SI + CC Versus 3:1 C:V Ratio During Neonatal CPR
SI + CC Versus 3:1 C:V Ratio During Neonatal CPR
Research question In newborn infants requiring CPR, does CC superimposed by sustained
 inflation compared to 3:1 compression to ventilation ratio improves return of spontaneous
 circulation?

 Overall objective: CC superimposed by sustained inflation will improve short- and long-term
 outcomes in preterm (>28 weeks or older) and term newborns.

 Hypothesis to be tested Primary hypothesis: By using CC superimposed by sustained inflation
 (CC+SI) during CPR the time needed to achieve return of spontaneous circulation (ROSC)
 compared to the current 3:1 compression to ventilation (C:V) will be reduced in asphyxiated
 newborns.
Background The majority of newborn infants successfully make the transition from fetal to
 neonatal life without any help. However, an estimated 10% of newborns need help to establish
 effective ventilation, which remains the most critical step of neonatal resuscitation.
 Fortunately, the need for chest compression (CC) or medications in the delivery room (DR) is
 rare. Although, only about 0.1% of term infants and up to 15% of preterm infants receive
 these interventions, this will result in approximately 1 million newborn deaths annually
 worldwide. A recent cohort study at a level III center with a trained resuscitation team
 showed that only 6 per 10,000 infants received epinephrine. However, the same study showed
 that those infants who received epinephrine during resuscitation had a high incidence of
 mortality (41%) and short-term neurologic morbidity (57% hypoxic-ischemic encephalopathy and
 seizures)4. A recent review of newborns who received prolonged CC and epinephrine but had no
 signs of life at 10 minutes following birth noted 83% mortality, with 93% of survivors
 suffering moderate-to-severe disability. The poor prognosis associated with receiving CC
 alone or with medications in the DR raises questions as to whether improved cardiopulmonary
 resuscitation (CPR) methods specifically tailored to the newborn could improve outcomes.

 The inability to predict which newborns need CPR, and the infrequent use of CPR in the DR
 have limited neonatologists' ability to perform rigorous clinical studies to determine the
 best method for delivering CC to newborn infants. The main cause of cardiovascular collapse
 in most newborns is asphyxia, which makes newborn infants distinctively different to the
 adult population. The International Liaison Committee on Resuscitation and the American
 Academy of Pediatrics/American Heart Association Neonatal Resuscitation Program have
 recognize this difference, however the guidelines almost exclusively rely on data from
 studies in the adult population or animal studies. Such data may not be wholly applicable to
 the neonatal population because the most common cause of cardiovascular collapse in the adult
 is ventricular fibrillation, not asphyxia. Thus, further studies are needed to determine the
 optimal method for improving hemodynamics and recovery during neonatal resuscitation.

 Research question In newborn infants (preterm infants >28 weeks' gestation and term infants)
 requiring CPR, does CC during sustained inflation compared to 3:1 compression to ventilation
 ratio improves return of spontaneous circulation (ROSC)?

 Primary objective: To prove that CC during sustained inflation will improve recovery from CPR
 in preterm and term newborns with faster ROSC than that by 3:1 CPR technique.

 Secondary objectives: To examine i) if CC during sustained inflation will improve short- and
 long-term outcomes in preterm and term newborns, and ii) the value of feedback devices (e.g.
 ECO2, ECG and/or SpO2) in determining ROSC.

 Hypotheses to be tested Primary hypothesis: By using CC during sustained inflation (CC+SI)
 during CPR the time needed to achieve ROSC compared to the current 3:1 compression to
 ventilation (C:V) ratio will be reduced in asphyxiated newborns.

 Secondary hypotheses: The benefits associated with CC+SI will i) decrease death; ii) improve
 short- and long-term outcomes in preterm and term newborns; and iii) feedback devices help to
 determine ROSC.

 Methodology Ethics This multi-center cluster randomized clinical trial will be reviewed by
 the appropriate Research Ethics Office at all participating sites. We will request a deferred
 consent model at all sites (see parental consent paragraph for details). Written consent will
 be sought from the parents of these infants as soon as possible after the birth so that
 acquired data could be utilized for research35.

 Inclusion criteria All infants (term or preterm infants >28 weeks' gestation) requiring CC in
 the delivery room will be eligible for the trial.

 Exclusion criteria Infants will be excluded if they have a congenital abnormality or
 condition that might have an adverse effect on breathing or ventilation (e.g. congenital
 diaphragmatic hernia), or congenital heart disease requiring intervention in the neonatal
 period. Infants will be also excluded if their parents refuse to give consent to this study.

 Randomization At the beginning of the trial participating sites will be randomized. Centers
 will be equally allocated to either CC+SI group ("intervention group") or 3:1 C:V group
 ("control group") for the first year. For the second year, after a wash out period of 2
 months, centers in the intervention group will be changed to control and vice versa.

 Sample Size and Power calculation Our primary outcome measure will be time to achieve ROSC.
 We hypothesize that the time to achieve ROSC will be reduced in the "CC+SI group". A sample
 size of 208 infants, 104 in each group, would be sufficient to detect a clinically important
 33% reduction in time to achieve ROSC using Cox proportional hazards regression with 80%
 power and a 2-tailed alpha error of 0.05. This 33% reduction represents 282 sec versus 420
 sec of CC (based on the database of 30 term infants requiring CC in 2014 and 2015 at the
 Royal Alexandra Hospital, Edmonton). To account for cluster randomization, the sample size is
 multiplied by design effect of 1.045, so the total sample size is 218 infants, 109 in each
 group.

 Blinding There will be no blinding during the recruitment phase. Each hospital is allocated
 to one treatment arm and therefore the CC technique used at each particular site is known to
 the clinical team. The outcome assessor will be unaware of the group allocation. This
 blinding will be maintained until the data is locked for the final analysis and un-blinded.

 Technique of Resuscitation Infants will only be eligible and randomized if they require chest
 compressions. The methods described below also describe resuscitation interventions for
 infants prior to the start of chest compressions.

 Description of general interventions Term infants The initial steps of the resuscitation will
 be according to the current neonatal resuscitation guidelines1. Delayed cord clamping should
 be performed as per local hospital policy (standard hospital practice guideline) at
 participating sites where eligible. Afterwards, dry and stimulate the infant, and open the
 airway. If heart rate is <100/min, the infant is gasping or has apnea, start positive
 pressure ventilation. At the same time consider attaching a pulse oximeter and also consider
 applying electrocardiography. If heart rate remains <100/min, perform MR. SOPA (Mask is
 tightly applied to the face, Re-position the head into the "sniffing" orientation, Suction
 the nares and the pharynx, Open the mouth, Pressure of PPV can be increased to a max of 40 cm
 H2O, Alternate airway (endotracheal tube or Laryngeal Mask Airway). If heart rate is <60/min
 or remains <60 despite all steps of MR. SOPA including alternate airway, start chest
 compressions and increase to 100% oxygen.

 Preterm infants The initial steps of the resuscitation will be according to the current
 neonatal resuscitation guidelines1. Delayed cord clamping should be performed as per local
 hospital policy (standard hospital practice guideline) at participating sites where eligible.
 Immediately afterwards, preterm infants (according to local hospital policy - standard
 hospital practice guideline) will be either placed (without drying) in a polyethylene bag
 under radiant heat44 or dried and placed under radiant heat.

 A pulse oximeter will be used to monitor oxygen saturation immediately after birth and oxygen
 delivery will be guided by published norm values for oxygen saturation in preterm infants45.

 If heart rate is <100/min, the infant is gasping or has apnea, start positive pressure
 ventilation. Also consider applying electrocardiography at that time. If heart rate remains
 <100/min, perform MR. SOPA (Mask is tightly applied to the face, Re-position the head into
 the "sniffing" orientation, Suction the nares and the pharynx, Open the mouth, Pressure of
 PPV can be increased to a max of 40 cm H2O, Alternate airway (endotracheal tube or Laryngeal
 Mask Airway). If heart rate is <60/min or remains <60 despite all steps of MR. SOPA start
 chest compression and increase to 100% oxygen.

 Mask Ventilation The clinical team will determine if an infant requires mask ventilation. If
 mask ventilation is required, it should be provided with a T-Piece device (e.g. Fisher &
 Paykel, Auckland, New Zealand or Giraffe Warmer, GE Health Care, Burnaby, BC, Canada), and
 appropriate face mask with default settings for peak inflation pressure (PIP) of 25 cmH2O, a
 peep expiratory pressure (PEEP) of 5 cm H2O, and a gas flow rate of 8-10L/min using a
 ventilation rate of 40-60 inflations/min. Initial respiratory support can include initial
 sustained inflations (instead of positive pressure ventilation alone) as per local hospital
 policy (standard hospital practice guideline).

 Cardiopulmonary Resuscitation If heart rate is <60/min or remains <60/min despite all steps
 of MR. SOPA start chest compressions. Prior to the start of CC intubation should be
 considered. However, in cases of intubation failure it is possible to start CC during mask
 ventilation. Compressions will be performed using the 2-thumb encircling technique as
 recommended by current resuscitation guidelines46. Epinephrine will be administered according
 to current resuscitation guidelines either via umbilical vein catheter (0.01 mg/kg per dose -
 first line) or via endotracheal tube (0.05 mg/kg to 0.1 mg/kg) or every three to five minutes
 as needed44. Chest compressions and epinephrine will be continued until ROSC.

 Determination of ROSC ROSC will defined as an increase in heart rate >60/min for 60sec
 determined by auscultation of the heart. Return of spontaneous circulation will be assessed
 after 60sec of a heart rate of >60/min.

 The current neonatal resuscitation guidelines do not recommend feedback devices (e.g. exhaled
 carbon dioxide (ECO2) monitors, electrocardiography (ECG), or pulse oximetry for detection of
 ROSC1. A secondary objective of the study will be to examine if either feedback device ECO2,
 ECG or pulse oximetry can be used for detection of ROSC. Participating centres (e.g.
 Edmonton, Graz, Winnipeg, Ulm, San Diego) are using either ECO2 monitors, ECG, and/or pulse
 oximetry routinely during neonatal resuscitation35,47-49.

 Interventions "3:1 C:V group" If the PIP has been increased >30 cm H2O during MR.SOPA, the
 PIP should be decreased to 30 cmH2O prior start of chest compression. Infants randomized into
 the "3:1 C:V group" will receive CC at a rate of 90/min and ventilations at a rate of 30 /min
 in a 3:1 C:V ratio as recommended by the current resuscitation guidelines1 (study flow
 diagram). During CC, the inflations should be delivered using a PIP of 25-30 cmH2O (As per
 local hospital policy /standard hospital practice guideline). Heart rate should be
 reevaluated every 60sec according to current neonatal resuscitation guidelines1.

 "CC+SI group" If the PIP has been increased >30 cm H2O during MR.SOPA, the PIP should be
 decreased to 30 cmH2O prior start of chest compression. Infants randomized into the "CC+SI
 group" will receive a SI with a PIP 25-30 cmH2O while receiving CC. CC will be performed at a
 rate of 90/min. The SI will be delivered over a period of 20 seconds. This will be followed
 by PEEP of 5-8 cm water as per local hospital policy (standard hospital practice guideline)
 for one second. Then the next SI for 20sec is started while CCs are continued. Again after
 20sec there will be a 1sec pause with PEEP, which will be followed by another SI for 20sec
 with continuous CC. After 3x20sec SI+CC (total of 60sec) an assessment of heart rate should
 be performed. If heart rate is >60/min continue with standard care as per local hospital
 policy (standard hospital practice guideline). If heart rate remains <60/min continue with
 CC+SI for another 60sec (3x20sec SI+CC (total of 60sec) at which time a further assessment
 should be performed. If heart rate remains <60/min continue with CC+SI. If CPR is ongoing for
 5 minutes using SI+CC, the clinical team must convert to the standard method of care using
 3:1 C:V ratio.

 Discontinuing Resuscitation Deciding how long resuscitative efforts should continue in any of
 the study infants will be solely at the discretion of the clinical team in accordance with
 the current neonatal resuscitation guidelines1 and per local hospital policy (standard
 hospital practice guideline) at every participating site.

 18-24 months follow-up SI+CC might result in improved pCO2 during CPR. A more favourable pCO2
 might be neuro-protective, and we hypothesize that SI+CC infants will have a better
 neurodevelopmental outcome compared to infants randomized to 3:1 C:V. Most infants who
 receive chest compressions are routinely assessed for neurodevelopmental outcomes at 18-24
 months of age in the outpatient clinic in the study hospital. A Bayley III assessment
 (reference) will be performed by a psychologist or other professional with the relevant
 competence at the study site. This participant data will be collected at the 18-24 month
 follow up visit if this is part of routine care; it is not mandatory to collect.

 Outcome measures Primary outcome Time to achieve ROSC is defined as a heart rate of >60/min
 for 60sec determined by auscultation of the heart. Return of spontaneous circulation will be
 assessed after 60sec of a heart rate of >60/min.

 Secondary outcomes Secondary outcomes amongst others will include neonatal mortality
 (Neonatal death <28 days) and morbidities, for example, rates of brain injury (reported
 either via magnetic resonance imaging (MRI) or head ultrasound), changes in regional cerebral
 oxygen saturation using near-infrared spectroscopy (NIRS) monitoring started in the DR and
 discontinued when clinical stabilization is achieved, results of amplitude-integrated
 electroencephalograpy (aEEG) monitoring until normalization of background patterns and
 occurrence of sleep-wake cycling, DR interventions (including the use of epinephrine),
 admission temperature, use of therapeutic cooling, mechanical ventilation, pneumothorax, use
 of inotropes, infection/sepsis, intraventricular hemorrhages, necrotizing enterocolitis,
 bronchopulmonary dysplasia, retinopathy of prematurity and long-term neurodevelopmental
 outcomes. We will also assess the value of ECO2, ECG and/or SpO2 in determining ROSC as
 described previously.

 Outcome assessment tools Mortality: All causes of mortality will be recorded. Cerebral injury
 will be assessed with either cerebral ultrasound or cerebral MRI if performed prior to
 discharge. If an infant dies prior to any neuro-imaging, a request for autopsy should be
 made. Autopsy could include imaging alone or full autopsy. Morbidity: Case history until
 discharge or term age (for preterm infants). Secondary outcome parameters: Case history and
 output from feedback devices.

 Compliance with the Protocol The clinical investigation will be conducted in compliance with
 this protocol. Modifications to the protocol will not be implemented without agreement from
 the Principal Investigators and relevant ethics committee approval are obtained.

 Investigators are not allowed to deviate from the protocol except as specified above. Any
 major or safety related deviations will be recorded, analyzed and the ethics committees
 notified. If an investigator refuses to comply with the protocol he/she will be disqualified.

 Data collection Infants will be recruited over a period of 26 months. Approximately another
 six months will be required to collect hospital data on all infants enrolled. Resuscitation
 data will be collected on a standard form (Neonatal Resuscitation Record) that will form part
 of each infant's hospital record. The Neonatal Resuscitation Record should be completed by
 the clinical team attending the resuscitation. Other medical data on each infant will be
 collected on an electronic Case Report Form (eCRF). The eCRF will be designed in
 collaboration with Women and Children's Health Research Institute, University of Alberta,
 Edmonton, Canada. Data will be entered into REDCap database from each site within one week
 after discharge or death of an infant. Long-term follow-up will be entered within two weeks
 after examination. All information entered into REDCap database will be used for analysis.

 Data analysis The Clinical Research Informatics Core and Biostatistics Core of the Women and
 Children's Health Research Institute, at the University of Alberta, will do data management
 and analysis. Additional information about the storage, management and analysis of study data
 is available.

 All analyses will be carried out according to the intention-to-treat principle. Statistical
 analyses will be performed using IBM SPSS Statistics Ver. 24 (IBM Corp.) and SAS version 9.4
 (SAS Institute Inc.) or later.

 Interim Analysis Interim safety analysis will be performed at 10%, 25% and 50% of enrolment
 to review the primary outcome, ROSC, and severe adverse events (SAEs).

 Thus, three interim analyses will be performed at 22 patients (11 patients per group), 54 (27
 patients per group), and 110 (55 patients per group) to evaluate the efficacy of the
 intervention. At each interim analysis posterior probability of SI+CC arm to reduce time to
 ROSC by 10% or more compared to the control arm will be calculated. If this probability is
 less than 0.5, the trial will be stopped for futility. If the posterior probability is
 greater than 0.98, the Data and Safety Monitoring Board will consider the trial to be stopped
 for superiority. Since no statistical tests will be performed at interim analyses, type I
 error (alpha) does not need adjustment.

 Final Analysis Primary Analysis The primary outcome is the time to achieve ROSC defined as a
 heart rate of >60/min for 60sec. Data will be analyzed on an intention-to treat basis and
 will include all randomized participants. A per protocol analysis will also be conducted
 using the data from the actual allocation of participating infants. A survival analysis will
 be used to analyze the difference in time to ROSC between intervention and control groups. To
 account for cluster randomization, Cox proportional hazards regression with time to ROSC as
 an outcome and allocation group as an independent variable will be created. Centers will be
 entered as clusters in the model and the statistical significance of the allocation group
 variable will be evaluated. The analysis will be 2-sided and p-value < 0.05 will be
 considered statistically significant.

 Secondary Analysis The data will be presented as mean (standard deviation, SD) for normally
 distributed continuous variables and median (interquartile range, IQR) when the distribution
 is skewed. The clinical characteristics and outcome parameters will be compared using
 Student's t-test for parametric and Mann-Whitney U-test for nonparametric comparisons of
 continuous variables, and χ2 for categorical variables. All p-values will be 2-sided and p
 <0.05 will be considered statistically significant.

 Assessment of safety Adverse and Serious Adverse Events Infants who require CPR in the DR are
 a very seriously ill patient group. Most adverse events may be of a serious nature with or
 without the SURV1VE-trial intervention, and both intervention groups are expected to have a
 very high proportion of serious adverse events (SAEs). Serious adverse events to be recorded
 are therefore mortality within the DR (e.g. did not achieve ROSC or did achieve ROSC but care
 was withdrawn), and within the NICU (any mortality).

 Expected adverse events Adverse events we expect to be related to the application of the
 treatment guideline include: No ROSC leading to death, accidental displacement of the
 endotracheal tube or extubation, accidental displacement of venous or arterial catheters, use
 of Nitric Oxide for pulmonary hypertension, sepsis, pneumothorax, and intraventricular
 hemorrhage (grades 1-4)52.

 Data and Safety Monitoring Board A Data and Safety Monitoring Board (DSMB) will monitor the
 study to: (1) protect all study patients, (2) safeguard the interests of all study patients,
 (3) monitor the overall conduct of the trial, (4) advise the investigators in order to
 protect the integrity of the trial, and (5) supervise the conduct and analysis of all interim
 analyses. To its end the DSMB will receive regular reports from the trial on any injuries or
 adverse events, any developments that jeopardize the continued success of the trial, and data
 by which to accomplish the evaluation of pre-determined early stopping rules. Serious Adverse
 Events to be reported (mortality) will be sent within 72 hours to the DSMB; reports of
 other/less serious adverse events and recruitment will be sent monthly; demographics and
 adverse events (including pneumothorax, and intraventricular hemorrhage grade 3 or higher
 according to Papile52) will be included with the interim and final safety and efficacy
 analyses. The DSMB will perform interim safety analysis at 10%, 25% and 50% of enrolment to
 review the primary outcome of ROSC, and SAEs. At the discretion of the DSMB further interim
 analyses can be requested.

 Members of the DSMB are Myra Wyckoff (current Chair of the International Liaison Committee on
 Resuscitation (ILCOR), Neil Finer (Professor Emeritus, University of California San Diego),
 and Maryna Yaskina (Statistician, Biostatistics Unit, Women and Children's Health Research
 Institute, University of Alberta, Edmonton, Canada).

 Suspension or premature termination of the clinical investigation The sponsor/principal
 investigator and the ethics committees can make decisions about trial discontinuation. If the
 trial is terminated or suspended the parents of all trial participants will be informed and
 appropriate follow-up will be assured. If sponsor/principal investigator terminates or
 suspends the trial the relevant ethics committees will be provided with a detailed written
 explanation of the termination or suspension.

 The sponsor/principal investigator can, upon completion of the analysis of the reason(s) for
 a suspension, decide to lift the suspension when the necessary corrective actions have been
 implemented. The investigators and ethics committees will be notified and provided with the
 relevant data supporting the decision.

 Stopping rules

 The DSMB will review the data at interim analyses at 10%, 25% and 50%. Predefined stopping
 rules will include:

 1. An increased mortality in the SI+CC group by 25% compared to the 3:1 C:V group at the
 predefined interim analysis at 10%, 25% and 50%.

 2. Increase in rate of morbidities including pneumothorax, intraventricular hemorrhage or
 the combination, in the SI+CC group by 25% compared to the 3:1 C:V group at the
 predefined interim analysis at 10%, 25% and 50%.

 3. Bayesian posterior probability of SI+CC group being better than the control is less than
 0.5 or greater than 0.98. (Posterior probability of SI+CC arm to reduce time to ROSC by
 10% or more compared to the control arm will be calculated. If this probability is less
 than 0.5, the trial will be stopped for futility. If the posterior probability is
 greater than 0.98, DSMB will consider the trial to be stopped for superiority. Since no
 statistical tests will be performed at interim analyses, type I error (alpha) does not
 need adjustment).

 Opt-out Rule In any cases were CPR is ongoing for 5 minutes, the clinical team must convert
 to the standard method of care using the 3:1 C:V ratio.

 Ethical Considerations The SURV1VE-trial will be conducted in compliance with the guidelines
 of the Declaration of Helsinki in its latest form, the International Conference on
 Harmonization of Good Clinical Practice Guidelines. In case of modifications in the study
 protocol that are not merely of a formal nature but contain changes pertinent to the study
 participants, a renewed vote of the relevant ethics committees will be obtained. If
 applicable, the patients (parents) will be informed in the patient information and consent
 form about changes in the terms and conditions of the trial. The SURV1VE-trial will only
 start the randomization of participants after approvals from the relevant ethics committees
 have been obtained.

 Data management Data handling and archiving Source data will be registered in the
 participant's medical records/CRF and into the eCRF. A common web-based eCRF will be devised
 to enable a central database (Women and Children's Health Research Institute, University of
 Alberta, Edmonton, Canada). Data entry into the central database and handling of medical
 records is the responsibility of the investigators. After the establishment of a 'clean
 file', the database will be locked; The data will be locked after completion of patient
 recruitment and data entry. After long-term follow-up data entry this portion of the database
 will locked. Data will be stored for statistical analysis at the Biostatistics Unit, Women
 and Children's Health Research Institute, University of Alberta, Edmonton, Canada. The trial
 database will hereafter be kept according to the respective national laws. After the end of
 trial, the data will be archived for 25 years according to good clinical practice guidelines.
 At each trial-site the data flow will be monitored according to the GCP principles by a
 locally appointed external monitoring committee. After completion of statistical data
 analysis, data will be pseudo-anonymized and stored at the University of Alberta, Edmonton,
 Canada.

 Data protection The investigator(s) permits trial-related monitoring, audits, and regulatory
 inspection(s) by providing direct access to the source data and other relevant documents.
 Trial data will be handled according to regulations of the data protection agency in the
 respective countries.

 Quality assurance The trial will be carried out in accordance with the Declaration of
 Helsinki in its latest form and the International Conference on Harmonization Good Clinical
 Practice (ICH-GCP) guidelines.

 Monitoring The chief investigator consents to data evaluation being performed by the person
 in charge of monitoring in accordance with the monitoring plan, in order to ensure
 satisfactory data collection and adherence to the study protocol. Furthermore, the chief
 investigator states that he/she is willing to cooperate with this person and shall provide
 this person with all required information whenever necessary. This includes access to all
 documents related to the trial, including study-relevant medical files of patients in
 original form. The tasks of the investigator include maintenance of these patients' medical
 files as comprehensively as possible; this includes information concerning medical history,
 accompanying diseases, inclusion in the trial, data about visits, results of investigations,
 dispensing of medication, and adverse events. The monitor will also be permitted to perform
 data evaluation and draw comparisons with the relevant medical files in accordance with the
 standard operating procedures and ICH-GCP guidelines at predetermined intervals, in order to
 ensure adherence to the study protocol and continuous registration of data. All original
 medical reports required as sources for the information given in the CRF or the database
 shall be inspected. The study participants will have given their consent to such inspection
 by signing the consent form. The person in charge of monitoring is obliged to treat all
 information as confidential and to preserve the basic claims of the study participants in
 respect of integrity and protection of their privacy.
Inclusion Criteria:

 -  Infants (term or preterm infants >28 weeks' gestation) requiring CC in the delivery
 room will be eligible for the trial.

 Exclusion Criteria:

 -  Infants will be excluded if they have a congenital abnormality or condition that might
 have an adverse effect on breathing or ventilation (e.g. congenital diaphragmatic
 hernia), or congenital heart disease requiring intervention in the neonatal period.
 Infants would be also excluded if their parents refused to give consent to this study.