Faecal cytokine levels of preterm infants coupled with microbiome profiles represent a potential non-invasive method to predict severity of necrotizing enterocolitis

Objectives Necrotizing enterocolitis (NEC) is a life-threatening disease, and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell’s staging is routinely used to guide diagnosis, but early diagnostic signs are non-specific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed in NEC infants. New techniques, using biomarkers as diagnostic tool to improve diagnosis of NEC, are emerging. Here we investigated faecal cytokine levels, coupled with gut microbiota profiles, as a non-invasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers. Study design Premature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary’s or Queen Charlotte’s & Chelsea) between January 2011 and December 2012. All but two babies received a first course of antibiotics from birth onwards. Faecal samples from diapers were collected consecutively during the NICU stay. Results Evaluation of microbiota profiles between the study groups revealed only minor differences. However, at later time points, significant changes in microbiota structure were observed for Firmicutes, with Enterococcus being the least abundant in Bell stage 2/3 NEC. Faecal cytokine levels were similar to those found in previous studies evaluating systemic cytokine concentrations in NEC settings, but measurement in faeces represents a non-invasive method to evaluate the early onset of the disease. For IL-1α, IL-5 and IL-10, a significantly rising gradient of levels were observed from healthy to NEC1 to NEC2/3. Conclusions Differences in certain faecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles, contribute to improving knowledge about NEC pathogenesis. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement LJH is supported by Wellcome Trust Investigator Awards (100974/C/13/Z and 220876/Z/20/Z); the Biotechnology and Biological Sciences Research Council (BBSRC), Institute Strategic Programme Gut Microbes and Health (BB/R012490/1), and its constituent projects BBS/E/F/000PR10353 and BBS/E/F/000PR10356. Work at Imperial College was supported by a programme grant from the Winnicott Foundation to JSK, and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial Healthcare NHS Trust and Imperial College London. KS was funded by an NIHR Doctoral Research Fellowship [NIHR-DRF-2011-04-128]. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This work is part of the study Defining the Intestinal Microbiota in Premature Infants (NeoM The Neonatal Microbiota study) (ClinicalTrials.gov Identifier [NCT01102738][1]), approved by West London Research Ethics Committee Two, United Kingdom (Reference number: 10/H0711/39). Parents gave written approval for their infants to participate in the study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Data availability: 16S rRNA gene amplicon data is available under BioProject accession number PRJNA889687. Cytokine data is provided in Supplementary Table 1. * NEC : Necrotizing enterocolitis NICU : neonatal intensive care unit VLBW : very low birthweight DOL : day of life OTU : operational taxonomic unit [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01102738&atom=%2Fmedrxiv%2Fearly%2F2022%2F10%2F26%2F2022.10.24.22281217.atom