Calprotectin and Gut Disease Study

Why is this research important in terms of improving the health of patients? Our study will ascertain whether there is a clear relationship between levels of faecal calprotectin and the true occurrence of necrotising enterocolitis in infants with congenital heart disease. This is important because infants with congenital heart disease are burdened with feeding difficulties and poor nutrition for a range of reasons, and they can be affected by necrotising enterocolitis because of abnormal circulation of the blood to all organs, including the gut. If necrotising enterocolitis could be identified correctly in a timely way, this would help clinicians manage the nutrition of these vulnerable infants, at the same time as helping clinicians to target necrotising enterocolitis treatment to babies who truly have this condition.

Considering the different disease processes for infants with congenital heart disease to other clinical populations, such as preterm infants, it is essential to investigate calprotectin concentration in this

1. An inaccurate diagnosis of NEC results in the withdrawal of an infant's milk feed, exacerbating parental stress in intensive care. Improved nutritional support has been associated with increased survival and a reduction in overall morbidity in infants with cardiac defects.10 2. An inaccurate diagnosis of NEC results in the unnecessary administration of antibiotics. Antibiotics cause antibiotic-related diarrhoea, which in turn affects expected growth.11,12 3. An inaccurate diagnosis of NEC results in the inappropriate use of intravenous nutrition as a substitute for enteral feeds during gut rest. Avoiding intravenous nutrition reduces the risk of line sepsis and liver disease.13 4. An accurate and timely diagnosis of NEC will reduce the length of hospital stay. Infants classified with suspected NEC have a longer hospital stay.5,9 5. The current per-patient marginal cost due to NEC is £13,900 and the cost due to feeding difficulties after surgery is £5,500,14, both of which could be reduced with a timely and accurate diagnosis of NEC, improving patient care.

3\. THEORETICAL FRAMEWORK Review of existing evidence Importance to the NHS and patients Congenital heart disease (CHD) is diagnosed in approximately 1 in 100 births.15 Significant CHD that required intervention in early childhood affected 3.6 per 1000 live births in England, 2004-2016.16 The unadjusted crude 30-day mortality rate amongst 9,586 paediatric cardiac operations undertaken between 2019-2022 was 2.1% but was highest in the youngest babies.7

The number one priority for the 'Research priorities in children and adults with congenital heart disease: a James Lind Alliance Priority Setting Partnership': how can damage to the gut during heart surgery in children with heart defects be minimised to reduce complications?17

Another priority listed in the same Priority Setting Partnership is 'What are the best approaches to feeding in babies with heart defects, to optimise growth, improve outcomes, and reduce parental distress?'17

In our national prospective study of NEC in 1819 infants who had cardiac surgery in 5 of the 10 UK specialist paediatric cardiac centres, we found 4% had NEC, with this proportion rising to 7.5% amongst neonates under 28 days of age.18

Necrotising Enterocolitis Infants with CHD are at high risk of developing NEC due to persistent diastolic flow reversal in the abdominal aortic, which reduces mesenteric blood flow.19 The aetiology of NEC is multifactorial with the interaction of cardiac surgery, gut hypoxia, poor mucosal integrity, and disruption of intestinal microbial dysbiosis.20,21

Management of NEC involves supportive care, empirical antibiotic therapy, parenteral nutrition, and bowel rest with gastric decompression initiated as soon as NEC is suspected.22 The exact duration of bowel rest in infants with nonsurgical NEC is based on disease severity and clinical judgment.23

Diagnostic Limitations of Necrotising Enterocolitis Interpretation of the clinical picture, including relevant plain abdominal radiographs in infants, is challenging and is one of the reasons for a wide variation in the reported incidence rate of NEC, ranging from 3 to 18%.5 Key markers of NEC, including intestinal dilation, ileus and pneumatosis intestinalis, are often absent on abdominal radiographs.25

The clinical presentation of NEC includes lethargy, temperature instability, recurrent apnoea, bradycardia, hypoglycaemia, and shock. Specific gastrointestinal symptoms include abdominal distention (70% to 98%), blood per rectum (79% to 86%), high gastric residuals after feeding (\>70%), vomiting (\>70%), and diarrhoea (4% to 26%).24

In an audit of 195 neonates at one cardiac centre (Great Ormond Street Hospital) 14% had feeds suspended during an evaluation for NEC for at least 24 hours but did not have a NEC diagnosis. This clinical path can involve inappropriate use of intravenous nutrition, unnecessary antibiotics administration and longer hospital stays.5,9 Of note, optimal nutritional support in infants requiring cardiac surgery has been associated with increased survival and a reduction in overall morbidity.10

Faecal Calprotectin- Biomarker Neutrophils are first-line responders of the innate immune system and generally combat infections by phagocytosis.26 Calprotectin is a neutrophil activation marker and is mainly exhibited in the cytoplasm of neutrophils and expressed on activated monocytes and macrophages.27 Calprotectin is secreted into the lumen in the early stages of intestinal inflammation; thus elevated faecal calprotectin concentrations are evident in infectious and inflammatory diseases such as inflammatory bowel disease.28 Faecal calprotectin is routinely used as a screening test in the evaluation of inflammatory gastrointestinal diseases and a faecal calprotectin value above 50μg/g should prompt review and a raised faecal calprotectin of greater than 200μg/g should prompt urgent assessment.29

Faecal calprotectin is used as a diagnostic marker for NEC in preterm infants.30 However, infants with CHD pose a unique clinical presentation in that their gut may be inflamed from birth due to compromised gut perfusion and therefore warrant specific investigation.5 Additionally, ischemia-reperfusion injury from cardiac surgery results in pronounced neutrophil activation followed by a hyperinflammation reaction that drives inflammation in NEC.31

Pilot Study Our published prospective single-centre observational pilot study monitored the relationship between faecal calprotectin concentration and gut inflammation in 30 infants admitted with cardiac defects requiring cardiac surgery.

Faecal calprotectin for infants who developed NEC (n=5) had a mean concentration of 3528μg/g (1004SD) compared with 1339μg/g (1008SD) in patients with suspected NEC (n=7) and 390μg/g (410SD) in those without NEC (n=18) (P=0.0001). Infants with suspected NEC had a significantly longer length of hospital stay, on average 18 days longer compared to infants without NEC (P=0.03).9

Research is warranted to establish whether faecal calprotectin can be a useful addition to the diagnostic algorithm for NEC in infants with heart defects.5,9 Important areas for further work include NEC prognostication, which includes earlier identification of the need for care escalation and the requirement for surgery.32 Considering the proposed different disease processes for cardiac infants, it is essential to investigate calprotectin levels independently from other diseases.5,9,31

Size of sample Total sample 300 infants Justification for sample size In a prior multi-centre observational study, the rate of true NEC in this age group was 7% and the rate of inconclusive suspected NEC was 14%. (Brown et al Incidence and risk factors for important early morbidities associated with paediatric cardiac surgery in a UK population, JTCVS 2019; 158;1185-1196)14 Therefore, to ensure faecal calprotectin is clinically useful, we define a minimum acceptable specificity of 80% with 5% precision, i.e. a threshold for the lower bound 95% CI of 75%.

Assuming the expected rate of NEC cases classified according to the gold standard is 7%, and the rate of no NEC cases is 93%, we would need 270 infants to be able to estimate the pre-defined specificity with the required precision. Given the observational nature of the study, lack of harm to the patient from participation and deferred consent, we expect few refusals and a low attrition rate (death), therefore assuming a loss to follow-up/drop-out rate of 10%, we plan to recruit 300 patients.

Buderer, N. M. F. (1996). Statistical methodology: I. Incorporating the prevalence of disease into the sample size calculation for sensitivity and specificity. Academic Emergency Medicine, 3(9), 895-900.37

10 SCREENING AND RECRUITMENT Children admitted to a participating centre will be screened for inclusion by the clinical care team in the cardiology wards and the paediatric intensive care units, as these babies will be admitted to either area depending on clinical need.

Children who meet inclusion and exclusion criteria will be recruited to the study as soon as possible after admission.

11 CONSENT

Consent Procedures As the passage of a first stool often occurs in the first 6-12 hours after admission to a specialist cardiac centre delaying collection of this stool would mean the loss of information critical to the study. Moreover, no harm will occur to participants related to their participation in this observational study nor from a sample of their stool being collected. We will commence data collection once the child's eligibility is confirmed. This is known as a deferred consent model ('research without prior consent'). This model, developed in line with the CONseNt methods in pediatric Emergency and Urgent Care Trials (CONNECT) study guidance,(38) has been found to be acceptable to parents/guardians and clinicians in several recent RCTs conducted in the UK pediatric critical care setting.( 39,40)

A trained member of the site research team will approach the parents/legal guardian to discuss the study as soon as practical and appropriate. This will usually be within 24-48 hours; however, this will depend on the patient's condition and will be left to the discretion of the clinical team. A Participant Information Sheet (PIS) will be provided. A modified consent procedure will be followed for 1) children in the care of the local authority, 2) where discharge occurs prior to consent being sought, or 3) the child dies before consent is sought. In cases where the local authority have full parental responsibility for a participant, the PIS and covering letter will be sent to the appropriate individual (e.g. the child's named social care worker or foster parent). If the local authority have shared responsibility for a participant, both parties with parental responsibility should be provided with the study information as described above. In the event where a participant is discharged from hospital before consent has been sought or confirmed, the most appropriate member of the site research team will attempt at least one phone call to the parents/legal guardians within five working days of hospital discharge to inform them of the participant's involvement in the study and provide more details. Following on from the call, as well as if there is no response to the call, the parents/legal guardians will be sent the covering letter for discharged patients and a copy of the PIS by post. If no objection is received within four weeks, then the participant's data will be included in the study. In the event where a participant has National Data Opt-out (NDOO) in place, only non-identifiable data will be retained and if there is no such decision recorded, all data will be included in the study.

In the situation where a participant dies before consent has been sought, a site research team member will obtain informatio