Study procedures and sample collection

Midwives, who were hired specifically for this trial, were based in labour and delivery complex of CHBAH throughout the year for the duration of the study. These

research midwives conducted trial procedures and collected samples and data.

Colonisation swab

A colonisation sub-study was included in the main PoPS trial to determine maternal vaginal colonisation rates with bacteria known to be pathogenic in infants, including Group B streptococcus, Escherichia coli and Klebsiella pneumoniae, and

transmission of colonisation to newborn. Knowledge of GBS-colonisation status of pregnant women in Soweto was limited, as the collection and testing of antenatal swabs and implementation of IAP is not routine, and not feasible in a setting like ours.

Women included in the colonisation cohort had a vaginal introitus swab collected prior to any interventional wipes being performed. Infants of women included in the colonisation cohort had a surface (skin) swab collected from the umbilical area, peri- auricular area and nares soon after birth, prior to the newborn receiving an

interventional wipe or a bath. Swabs for colonisation were placed into Amies without charcoal transport medium and transported at room temperature to the laboratory of RMPRU for processing within 48 hours of collection.

Trial intervention

Per vaginal (PV) examinations to assess progress of labour are performed approximately 4 hourly in labouring women by CHBAH-employed attending

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by research midwives immediately after the routine PV examinations to avoid increasing the numbers of times the participant had to be repositioned for

procedures. Maternal wipes were performed approximately 4 hours apart, but not more frequently than 3-hourly. The research midwives monitored participants during and after study wipes for possible reactions to wipes. A decision to terminate wipes early could have been made by the study midwife or attending physician, mainly in the event of a change in eligibility for trial continuation or moderate or severe reaction to chlorhexidine.

Chlorhexidine Interventional wipes:

Commercially-available 5% chlorhexidine gluconate was diluted weekly to 0·5% with autoclaved drinking-quality tap-water and stored in one litre opaque bottles at room temperature. Autoclaved tap-water was used for control-arm wipes. Chlorhexidine was tested for activity by RMPRU laboratory using spectrophotometric and biological activity tests, and both solutions were tested for sterility before and after a week’s use.

Study midwives wrapped cotton wool soaked in 0.5% chlorhexidine solution around their index and middle fingers of their gloved examining hand, and gently wiped the cervix and vaginal walls in a clockwise motion. The presenting part of the foetus and mother’s external genitalia were also wiped with chlorhexidine-soaked cotton wool pads. Following birth and collection of colonisation cohort skin swabs (if applicable), infants of participants randomised to chlorhexidine interventional wipes were wiped from head to toe (avoiding face and ears) with 0.5% chlorhexidine-soaked cotton wool swabs.

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Placebo/ control wipes: Study midwives wiped the external genitalia of women randomised to control arm with sterile water- soaked cotton swabs. Chlorhexidine was not used for control wipes in these maternal participants, as disinfection of external genitalia may have had some benefit to mother and infant, and may have raised questions on the true impact of internal vaginal wipes. Infants of participants randomised to control arm had their feet wiped with chlorhexidine-soaked cotton swabs. Although an infant wipe was not essential in the placebo arm, it assisted in maintaining blinding of study investigators, who were reviewing case report forms of wipe dates and times. Before neonatal wipes all babies received a water bath per standard-of-care.

Participant follow up

Neonatal participants were followed up for hospitalisation for sepsis until 28 days of life, and maternal participants until 14 days post-delivery. Active monitoring of

admissions to the neonatal unit and paediatric department at CHBAH was conducted daily by research trial staff members. Medical officers employed for the trial, and blinded to interventional arm, reviewed medical records of all admitted neonates, and abstracted information related to sepsis end-point.

Sterile site cultures from admitted trial participants were collected at the discretion of attending physicians and processed per standard practice at the hospital

microbiology laboratory. Routine methods were used for culture and identification of invasive pathogens from sterile sites, including the BacT/Alert microbial system (Organon Teknika, Durham, NC) for blood culture. Active laboratory-based surveillance was also conducted to confirm that all sterile site cultures from study

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neonates were captured. Information related to the neonatal sepsis endpoints was abstracted from medical records by trained study physicians. Hospital mortuary logs were also reviewed regularly to ensure deaths among trial participants were

captured.

Due to the complexity of diagnosis in different settings, with variable access to diagnostic capacity or tests, no standardised definition of clinically-diagnosed neonatal sepsis was universally used, and the PoPS team developed its own definitions with the assistance of a team of neonatologists, which were used to determine rates of clinical- and culture-confirmed neonatal sepsis in study

participants 229, 230. Neonates of women who continued participation on PoPS at the time of labour were assessed for culture-confirmed- and clinically-confirmed neonatal sepsis endpoints.