Study design
All 156 infants included in the FortiLat trial (gestational age <32 weeks and/or birthweight ≤1500 g., exclusively fed with human milk) were considered for the present study.
After informed written parental consent was obtained, infants were randomized 1:1 by a software-generated list in one of the following groups: the control group (BF-arm) underwent adjustable fortification with fortifier derived from bovine milk; the FortiLat group (DF-arm) underwent adjustable fortification with fortifier derived from donkey milk. The different composition of the two types of fortifiers can be observed in table 1.
Table 1. Composition of BF and DF – Values per 100 g of product
|
BF
|
DF
|
Protein g (Nx6.25)
|
20.0
|
22.5
|
Carbohydrate (g)
Of which:
|
66.0
|
59.0
|
Lactose (g)
|
6.0
|
59.0
59
|
Maltodextrine (g)
|
60.0
|
0.0
|
Fat (g)
|
0.4
|
3.6
|
Kcal
|
385
|
390
|
Kcal/g protein
|
18.8
|
15.6
|
Calcium (mg)
|
1500
|
938
|
Phosphate (mg)
|
900
|
734
|
Osmolality (mosm/kg)
|
453
|
441
|
pH
|
6.8
|
6.7
|
Buffering capacity (mmol l-1/dph)
|
12.2
|
9.2
|
BF: bovine milk-derived human milk fortifier
DF: donkey milk-derived human milk fortifier
Details on eligibility criteria, sample size calculation, settings and randomization and are specified in the FortiLat trial.(15) This study follows ed the Consolidate Standards of Reporting Trials.
The detailed protocol of the FortiLat trial is described in our previous paper.(15)
At day 21 since beginning of fortification, infants showing typical (excessive regurgitations, vomiting, blenching) signs of GER and CR symptoms (apneas/desaturations/bradycardia, paleness, cyanosis) were enrolled into the present study. Enrolled infants underwent synchronized MII/pH and CR monitoring and then gastric emptying US assessment as specified below.
MII/pH monitoring
The evaluation of GER events was performed through MII/pH monitoring (Sleuth monitoring system). The recommendations for the procedure and the identification of GER events have already been described in a previous work by our group.(16) A MII-GER event was defined as a retrograde drop of impedance to 50% of the basal value for at least 5 seconds, starting in the most distal channel, proceeding to one or more proximal channels and followed by a recovery of the impedance baseline values. A reflux reaching the two most proximal channels is defined as proximal GER; pH-GER events, defined as drop of pH value below 4 longer than 5 seconds and not associated with a MII-GER event, were considered in the analysis as well.
The following features were evaluated:
- MII-GER frequency, expressed as reflux events/h;
- Bolus reflux extent (BRE): the proximal extent reached by the refluxate, indicated by the number of channels sequentially involved, expressed as number of channels;
- Bolus clearance time (BCT): duration of a reflux from the drop to 50% of the impedance baseline value to its recovery recorded in the distal impedance channel, expressed as seconds;
- Bolus exposure index (BEI): total percentage of time bolus reflux was detected by MII, expressed as a percentage;
- pH-GER frequency, expressed as reflux events/h;
Reflux index (RI): total percentage of time with pH <4, expressed as a percentage. Each MII-GER event was defined as weakly acidic (4 ≤ pH > 7), acidic (pH <4), or weakly alkaline (pH ≥7) according to the minimum pH value reached during each event.(16–18)
CR monitoring
The VitaGuard VG3100 system (Getemed Medizin und Information stechnik AG, Teltow, Germany), equipped with Signal Extraction Technology (Masimo Corp. Irvine, CA, USA), was used to perform CR monitoring, heart rate, transcutaneous blood oxygen saturation, and respiratory rate were measured during synchronized CR and MII/pH monitoring by a pulse-oximetry sensor placed on the right wrist or foot and three cardiac electrodes placed on the chest. CR tracings were “visually” analyzed by a trained operator blinded to the MII/pH tracings using VitaWin3® evaluation software.
CR events were defined as episodes of apnoea lasting more than 20 seconds or over 5 seconds if followed by desaturation or bradycardia, episodes of desaturation with blood oxygen saturation below 80%, and episodes of bradycardia with heart rate below 80 beats per minute.(7,19) Minimum duration of bradycardia and blood oxygen desaturation events to be considered for the analysis was 4 seconds.
Synchronized MII/pH and CR monitoring
CR and MII/pH monitors were synchronized using an external reference (i.e. Internet time) and digitally marking each tracing at the beginning of the study. The specific times of each CR event were adjusted for the offset between the clocks of the CR and MII/pH monitors. The symptom association probability (SAP) index was calculated to evaluate temporal associations between GER and CR events.(20) SAP index value >95% identified patients with a significant number of GER-CR associations.
The primary outcome of the study was the MII-GER frequency, secondary outcomes were reflux characteristics, half gastric emptying time (T/2) and the SAP index.
Gastric US
Gastric antral transit was used as a proxy of gastric emptying. This was determined by measuring ultrasonically the changes in the antral cross-sectional area (ACSA) which occur after a feed (21). Serial measurements of ACSA were made before, during and after administration of feeds every 10 minutes for 90 minutes.
The gastric emptying curve was calculated from the ultrasound series and represented by the best fitting polynomial function (R2 > 0.98. P < 0.001) of the ACSA values and on these bases the half gastric emptying time (T/2), calculated as the time required to achieve a 50% reduction in ACSA values was obtained.(21) Half gastric emptying time was evaluated as a secondary outcome.
Statistical analysis
Statistical analyses were performed using the STATISTICA software package for Windows (StatSoft, Inc., Tulsa, Oklahoma, USA). Results are expressed as median and interquartile range (IQR) if not otherwise specified, and the p value was set at 0.05. The distribution of the variables was evaluated by the Shapiro-Wilk test. The Student's t-test was used to evaluate differences between normally distributed variables, and the Mann-Whitney test for unpaired data was used for non-normally distributed variables.