After necrotizing enterocolitis in preterm infants: The time to reach full enteral feeding and its relation to intestinal recovery and neurocognitive development.

Necrotising enterocolitis (NEC) is a common but devastating disease mainly
occurring in preterm infants. Neurodevelopmental outcome is significantly
poorer in NEC survivors when compared to their peers without NEC. Reduced
(cerebral) growth due to feeding problems after NEC might add to this impaired
development. Several studies reported on the importance of enhanced nutrition
in very preterm infants to stimulate brain growth and neurodevelopmental
outcomes.
A major concern for neonatologists and surgeons is the timing of reintroducing
enteral feeding after the infant had developed NEC, during which nil per os
(NPO) is prescribed. It is not clear how to approach the delicate balance
between undernutrition, delayed growth and impaired neurodevelopmental outcomes
later on one hand, and the risk of re-introducing enteral feeds after NEC too
early, with the intestines still damaged and unable to digest feeding on the
other. Refeeding too early might lead to an aggravation of the inflammation of
the bowel wall or recurrent episodes of NEC and possibly gut-related
septicaemia. Due to the lack of any evidence regarding optimal time and pace of
reintroducing enteral feeding after NEC, current refeeding protocols are only
consensus based.
Several methods might help to assess the quality of the intestines during and
after NEC, of which Near-InfraRed Spectroscopy (NIRS), the measurement of
urinary intestinal fatty acid binding proteins (I-FABPs), and plasma Citrulline
levels, are three validated methods for assessing bowel wall damage. NIRS
measures the regional intestinal oxygen saturation as surrogate for perfusion.
Because NEC is associated with low intestinal perfusion and ischemia, NIRS may
provide information for intestinal recovery. I-FABP is a protein present in the
epithelial cells of the small and large intestines, and is released immediately
after intestinal epithelial cell damage and excreted by the kidney. This makes
I-FABP a potential reliable marker for the detection and progression of NEC.
Citrulline is a free amino acid synthesized in the liver and small intestine
and found in human plasma. Lower plasma Citrulline levels are associated with a
reduced function of enterocytes and lower absorptive capacity of the small
intestine. Recent studies showed low plasma Citrulline levels in preterm
infants with NEC. This makes Citrulline another potential marker for the
evaluation of the recovery of the intestine.
A method to evaluate the neurodevelopmental state of those infants who
developed NEC, is the assessment of the quality of general movements of the
infant (to predict long-term outcome). With the assessment of the general
movements a motor-optimality score (MOS) can be calculated and compared with
the norm.
The aim of this study is to gain more insight in the intestinal recovery after
NEC, measured by NIRS, plasma Citrulline levels, and urinary I-FABPs, in
relation to the period to reach full enteral feeding after NEC. Furthermore, we
will relate this period to reach full enteral feeding to neurodevelopmental
outcome, measured by the assessment of general movements. **

The aim of the study is to gain more insight in the course of intestinal
oxygenation, urinary
I-FABP concentration, and plasma Citrulline levels (as markers for intestinal
damage and recovery) after NEC in relation to time to full enteral feeding and
to relate this time to full enteral feeding to neurocognitive outcome.
Our primary objective is to assess the relation between cerebral and intestinal
oxygen saturation and extraction measured with NIRS, the measurement of urinary
I-FABP concentration, plasma Citrulline levels, and the time it takes to reach
full enteral feeding.
Our second objective is to assess the relation between the time it takes to
reach full enteral feeding after developing NEC and neurodevelopmental outcome
measured with the assessment of the quality of general movements.

This study will be a prospective observational cohort study. Thirty-two preterm
born infants admitted to our neonatal intensive care unit in the UMCG,
Groningen, with a gestational age below the 37 weeks who develop NEC with
radiological signs of pneumatosis intestinalis will be enrolled.

When radiological signs of pneumatosis intestinalis are confirmed by the
radiologist, urine samples from the diaper and intestinal near-infrared
spectroscopy measurements (NIRS) during two hours will be performed every day
during the NPO period. After reintroducing enteral feeding, urine samples from
the diaper and intestinal NIRS measurements during two hours will also be
performed once every day. Both, urinary sampling and the measurements with NIRS
will continue until the infants reached full enteral feeding or to day 36 after
inclusion, whichever comes first.
To measure the plasma Citrulline levels during the period after NEC, we need to
sample 100 microliter blood. This will only be performed when blood will be
drawn for clinical purposes. Measurement of plasma citrulline will ideally be
done within 48 hours after radiologically confirmed pneumatosis intestinalis as
a radiological sign of NEC, once/twice during the nil per os period, at the
time when minimal enteral feeding will be reintroduced followed by a
measurement once a week, with the last measurement on the day that full enteral
feeding is reached or on day 36 after inclusion, whichever comes first.
Furthermore, in enterally fed infants, blood samples should be obtained one
hour before feeding to avoid postprandial fluctuations of plasma amino acids.
Citrulline levels will be assessed with LC-MS analysis.
Finally, a video record will be made to assess the quality of general movements
at term age and at the age of three months post term.

This pilot study is an observational study, implying minimal extra care;
therefore there is almost no burden or risk associated with participation. The
study contains four major parameters, i.e. intestinal NIRS measurements during
two hours daily, urinary I-FABP samples, plasma citrulline concentration and
the motor-optimality score from the assessment of the quality of GMs.
Firstly, NIRS is continuous and non-invasive method and monitoring rcSO2 is
already routine clinical care in infants with NEC admitted to the NICU at the
UMCG. For the purpose of this study a second sensor will be placed
infra-umibilical on the abdomen of the infant using special gauze (Mepitel®) so
that skin irritation is avoided. The sensors will be placed and removed only
during routine handling moments, so the infant will not be disturbed. Previous
studies have demonstrated that this does not hamper routine care and that it is
not a burden for the child. We previously showed the benefit of using NIRS to
assess cerebral and intestinal oxygen saturation in premature infants with NEC.
Starting to measure the rintSO2 within several hours after NEC diagnosis can
distinguish infants who might develop severe NEC, needing surgical treatment
(registered in the Dutch Trial Registry under NTR3239, METc 2010/038) (10).
Additionally, cerebral oxygen saturation is possibly an indicator for the level
of illness of the infant. This is why cerebral oxygen saturation measurements
are standard clinical care.
This confirms the diagnostic additive value of starting directly with the
measurements. Furthermore, it is not known when the intestine starts to recover
or when the intestine gets even worse. Without measuring from the start,
baseline values are missing, and the point of recovery or worsening can be
missed very easily.
Given the minimal burden and risks for the patient and the proven additive
value for predicting the severity of the diagnosed NEC, we consider this
non-invasive method to be very important in the period after the onset of NEC.
Secondly, collecting samples of urinary I-FABPs will be performed by a little
gauze in the diaper. This is non-invasive and will not be a burden to the
infant. The samples will be collected during routine clinical care to not
disturb the infant. Again, we have wide experience in this method of collecting
urine, which does not affect the infant in any way. I-FABP concentration will
be determined using a standard commercial ELISA.
Thirdly, we intend to measure plasma Citrulline levels during the period after
NEC by sampling 100 microliter extra blood, only when blood will be drawn for
clinical purposes. Measurement of plasma citrulline will ideally be done within
48 hours after radiologically confirmed pneumatosis intestinalis, once or twice
during the NPO period, at the time when minimal enteral feeding will be
reintroduced followed by a measurement once a week, with the last measurement
on the day that full enteral feeding is reached or on day 36 after inclusion,
whichever comes first. Citrulline levels will be assessed with Liquid
chromatography-mass spectrometry (LC-MS).

Finally, GMs is a widely accepted non-invasive method to assess neurological
neonatal outcome. At the age of term and three months post term the infant will
be filmed during an outpatient clinical visit or at home. The infant has to be
relaxed and comfortable for a successful GMs analysis. The infant has to have
little clothes on (romper) so that every movement is visible. Temperature of
the environment will be adapted to this situation. The camera will be placed in
a way that parents or the infant will not be hindered.

This pilot study may provide more insight into intestinal recovery after
developing NEC in preterm infants, which might be of importance to determine
the best time to start reintroducing enteral feeding, and to improve growth and
eventually neurodevelopmental outcome in those infants.