graz, includes Gentamycin 7mg/kg every 12h, Nystatin
    10.000U/kg every 6h, Probiosis usingLactobacillus rhamnosus 1g = 1x10^9 CFU/dtwice daily and pooled pasteurized breastmilk 1ml/kg every 3h with an increase of 1ml/kg/d within the first week of life.
    Stool samples VLBW preterm infants will be collected every two days within the first weeks of life. 7 samples will be taken of each infant.
  •   Preterm infants <1500g, treated at the Neonatology ward, Department of Pediatrics, General Hospital of Klagenfurt am Wörthersee.
    There exists no special NEC prophylaxis at the Neonatology ward, Department of Pediatrics, General Hospital of Klagenfurt am Wörthersee. Probiosis using Bifidobacterium infantis 2x10^9 CFU/d and Lactobacillus acidophilus 2x10^9 CFU/d is applied. No standardized application of antibiotics or antimycotics is applied. In case of bacterial infection Ampicillin and Trobramycin are given. Infants <1000g birthweigt receive candidaprohylaxis using Fluconazol 6mg/kg every 72h. Nutrition is based on pasteurized breast milk or formula.
    Stool samples VLBW preterm infants will be collected every two days within the first weeks of life. 7 samples will be taken of each infant.
  •   Preterm infants <1500g, treated at the Neonatology ward, Department of Pediatrics, General Hospital Leoben.
    There exists no special NEC prophylaxis at the Neonatology ward, Department of Pediatrics, General Hospital of Leoben. No probiotics are used. No standardized application of antibiotics or antimycotics is applied. In case of bacteria or fungall infection specific treatment is initiated. Nutrition is based on pasteurized breast milk or formula.
    Stool samples VLBW preterm infants will be collected every two days within the first weeks of life. 7 samples will be taken of each infant.
end of 1:N-Block interventions
start of 1:1-Block design

Characteristics

  •   Non-interventional
  •   Other
  •   Non-randomized controlled trial
  •   Open (masking not used)
  •   [---]*
  •   Other
  •   Basic research/physiological study
  •   Other
  •   N/A
  •   N/A
end of 1:1-Block design
start of 1:1-Block primary endpoint

Primary Outcome

Monitoring changes in composition and diversity of intestinal microbiota in preterm born infants <1500g birth weight within the first weeks of life receiving NEC (necrotizing enterocolitis)-prophylaxis vs not receiving NEC-prophylaxis. Collection of stool samples every 2 days within the first two weeks of life and analysis from microbiota diversity and abundance.

end of 1:1-Block primary endpoint
start of 1:1-Block secondary endpoint

Secondary Outcome

Differences in NEC-incidence between the two groups.

end of 1:1-Block secondary endpoint
start of 1:n-Block recruitment countries

Countries of Recruitment

  •   Austria
end of 1:n-Block recruitment countries
start of 1:n-Block recruitment locations

Locations of Recruitment

  • University Medical Center 
  • Medical Center 
  • Medical Center 
end of 1:n-Block recruitment locations
start of 1:1-Block recruitment

Recruitment

  •   Actual
  •   2015/10/29
  •   60
  •   Multicenter trial
  •   National
end of 1:1-Block recruitment
start of 1:1-Block inclusion criteria

Inclusion Criteria

  •   Both, male and female
  •   no minimum age
  •   2   Weeks
end of 1:1-Block inclusion criteria
start of 1:1-Block inclusion criteria add

Additional Inclusion Criteria

All infants treated at the Neonatology ward, Department of Pediatrics, Medical University Graz, and at the Neonatology ward, Department of Pediatrics, General hospital of Klagenfurt, or general hospital of Leoben between October 2015 and March 2017, having a birth weight <1500g.

end of 1:1-Block inclusion criteria add
start of 1:1-Block exclusion criteria

Exclusion Criteria

Non viable at birth, death within the first two weeks of life, genetic syndrome disease, malformation, meconium transit disorder.

end of 1:1-Block exclusion criteria
start of 1:n-Block addresses

Addresses

  • start of 1:1-Block address primary-sponsor
    • Division of Neonatology, Department of Pediatrics, Medical University Graz, Austria
    • Mr.  Univ. Prof. Dr.  Bernhard  Resch 
    • Auenbruggerplatz 34/2
    • 8036  Graz
    • Austria
    end of 1:1-Block address primary-sponsor
    start of 1:1-Block address contact primary-sponsor
    end of 1:1-Block address contact primary-sponsor
  • start of 1:1-Block address other
    • Department of Pediatrics, General Hospital of Klagenfurt am Wörthersee
    • Mr.  Dr.  Raimund  Kraschl 
    • Feschnigstraße 11
    • 9020  Klagenfurt am Wörthersee
    • Austria
    end of 1:1-Block address other
    start of 1:1-Block address contact other
    end of 1:1-Block address contact other
  • start of 1:1-Block address scientific-contact
    • Division of Neonatology, Department of Pediatrics, Medical University Graz, Austria
    • Mr.  Univ. Prof. Dr.  Bernhard  Resch 
    • Auenbruggerplatz 34/2
    • 8036  Graz
    • Austria
    end of 1:1-Block address scientific-contact
    start of 1:1-Block address contact scientific-contact
    end of 1:1-Block address contact scientific-contact
  • start of 1:1-Block address public-contact
    • Division of Neonatology, Department of Pediatrics, Medical University Graz, Austria
    • Mr.  Univ. Prof. Dr.  Bernhard  Resch 
    • Auenbruggerplatz 34/2
    • 8036  Graz
    • Austria
    end of 1:1-Block address public-contact
    start of 1:1-Block address contact public-contact
    end of 1:1-Block address contact public-contact
end of 1:n-Block addresses
start of 1:n-Block material support

Sources of Monetary or Material Support

  • start of 1:1-Block address materialSupport
    • Kleine Helden - Initiative für Frühgeborene
    • 8010  Graz
    • Austria
    end of 1:1-Block address materialSupport
    start of 1:1-Block address contact materialSupport
    •   [---]*
    •   [---]*
    •   [---]*
    •   [---]*
    end of 1:1-Block address contact materialSupport
  • start of 1:1-Block address otherSupport
    • Division of Neonatology, Department of Pediatrics, Medical University Graz
    • Auenbruggerplatz 34/2
    • 8036  Graz
    • Austria
    end of 1:1-Block address otherSupport
    start of 1:1-Block address contact otherSupport
    •   [---]*
    •   [---]*
    •   [---]*
    •   [---]*
    end of 1:1-Block address contact otherSupport
end of 1:n-Block material support
start of 1:1-Block state

Status

  •   Recruiting complete, follow-up complete
  •   2018/06/20
end of 1:1-Block state
* This entry means the parameter is not applicable or has not been set.