Onderzoek naar nieuwe diagnostische markers voor vroeg-diagnostiek van necrotiserende enterocolitis bij pasgeborenen.

Necrotising enterocolitis is the most prevalent acute gastro-enterological disease in the Neonatal Intensive Care Unit (NICU). The disease often progresses rapidly, with potentially life-threatening complications, e.g. bowel perforation necessitating laparotomy. Morbidity and mortality are therefore high, in some series up to 40%. However, diagnosis is often difficult as signs are often non-specific, and to date specific markers for the disease or its complications are not available. Early diagnosis allows for early treatment, which may improve outcome in patients with NEC.
Promising markers for the diagnosis of NEC are Fatty Acid Binding Proteins (FABPs). FABPs are proteins which are important in the fat-metabolism of the enterocyte. When enterocytes are damaged, FABPs are released in the circulation and subsequently secreted by the kidney. This also holds true for parts of the tight junctions, the bridges between enterocytes. When these are damaged, Claudin-3 is released. Claudin-3 can also be assessed in urine. Preliminary data suggest that both urine FABP and Claudin levels (expressed as ratios to urine Creatinine) are early indicators for loss of gut integrity and therefore possible early indicators for NEC and its complications. Although I-FABP and Claudin have been suggested as early markers for NEC, little is known about the location and quantification of FABP’s in the bowel wall during the development of (complications of) NEC.
As ischemia and reperfusion are supposedly involved in the pathophysiological mechanisms underlying NEC, early identification of ischemia of the gut can be of vital importance. This is possible using near-infrared spectroscopy (NIRS). NIRS is a continuous, non-invasive portable technique allowing for the determination of regional tissue oxygenation and thus the presence of ischemia. NIRS is increasingly being used to monitor cerebral oxygenation, but can also be used to monitor oxygenation of abdominal tissues. As the cerebral oxygenation of the brain remains relatively constant, the ratio between local oxygenation and cerebral oxygenation has been shown to provide an adequate estimate of local tissue oxygenation.
It is known that neonates with NEC have a disturbed intestinal flora. As yet, it is unknown whether this is cause or effect of the disease. Although regular culture is able to identify most pathogens, there are pathogens which cannot be cultured using the routine techniques. Recently a new technique has been developed to investigate the presence of bacteria on the DNA/RNA level using the so-called denaturing gradient gel electrophoresis (DGGE) GGE-16SrRNA-technique. This offers the possibility to investigate pathogens which cannot be identified using routine culturing techniques.

Finally, cytokines may allow for early diagnosis of NEC. Increased serum levels of cytokines are found in early phases of inflammatory conditions. As yet, many assays for cytokines can only be performed on serum samples. Recently, new techniques have become available which make it possible to analyse urine cytokine profiles. When this method would also be possible in neonates, obtaining blood samples for these assays will not be necessary anymore.



Objective:

The overall objective of this study is an improvement in (early) diagnosis of NEC and its life-threatening complications, thus to be able to detect children who are “at risk” for NEC and its complications as early as possible.

To this end we will as a first objective assess urine iFABP/Claudin to Creatinine ratio as early markers for NEC and its possible complications and compare them with regional tissue oxygenation as measured by NIRS. This should give insight in the relation between tissue oxygenation and enterocyte damage. It also should provide insight in the use of NIRS and iFABP/Claudin levels as diagnostic markers for (complicated) NEC and might offer new modalities to guide therapy based on these results.

Second objective is to correlate the iFABP/Claudin levels and NIRS data with the other serum markers of the inflammatory and metabolic response such as serum IL1, IL6, IL10, TNFa and lactate and base excess.

Third objective is - when surgery has become necessary - to analyse resectional specimens for the exact location and quantification of iFABPs.

Fourth objective is to validate the Biosource™ Multiplex Assay (Invitrogen), a novel assay to test multiple cytokines and chemokines in small volumes of urine, by comparing urine results with results obtained in serum.

Fifth objective is to gain insight in the inflammatory and metabolic process, including gut wall integrity and cytokine profiles during the development of NEC and its complications.
Sixth objective is to assess faecal bacterial flora using both routine cultures and the DGGE-16SrRNA-technique.



Study design:

Prospective cohort study.



Study population:

(>)40 premature infants with (suspected) NEC.

Inclusion criteria: Patients in the NICU with (suspected) NEC: (Bell stage I based on clinical parameters such as bowel distension, bloody stools, causing sufficient clinical concern to withdraw enteral feeding and/or to perform an abdominal X-ray).

Exclusion criteria:

1. Congenital bowel defects;

2. Intensive light therapy for hyperbilirubinaemia necessitating the use of three phototherapy lamps (as this interferes with NIRS measurements).



Intervention:

When there is a suspicion of NEC, urine samples and blood will be collected every eight hours and abdominal X-rays will be performed upon the first suspicion of NEC until confirmation of the diagnosis by either clinical or surgical means. This is, except for the urine samples, according to routine clinical practice. The diagnosis NEC will be established using the Bells criteria, with the presence of NEC defined as signs of pneumatosis intestinalis on the abdominal X-ray as assessed by an independent pediatric radiologist.

For the serum analysis 100ìl of blood will suffice. This can be obtained during the daily routine bloodtests without any further burden for the child. Urine (preferably >3 ml) can be directly obtained from the urine catheter or is collected via a small cotton wool placed into the diaper for at least one hour. Urine is subsequently removed from the cotton wool by compressing the cotton wool in a syringe.

At the same time as the interventions mentioned above, regional tissue oxygenation of the gut and the brain is evaluated using the NIRS system during the first 48 hours (i.e. after diagnosing NEC with Bell stage >1).

Blood and urine samples will be collected at ten different intervals (T1-T10), without regard to the definitive diagnosis. If at certain intervals no routine blood samples are obtained, only urine is collected. In this way, the patients are not burdened.

Surgery: All surgical procedures will be performed in the UMC Groningen. When surgery becomes necessary and bowel is resected, small parts of the resected bowel and adjacent normal tissue will be collected. One part is snap frozen, the other part is stored in Formalin. We will use routine Hematoxin-Eosin staining for histologic evaluation and use immunohistochemistry for the determination of the localisation of the FABPs. These proteins will also be assessed on the mRNA and/or protein level. We will also perform immunohistochemistry for carbohydrases to examine the gut maturity.

During the operation, Visible Light Spectroscopy (VLS) will be used to assess the blood circulation and tissue oxygenation of the bowel at risk. This will help the surgeon in deciding which bowel segments to resect and whether it is safe to perform a primary anastomosis instead of a enterostomy.



Main study parameters/endpoints:

Urine I-FABP/Claudin to Creatinine ratio, tissue oxygenation as measured via NIRS.

Secondary outcome: Inflammatory/metabolic markers such as WBC, CRP, TNFa, IL6 and IL10 and lactate/base excess as measured in blood and urine, location/quantity of FABP’s in the resected bowel and faecal cultures. Bowel oxygenation as measured with VLS.



Nature and extent of the burden and risks associated with participation, benefit and group relatedness:

Urine (preferably >3 ml) will be collected via tissues placed in the diaper or directly from the urine catheter. Patient will therefore not undergo extra procedures. Regarding the bloodtests: 0.25ml will be enough, which can be obtained during the daily routine bloodtests without any further burden for the child. NIRS (and VLS) measurements are non-invasive and do not interfere with routine care.

Data from this study cannot be obtained in another population, as (premature) neonates are the only children who develop NEC. The results from this study might offer new and non-invasive tools for the early diagnosis of NEC, or complications of NEC, thereby improving allowing for early treatment and improving outcome.

1. Intestinal fatty acid binding proteins (FABP) are diagnostic markers for necrotizing enterocolitis (NEC) and possibly also for complicated NEC;

2. The cerebro-splanchnic oxygenation ratio ia a diagnostic marker for NEC and possibly also for complicated NEC.

When a patient is suspected of NEC, blood and urine samples are being collected at regular intervals: if possible every 8 hours. NIRS is performed during the first 48 hours.
VLS is performed only if the patient is being operated on.

After having collected blood and/or urine samples at 10 subsequent intervals, study participation is completed.

When patient is suspected of NEC:

1. Collecting of blood and urine samples at 10 subsequent intervals;

2. Measuring cerebral and abdominal tissue oxygenation by near infrared spectroscopy (NIRS);

3. When patients are being operated on: Measuring bowel oxygenation by visible ligth spectroscopy (VLS).