Methodologies

1 Study Design:

This study is conducted in the Department of Neonatology across multiple tertiary-referral

The study focuses on Infants under 28 days (neonates) of age has suspected sepsis who were
treated with intravenous antibiotic therapy . Blood cultures were obtained to confirm the
presence of infection, allowing for an accurate assessment of pathogen profiles and
treatment outcomes.

Total sample size of approximately 3,200 infants across all sites upto 200 infants per hospital
site were enrolled across on all sites . The inclusion criteria has hospitalised infants under 60
days of age with a new episode of clinically suspected sepsis and were enrolled between
2018 and 2020 and included 19 hospital sites across 11 countries in Asia (Bangladesh, China,
India, Thailand, Vietnam), Africa (Kenya, South Africa, Uganda), Europe (Italy, Greece) and
South America (Brazil).

The sites were selected to ensure the diversity in geographical region and type of health
facility including secondary and tertiary referral hospitals, public health facilities, and
hospitals with varying proportions of in-born and out-born neonates . All participating
facilities had access to microbiology services , which was essential for accurate pathogen
identification and outcome monitoring.
Infants were eligible for inclusion if a treating physician had initiated antibiotic therapy for a
new suspected sepsis episode, and a blood culture was obtained. A minimum of 2 clinical
signs of sepsis or 1 clinical sign combined with 1 indicating laboratory sign of sepsis was
required for eligibility. Laboratory values is not mandatory in all settings to ensure
generalizability across facilities with different diagnostic capacities.
Controls: The study did not include a separate control group. It only focused exclusively on a
cohort of infants with sepsis to analyze patterns of antibiotics use , pathogen distribution
and clinical outcomes .
Data Collection: Data collection was done by using standardized protocols at all participating
centres . Information that were collected includes demographic details, clinical
presentation, laboratory results, antibiotic usage patterns, identified pathogens, and
outcomes.
Enrolment per site was capped at approximately 200 infants, adapting to local case volume
and hospital capacity. This sampling frame allowed for manageable recruitment while
ensuring enough representation per site.
Exclusion criteria included infants for whom a non-infectious diagnosis was considered more
likely, or those with serious non-infective comorbidities expected to cause death within 72
hours. Infants who had previously received antibiotics were eligible if they were starting a
new regimen for a distinct sepsis episode, provided a blood culture was obtained. Sepsis
episodes occurring more than 48 hours after hospital admission were classified as hospital-
acquired infections (HAI).
Participants were followed up for 28 days after enrolment, whether they remain
hospitalized or were discharge (by telephone follow up s conducted if necessary). Baseline
demographics, clinical presentation, microbiological results, antibiotic regimens and
outcomes (including mortality, duration of IV antibiotics, clinical deterioration) were
collected.
Ethical approval was obtained from relevant institutional review boards or ethics
committees at each participating centre. Informed consent was obtained from parents or
guardians of the enrolled infants ensuring adherence to ethical standards in research
involving vulnerable populations.
2 Sample Size
The study was conducted in neonatal intensive care units (NICUs) and paediatric wards
across 12 countries, including India, Bangladesh, Kenya, and Ethiopia. The study consisted of
neonates and young infants aged 0–59 days who were admitted with suspected or
confirmed sepsis. The inclusion criteria encompassed both term and preterm infants,
without any restrictions based on sex, birth weight, or underlying conditions. Exclusion
criteria involved infants with congenital anomalies incompatible with life and those whose
parents declined participation.
Sample Size and Power Calculation
The sample size and power calculations were designed to achieve sufficient statistical power
to identify meaningful associations between antibiotic use, pathogen profiles, and mortality
outcomes. While the exact number of participants was not specified in the provided
information, the multi centre nature of the study and the diverse geographical
representation suggest a substantial sample size, enhancing the generalisability of the
findings.
Operational Definitions
Key operational definitions were employed across all sites to ensure consistency in the
study which included:
 Sepsis: Defined according to the International Paediatric Sepsis Consensus
Conference criteria, including the presence of clinical signs of infection and systemic
inflammatory response syndrome (SIRS) in the presence of a suspected or confirmed
infection.
 Antibiotic Use: Categorised based on the type of antibiotic administered, the time of
initiation (empiric vs. targeted), and adherence to local or international guidelines.
 Pathogen Identification: Utilised blood cultures and other relevant microbiological
tests to identify causative organisms, with results classified according to standard
microbiological nomenclature.
 Mortality Outcome: Recorded as in-hospital death or survival, with stratification
based on time to death following admission.
Flowchart of Study Design
The study’s design can be visualised through a flowchart illustrating sequential stages:
screening, eligibility assessment, enrolment, data collection, follow-up, and analysis.
1. Screening: Neonates and young infants were identified upon admission to
participating NICUs and paediatric wards.
2. Eligibility Assessment: It is conducted to verify inclusion and exclusion criteria
ensuring only suitable participants were considered.
 3. Enrolment: Obtaining informed consent from parents or guardians before officially
including participants in the study .
4. Data Collection: Detailed demographic, clinical, microbiological and treatment
information was recorded systematically throughout the period of hospitalisation.
5. Follow-up: Participants were monitored until either discharge or in-hospital death,
with careful documentation of any adverse events or complications.
6. Analysis: The gathered information underwent analysis , where data was complied
and statistically examined to identify key patterns and predictors of outcomes.
This flowchart provides a clear visual and methodological pathway ,, ensuring that the
research remains transparent , reliable and reproducible in the research process.
3 Implementation Plan
1. Study Design and Methodology
The NeoOBS study was designed as a prospective, observational cohort study, carried out
across multiple neonatal units worldwide. By including sites from Africa, Asia, and Latin
America, the researchers were able to capture a wide spectrum of clinical practices, pathogen
profiles, and outcomes related to neonatal sepsis, offering a truly global perspective.
2. Data Collection and Management
Data collection and management were carefully structured. Standardised case report forms
were used to gather detailed information on patient demographics, clinical presentation,
microbiological results, antibiotic use, and treatment outcomes. To ensure consistency, local
investigators received rigorous training, while regular monitoring visits helped maintain data
quality. A centralised data management team oversaw data entry, validation, and analysis,
ensuring reliability across all sites.
3. Ethical Considerations and Approvals
Approvals were obtained from institutional review boards in each participating country, and
written informed consent was secured from the parents or guardians of all enrolled neonates.
This careful process followed established guidelines for research involving vulnerable
populations.
4. Stakeholder Engagement and Collaboration
Partnerships were established with academic institutions, healthcare providers, and
international health organisations. Regular meetings and open communication channels
helped maintain coordination and ensured that challenges were addressed quickly and
effectively.
5. Monitoring and Evaluation
To maintain scientific and ethical integrity, robust monitoring and evaluation processes were
put in place. Continuous monitoring mechanisms tracked the progress of the study, while data
safety and monitoring boards oversaw adherence to ethical standards. Periodic evaluations
allowed the team to identify trends early and apply corrective measures when necessary.
[Link] of Findings
The findings of the NeoOBS study were widely shared through peer-reviewed journals,
international conferences, and reports to participating institutions and stakeholders. This
ensured that the knowledge generated contributed not only to advancing global understanding
of neonatal sepsis but also to shaping future research and policy directions.
7. Sustainability and Future Directions
Efforts are focused on improving antibiotic stewardship, enhancing diagnostic capacity, and
ultimately reducing neonatal sepsis mortality. In addition, there is a strong commitment to
building capacity within local healthcare systems and fostering long-term collaborations to
address ongoing and emerging challenges in neonatal health.
4 Ethics Review
Informed Consent Process
Before enrolling neonates and young infants into the study, written informed consent was
obtained from their parents or legal guardians. Trained study staff carefully explained the
purpose of the research, the procedures involved, and any potential risks or benefits. Parents
or guardians were assured that participation was entirely voluntary and that they could
withdraw their child from the study at any time without it affecting the infant’s medical care.
Ethical Approvals
The study was reviewed and approved by multiple recognised ethics committees and
institutional review boards before it began, ensuring that all research activities were
conducted in accordance with international ethical standards.
 The Kilimanjaro Christian Medical University College of Tumaini University Health
Research Ethics Committee (Certificate number: 2577)
 The Tanzania National Institute for Medical Research National Health Research Ethics
Coordinating Committee (NIMR/HQ/R.8a/Vol. IX/4085)
 An Institutional Review Board of the Duke University Health System (Pro00109919)
Participant Safety and Risk Management
The study was carefully designed to prioritise the safety of all participants. Blood samples
were collected using sterile techniques, and the volume drawn was strictly based on the
infant’s weight, with a minimum of 2 mL required for culture. This approach reduced the risk
of excessive blood withdrawal. In addition, participants’ vital signs and overall clinical
condition were closely monitored at regular intervals, allowing the study team to quickly
identify and manage any adverse events.
Benefits to Participants
Although the main purpose of the study was to generate data to inform global health
strategies, participants could also experience direct benefits. Early detection and treatment of
infections were possible through close monitoring, and in the long term, the study’s findings
are expected to improve clinical practices and health outcomes for neonates and young
infants worldwide.
The study followed strict ethical standards throughout—ensuring that informed consent was
obtained transparently, participant safety was prioritised at every step, and the potential
benefits were clearly explained to parents and guardians.
5 Data Collection and statistical analysis plan
The data collection strategy was designed to provide a detailed overview of the study
population, covering aspects such as patient characteristics, antibiotic usage patterns,
pathogen distribution, and mortality rates.
Key Variables and Measurement
The study focused on a set of well-defined key variables, which included:
 Antibiotic Use: Recorded as by type, duration, and adequacy of antibiotic therapy
administered to neonates.
 Pathogens: Identified through blood cultures, with specific antimicrobial resistance
patterns.
 Mortality: Recorded as a binary outcome (alive or deceased) during hospitalisation.
Data Collection Methods and Tools
Data were collected at all study sites using standardised case report forms (CRFs). These
forms recorded essential details such as patient demographics, clinical signs, laboratory
findings, prescribed antibiotics, and treatment outcomes. To maintain accuracy and
consistency, all information was entered into a central electronic database, where it was
carefully managed and monitored.
Data Entry and Analytical Platforms
The collected data were managed using REDCap (Research Electronic Data Capture), a
secure web-based application designed for data collection and management. Statistical
analyses were conducted using R software, version 4.4.1, which facilitated comprehensive
data analysis.
Descriptive Data Analysis Plan
Descriptive statistics were employed to summarise the data effectively.
Continuous Variables weee expressed as medians with interquartile ranges (IQRs) or means
with 95% confidence intervals (CIs), depending on the distribution of the data.
Categorical Variable were presented as frequencies and percentages which provides a clear
overview of the study population and outcomes.