Selective Antibiotics When Symptoms Develop Versus Universal Antibiotics for Preterm Neonates At-risk of Early-onset Bacterial Sepsis: a Multicentric, Randomized, Controlled, Non-inferiority Trial (the SAUNA Trial)
Phase: PHASE3
Conditions: Sepsis, PROM, Preterm (Pregnancy), Early-Onset Neonatal Sepsis, Preterm Premature Rupture of Membrane, Preterm Birth
Sponsor: Indian Council of Medical Research

 
Selective Antibiotics when symptoms 
develop versus Universal antibiotics for 
preterm Neonates At-risk of early-onset 
bacterial sepsis: a multicentric, randomized, 
controlled, non-inferiority trial (the SAUNA 
trial) 
 
 
 
 
 
 
 
 
NCT number. To be allotted 
Date: 2nd April 2024 
 
 
 

PROJECT PROTOCOL 
 
Title of the project:  
Selective Antibiotics when symptoms develop versus Universal antibiotics for preterm Neonates At-
risk of early-onset bacterial sepsis: a multicentric, randomized, controlled, non-inferiority trial (the 
SAUNA trial) 
 
Objectives 
• To determine whether antibiotics administered selectively to at-risk preterm neonates 
[<35 weeks gestation with prolonged rupture of membranes (PROM) or preterm 
premature rupture of membranes (pPROM)] when they develop signs of sepsis compared 
to administering antibiotics from birth to all at-risk neonates is non-inferior with respect 
to the primary outcome of “mortality or any episode of culture-positive sepsis or severe 
sepsis” in the 1st week of life 
• To determine whether neonates receiving selective antibiotics (as above) compared to 
those receiving antibiotics from birth (as above) are superior with respect to the co-
primary outcome of fewer antibiotic courses of 48 hours duration or more in the 1st week 
of life 
• To determine whether neonates receiving selective antibiotics (as above) compared to 
those receiving antibiotics from birth (as above) are significantly different with respect to 
a wide range of secondary outcomes (listed under “Outcomes”) 
 
Summary of the proposed research project: 
Background:  
 
Sepsis is the major cause of neonatal mortality and early-onset neonatal sepsis (EONS) accounts 
for more than two-thirds of all cases of neonatal sepsis. Prolonged rupture of membranes (PROM) 
and preterm premature rupture of membranes (pPROM) are important risk factors of EONS. There 
is equipoise in the published literature whether antibiotics must be immediately initiated among 
all preterm neonates (<35 weeks gestation) delivered following PROM or pPROM who are 
asymptomatic at birth or whether antibiotics can be selectively administered if and when the at-
risk neonates become symptomatic.  
Research question 
Among neonates <35 weeks gestation born with PROM >18 hours or pPROM and who are either 
asymptomatic or have no symptoms of sepsis at 4 hrs postnatally (P), is selectively administering 
antibiotics to neonates who later develop clinical sepsis [I] compared to administering antibiotics 
pre-emptively to all at-risk neonates [C] non-inferior with respect to the composite outcome of 
“mortality and/or culture-positive sepsis and/or severe sepsis” [O] within 7 days after enrolment 
[T] by an absolute margin of 7% [E] in a randomized controlled trial (S)? 
The trial will also have a superiority outcome: “need for antibiotic treatment lasting greater than 
48 hours within 7 days after enrolment”. The absolute superiority margin will be 50%. 
  
Methodology 
 
 
At-risk inborn infants <35 weeks gestation as above will be enrolled in the study at 4 hours 
after obtaining parental consent. Eligible neonates will be randomly allocated to (a) receive further 
antibiotics only if they develop symptoms of sepsis [criteria defined a priori] or (b) start receiving 
antibiotics immediately. They will be monitored closely for the primary outcomes until 7 days after 
birth and followed up until discharge or day 30 of life.         
Proposed outcome. 

 
Primary outcome (non-inferiority): Composite of “mortality or blood culture proven sepsis or 
episode of severe sepsis” in the 1st 7 days. Co-primary outcome: Need for antibiotic treatment for 
greater than 48 hours in the 1st 7 days. 
 
Detailed research plan: 
Methodology 
Study design: multi-centric, 2-armed, randomized, active-controlled, stratified, block randomized, 
non-inferiority trial with outcome assessment blinded. Before the start of the study, all centres will 
share the empiric obstetric antibiotic policy for mothers delivering with PROM or pPROM and empiric 
neonatal antibiotic policy for suspected EONS followed in their centres and the basis for the policy. 
Centres will be permitted to follow their own written down policies as these are likely to be based on 
local antibiotic sensitivity patterns. 
Screening criteria: All mothers and newly born infants will be screened if delivery occurs at a 
gestational age of 260/7 to 346/7 weeks with prolonged rupture of membranes >18 hours or pre-labour 
rupture of membranes. Placental and umbilical cord histopathology, high vaginal swab culture and 
amniotic fluid culture (by amniocentesis) are not standards of care in all the participating centres, but 
if any of these tests is performed, its result will be considered. For neonates requiring any form of 
respiratory support, a chest x-ray will be obtained prior to 4 hours after birth.  
 Inclusion criteria:  
• 
Gestational age of 260/7 to 346/7 weeks 
●     Chronological age 4 hours  
●     Have any one or both of the following risk factors of EONS: 
■       Prolonged rupture of membranes >18 hours 
■       Pre-labour rupture of membranes [as all subjects will be preterm, this is effectively 
pPROM] 
●     Are either asymptomatic or have no signs attributable to sepsis at 4 hours. This will be defined 
as absence of the following clinical signs or need for interventions mentioned below: 
1. Apnea (Standard definition) requiring intervention at any time until enrolment. 
2. Need for a fluid bolus or inotropic support at any time until enrolment. 
3. Seizures or seizure-like activity at any time until enrolment. 
4. Upper GI bleed in the absence of a history of ante-partum hemorrhage at any time 
until enrolment. 
5. Pus from any site at any time until enrolment. 
6. Need for CPAP >6 cms of water with FiO2 >35% at 6-8 hours OR need for CPAP £6 
cms and FiO2 £35% but with increasing requirement of support** 
7. Chest Xray (if performed) with radiological features of pneumonia. 
8. Need for intubation and mechanical ventilation. 
9. Temperature >37.5°C or <36°C, unexplained by environmental causes 
10. Feed intolerance [bilious or bloodstained vomiting (or gastric residuals) or visibly 
distended abdomen or >50% of the previous feed volume as gastric residuals] 
11. Lethargy or unarousability 
12. Sclerema 
 
Exclusion criteria 
  
Subjects will be excluded if they have any 1 of the following: 
1. Life-threatening congenital malformation 
2. Severe perinatal asphyxia (Apgar score <5 at 10 minutes or cord pH <7.0) 
3. Clinical chorioamnionitis# [see definition below] 
4. Foul-smelling liquor 

5. Multiple gestation 
6. Received a dose of antibiotics 
7. Positive amniotic fluid culture (if performed and available prior to randomization) 
8. Treating neonatologist unwilling to enroll the patient in the trial on the grounds 
that the patient needs antibiotics. 
 
#Fever: (maternal temperature 38.0°C) PLUS any 2 of the following: (a) maternal tachycardia (HR 
>100 bpm), (b) baseline fetal tachycardia [FHR >160 bpm for 10 minutes or longer, excluding 
accelerations, decelerations and periods of marked variability; OR an FHR >160 bpm during and after 
at least 3 consecutive contractions], (c) purulent fluid from the cervical os, (d) uterine tenderness, (e) 
maternal WBC count 15,000/microlitre in the absence of corticosteroids. 
 
Informed consent and enrolment 
Parents of eligible neonates would be approached for possible participation in the study. A parent 
information sheet would be provided to them and they would be given the opportunity to ask 
questions and clear their doubts. Neonates will be enrolled after obtaining written informed consent. 
The research nurses and project scientist will be responsible for enrolling and assigning participants 
to interventions. 
 
Baseline characteristics: 
The following baseline characteristics will be recorded: 
Maternal: age, educational status, parity, duration of rupture of membranes, duration of labour, 
spontaneous premature onset of labour, placental or umbilical cord inflammation on pathology (if 
tested), high vaginal swab or placental or amniotic  
fluid 
culture 
(If 
tested), 
Intrapartum 
antibiotics if administered (names, duration before delivery), mode of delivery, duration of labour (if 
any) prior to Cesarean section.  
Neonatal: gestational age, birth weight, sex, Apgar score at 5 minutes, age at enrolment, clinical signs 
and laboratory parameters, blood culture (at baseline sent for all eligible neonates).  
 
 Randomisation: 
Neonates will be randomized to 1 of the following groups: 
1. Group 1: Intervention group (Selective antibiotic group) 
2. Group 2: Comparison group (Universal antibiotic group) 
A stratified, blocked randomization scheme with 1:1 allocation ratio will be adopted. Stratification will 
be performed for gestational age (280/7-316/7 weeks and 320/7-346/7 weeks), and for centre. Randomly 
varying, permuted, even-numbered blocks (Sizes 4, 6, 8) will be used. The randomization sequence 
will be generated from the website www.randomization.org. The statistician at the nodal center will 
generate the randomization sequence and will not be involved in patient recruitment. 
 
Intervention groups: 
In both groups 1 and 2, the choice of antibiotics will be as per the empirical antibiotic policy of the 
centre. The storage, dose, reconstitution, and mode of administration of antibiotics will be as per 
Neofax (Micromedex, June 2020) to make the processes uniform across centres. The duration of 
antibiotics will be as follows: 
 
 
 
 

Intervention arm 
Control arm 
Antibiotics 
immediately 
after 
enrolment and randomization 
• No antibiotics started. 
• Start administering antibiotics. 
Neonate 
remains 
asymptomatic 
over one week of observation, AND 
placental/cord histopathology (if 
sent) 
not 
suggestive 
of 
chorioamnionitis and/or amniotic 
fluid culture (if sent) sterile 
• If 
pre-randomization 
blood 
culture reported positive with 
non-contaminant 
organism*, 
start antibiotics according to 
sensitivity report for 7-14 days. 
•  If pre-randomization blood 
culture is sterile, no antibiotics 
• If 
baseline 
blood 
culture 
reported positive with non-
contaminant 
organism*, 
administer 
antibiotics 
(or 
change antibiotics, if required 
according to sensitivity report) 
for 7-14 days. 
● If 
pre-randomization 
blood 
culture 
sterile, 
stop 
ongoing 
antibiotics. 
Neonate 
remains 
asymptomatic 
over one week of observation, BUT 
amniotic fluid culture positive (if 
sent), 
and/or 
placental 
histopathology (if sent) suggestive 
of chorioamnionitis 
• If 
pre-randomization 
blood 
culture reported positive with 
non-contaminant 
organism*, 
start antibiotics according to 
sensitivity report for 7-14 days. 
• If 
pre-randomization 
blood 
culture is sterile, administer 
antibiotics for 5-7 days. 
• If 
baseline 
blood 
culture 
reported positive with non-
contaminant 
organism*, 
administer 
antibiotics 
(or 
change antibiotics, if required 
according to sensitivity report) 
for 7-14 days. 
• If 
pre-randomization 
blood 
culture 
sterile, 
continue 
ongoing antibiotics for 5-7 
days. 
Neonate becomes symptomatic** 
for sepsis within 1st week of life 
  
• Start antibiotics.  
• If 
pre-randomization 
blood 
culture is sterile, send fresh 
blood culture for symptomatic 
episodes. If that is also sterile, 
administer antibiotics for 5-7 
days. If blood culture for 
symptomatic 
episode 
is 
positive, treat accordingly for 
7-14 days. 
• If 
pre-randomization 
blood 
culture 
positive 
report 
available around the time 
neonate 
becomes 
symptomatic, treat accordingly 
for 7-14 days. 
• If 
pre-randomization 
blood 
culture is sterile, send fresh 
blood culture for symptomatic 
episodes. If that is also sterile, 
continue 
antibiotics 
for 
a 
cumulative duration of 5-7 
days. If blood culture for 
symptomatic 
episodes 
is 
positive, treat for 7-14 days. 
• If 
pre-randomization 
blood 
culture positive report available 
around 
the 
time 
neonate 
becomes symptomatic, treat 
accordingly for 7-14 days. 
 
 
* Contaminant defined as onset of growth beyond 72 hours or growth of aerobic spore bearers or 
Propionibacterium spp or Corynebacterium spp or diphtheroids or CONS in asymptomatic neonate 

** “Symptoms of sepsis” in the 1st week of life that merit antibiotics will be clearly defined. A standard 
operating procedure (SOP) with clusters of clinical signs and the threshold above which they will be 
considered clinical signs of sepsis, will be made after evolving a consensus across all the participating 
centres. The research staff will closely monitor the administration of antibiotics and will ensure 
adherence to the protocols. 
Concealment of allocation: 
Allocation will be concealed using serially numbered opaque sealed envelopes. Envelopes will bear 
slips of paper mentioning the group of allocation. Investigators will open the envelopes only after the 
name of the enrolled patient has been written outside the envelope. 
 
Blinding: 
Project staff, nurses and resident doctors looking after the neonate will not be blinded. The 
assessment of the primary outcome will be performed by a blinded adjudicator, who is not involved 
in the recruitment and monitoring of subjects. A part of the case report form (CRF) containing relevant 
details of all episodes of sickness in the 1st week of life will be detached from the main form and will 
be sent to the blinded adjudicator. This part will be linked to the main form only by a unique 
identification number. No patient identifiers or allocation group will be mentioned on the part sent to 
the blinded adjudicator.  
 
Monitoring: 
All enrolled subjects will be monitored for the following until discharge or day 30 of life, whichever 
is later: 
a. Clinical signs of sepsis including date of onset: As mentioned earlier, a standard operating procedure 
(SOP) with clusters of clinical signs and the threshold for designating clinical signs of sepsis, will be 
made after evolving a consensus across all the participating centres. 
b. In case the subject develops clinical signs of sepsis during the 1st week of life, the following 
investigations will be mandatorily performed:  
• complete blood counts (CBC) [including total leukocyte count (TLC), absolute 
neutrophil count (ANC)],  
• C-reactive protein (CRP),  
• Procalcitonin (if facilities available),  
• Chest x-ray (if respiratory signs present),  
• blood culture and  
• cerebro-spinal fluid (CSF) examination (including, but not limited to, TLC, ANC, 
glucose, protein, Gram stain and culture).  
The results of these investigations will be recorded. Age-appropriate nomograms of CBC, CRP and 
Procalcitonin will be used to identify abnormal values. Investigations for clinical signs beyond the 
1st week until discharge will be left to the discretion of the treating team. 
c.    If antibiotics are started at any time during hospital stay, the name of antibiotics, start and end 
date will be recorded for each course of antibiotics. 
d.    If antibiotics are changed at any time during hospital stay, name of antibiotics, start and end 
date will be recorded for each course of antibiotics. 
e.    Need for the following during any episode of clinical sepsis during hospital stay: 
• Fluid boluses 
• Inotropes 
• CPAP 
• Mechanical ventilation 
• Blood product transfusion 
• Exchange transfusion 

f.     Duration of hospital stay 
g.    Mortality [if so, date and whether cause of death ascribed to sepsis] 
An asymptomatic neonate who is otherwise fit to be discharged home, can be discharged, with 
instructions to report back to the research team in case of any symptoms. The research team will 
make a follow-up telephonic call on day 7 of life, and weekly thereafter until day 30 of life.  
 
Co-Interventions 
Co-interventions after randomization until discharge will be recorded. An SOP of management of all 
common neonatal conditions will be evolved as a consensus between the participating centres before 
the start of the trial. This will be based upon the NNF clinical practice guidelines and the ICMR standard 
treatment workflows. There are no specific co-interventions permitted or prohibited during the trial.  
Outcomes 
Primary (Non-inferiority outcome) 
Composite of all-cause mortality and/or any episode of culture-positive sepsis and/or severe sepsis* 
within the 1st 7 days after randomization 
*Severe sepsis will be defined as: 
• Clinical signs of sepsis AND 
• Either a positive blood culture or laboratory evidence of sepsis (either CRP OR 
Procalcitonin above the age-appropriate cut-off value OR any two of the CBC 
parameters outside the age-appropriate ranges OR chest x-ray suggestive of 
pneumonia) AND 
• One or more of the following indices of severity: 
o Need for intubation and mechanical ventilation 
o Need for inotropes >10 mic/kg/min dopamine or >10 mic/kg/min dobutamine 
or adrenaline > 0.05 mic/kg/min 
o Need for exchange transfusion 
o Need for platelet concentrates or FFP 
o Meningitis (defined as either CSF culture positive or Gram stain positive or Cell 
count >25/microlitre or glucose <25 mg/dl or protein >180 mg/dl) 
Co-primary outcome 
The co-primary outcome will be need for intravenous antibiotics for ≥ 48 hours within the 1st 7 days 
of life. 
 
Secondary outcomes 
• Individual components of the composite outcome 
o All-cause Mortality within 1st 7 days of life 
o Blood culture positive sepsis of any severity within 1st 7 days of life 
o Episode of severe sepsis within 1st 7 days of life 
• Outcomes within the 1st 72 hours 
o Composite of mortality/blood culture positive sepsis/severe sepsis 
o individual components of composite outcome within 1st 72 hours 
• Outcomes during hospital stay 
o Composite of mortality/blood culture positive sepsis/severe sepsis 
o individual components of composite outcome during hospital stay 
o Necrotizing enterocolitis, stage II-III by modified Bell’s staging criteria 
• Outcomes during 1st 30 days of life 
o Composite of mortality/blood culture positive sepsis/severe sepsis 
o Individual components of composite outcome during 1st 30 days 

o Necrotizing enterocolitis, stage II-III by modified Bell’s staging criteria 
• Cause-specific mortality 
o Sepsis-related mortality within 1st 72 hours of life, 7 days, during hospital 
stay and during 1st 30 days of life 
• Episodes of clinical EONS (as per definition from a repertoire of clinical signs with 
normal lab parameters) 
o clinical sepsis within 1st 72 hours of life, 7 days, during hospital stay and 
during 1st 30 days of life 
• Episode of Probable EONS [as per definition from a repertoire of clinical signs, AND 
with abnormal age-appropriate values of one or more of TLC, ANC, CRP, PCT, chest 
x-ray suggestive of pneumonia AND with sterile blood culture] 
• Episode of asymptomatic proven EONS within 72 hours of life [asymptomatic but 
baseline blood culture positive with non-contaminant organism] 
• Need for sepsis workup [“Sepsis workup” defined as one or more of CBC, CRP, 
Procalcitonin, blood culture] during 1st 72 hours of life, during 1st 7 days of life, 
during hospital stay and during 1st 30 days of life 
• Need for antibiotics during hospital stay 
o Cumulative duration of antibiotic therapy during 1st 7 days of life 
o Cumulative duration of antibiotic therapy during 1st 72 hrs 
o Cumulative duration of antibiotic therapy during hospital stay 
• Duration of hospitalization 
• Episodes of healthcare associated infection during hospital stay [Defined as any 
episode of culture positive sepsis with onset after 72 hours of life or any episode of 
culture-positive sepsis if baseline blood culture was sterile] 
• Adverse effects (AE): An AE log will be maintained for all participants in the RCT. 
The log includes start and end date, severity, outcome, and action taken. A detailed 
AE chart with definitions of the AE and criteria for classifying the level of severity 
has been made. These have been adapted from the Common Terminology Criteria 
for Adverse Events (CTCAE) version 5. 
SAEs will be reported to and independent safety monitoring committee appointed 
by the institutional ethics committee and to the DSMB. Adverse effects attributed 
to antibiotic therapy [WHO-UMC scale will be used for estimating causality]. 
 
 
Sample size calculation 
An estimate of the primary outcome was made from the results of the DeNIS study (14). If there is 
truly no difference in the primary outcome between the intervention and standard treatment groups 
(20% in both groups), then 1374 patients are required to have 90% power that the upper limit of a 
one-sided 97.5% confidence interval will exclude a difference in favour of the standard treatment by 
a noninferiority margin of more than 7%. Accounting for losses to follow up, approximately 1500 
patients will be required, with 750 in each arm. 
Raw data from the DeNIS study shows that 1.6 % of all live births would be available for enrolling in 
the study. Thus, to get 1500 patients, we would have to screen 92,000 live births. If any given centre 
has approximately 5000 live births per annum, assuming that at least 6 months in the beginning and 
at the end are excluded for the run-in period of the trial and data analysis and writing respectively, 
then to get 92,000 live births available for screening over an effective recruitment period of 3.5 years 
in a 4-year study, we will need 9 centres. 

 
Data Safety Monitoring Board (DSMB) and Interim analysis 
A DSMB will be constituted, which will be independent of the funding agency and the trial centres and 
will have no competing interests. The DSMB charter will be drawn up after the constitution. The DSMB 
will monitor serious adverse events and data accrued in the trial. 2 interim analyses will be performed. 
The first interim analysis after 33% of the planned sample size has been enrolled or 33% of the 
anticipated primary non-inferiority outcome events have occurred, whichever is earlier. The 2nd 
interim analysis after 66% of the planned sample size has been enrolled or 66% of the anticipated 
outcome events have occurred, whichever is earlier. Sample size would be recalculated at the 1st 
interim analysis. After any of the interim analyses, the DSMB can advise that the trial should be 
stopped for unequivocal benefit (noninferiority demonstrated), harm or futility. O’Brien Fleming 
stopping criteria will be used for the primary outcome. Pocock’s stopping criteria will be used for harm. 
 
Statistical analysis plan 
Categorical variables will be described as frequencies and percentages, normally distributed 
numerical variables as mean (standard deviation) and variables with skewed distributions as 
median (1st, 3rd quartile). Dichotomous variables will be compared between the 2 groups by 
chi-square test and Fisher’s exact test as appropriate. Normally distributed numerical 
variables will be compared by unpaired student’s t-test. Variables with skewed distributions 
will be compared by Mann-Whitney U test. Magnitude of the effect size will be expressed as 
unadjusted odds ratios (95% confidence interval). Missing data will be replaced by a process 
of multiple imputation.  
As it is a noninferiority trial, the primary analysis will be as per protocol and the secondary 
analysis will be as per intention-to-treat.  
 Multi-variable logistic regression analyses will be performed with the primary outcome as the 
outcome variables, and the group of randomization as the primary predictor variable, adjusted 
for the following baseline covariates decided a priori: gestation strata, neonate received no 
doses of antibiotics versus received one dose of antibiotics prior to enrolment, center, 
duration of maternal intrapartum antibiotic prophylaxis (0-4 h vs >=4 h), was completely 
asymptomatic until enrolment versus had any transient symptoms prior to enrolment, was on 
CPAP at enrolment versus no CPAP at enrolment, Apgar score (<7 at 5 min vs. >=7), HVS culture 
positive for pathogenic bacteria or not, placental histology suggestive of infection or not. 
Variables will be checked for multicollinearity using Eigenvalues and Variance Inflation Factor. 
Variables that are not multicollinear will be forced into the model. No software-based variable 
selection algorithm will be used.  
Subgroup analyses will also be performed for all the covariates mentioned above under 
multivariable logistic regression analysis.