Study design and Methods: Sepsis surveillance study

2.2.1. Study design and enrolment

A bacterial surveillance system was established at CHBAH in the neonatal and paediatric wards in collaboration with the staff in the Departments of Paediatrics and Microbiology to determine incidence, aetiology and clinical spectrum of culture- confirmed sepsis in young infants and the impact of maternal HIV-infection. Pathogenic bacteria isolated from sterile site samples (blood, cerebrospinal fluid) collected by attending physicians during admission of young infants (≤90 days old), were identified by surveillance and review of clinical and microbiology records at CHBAH between January 2004 and December 2008.

Pathogenic bacteria were defined as organisms which are capable of causing disease, and are uncommonly considered to being contaminants. Bacterial contaminants included, but were not limited to, the following: Bacillus species,

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negative Staphylococcus unless identified from 2 or more cultures obtained at separate times. Additional isolates which were potential contaminants were

evaluated for clinical significance by the investigational team based on associated clinical and laboratory signs.

Young infants with GBS were identified through daily screening of admissions to neonatal and paediatric wards and microbiological reports. Additionally, all GBS isolates from sterile sites of any patient at CHBAH were kept aside by microbiology staff for collection by RMPRU staff. Young infants with pathogenic bacteria other than GBS were identified predominantly retrospectively through the surveillance system of microbiological records.

The parents or legal guardians of prospectively-enrolled infants were informed about the surveillance study, and signed written, informed consent forms. If the infant had died or been discharged prior to identification in the wards and obtaining parental consent, data were collected retrospectively. An audit of the NHLS database of all invasive pathogens isolated from paediatric patients over the study period was conducted to ensure that all patients were identified.Study investigators obtained information about each patient identified as having an invasive bacterial infection from their medical records and microbiological reports.

2.2.2. Routine Blood Culture Sample collection and processing

Blood samples for culture obtained by attending physicians were placed into

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culture bottles and evaluated by the microbiology department of the National Health Laboratory Services (NHLS) at CHBAH. Cerebrospinal fluid (CSF) samples were obtained from infants admitted from the community with suspected sepsis, and from infants identified to have GBS-bacteraemia from blood sample collected at birth. CSF sample analysis included tests in clinical chemistry, haematology and

microbiology as per routine practice by the NHLS. GBS isolates from blood or CSF were plated onto on blood agar plates by NHLS staff and were kept aside for

collection by RMPRU staff. Pure cultures of these GBS isolates were stored at -70°C in RMPRU laboratory and serotyped in batches by latex agglutination as decribed228. Serotyping of GBS is not performed routinely by NHLS.

Antimicrobial susceptibility testing of isolated bacteria is performed routinely by the NHLS Department of Microbiology. A Gram stain is performed on samples from BacT/Alert blood culture bottles which display positive results, and on pathogens isolated from CSF samples. The results of the Gram stain guide the decision of which antimicrobial profile to select for susceptibility testing.

The Kirby-Bauer disk diffusion susceptibility test method is used in NHLS.

Pathogenic organisms identified from blood or CSF cultures are grown on Mueller- Hinton agar, in the presence of antimicrobial-infused filter paper disks. A zone of absence of growth around the disk indicates inhibition of the organism by the specific antimicrobial. An automated Microscan Microbiology System (Beckman Coulter, previously Siemens) is also utilised for both the identification of the organism and the susceptibility testing using relevant panels in cases where the Kirby-Bauer method has not been utilised. Etest® strips are used to supplement the Kirby-Bauer and

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Microscan results for certain pathogens and antibiotics (e.g. Vancomycin Etest® used for Vancomycin-resistant Enterococci and methicillin resistant Staphylococcus

aureus.)

Minimum inhibitory Concentration (MIC) of antimicrobials is reported according to the Clinical Laboratory Standards Institute (CLSI) standards (www.clsi.org ).

Invasive disease was categorized as bacteraemia if identified in blood only or as meningitis if (i) identified from CSF or (ii) there was CSF cytological evidence of purulent meningitis (>5 leucocytes/mm3, adjusted in traumatic lumbar punctures to allow 1 leucocyte per 500 erythrocytes) in an infant with bacteremia.

2.2.3. Data collection and analysis

Data, including demographic and admission information, were abstracted from medical records of infants admitted to the neonatal or paediatric wards of CHBAH and entered onto paper case report forms (CRFs) by study doctors. Delivery information and maternal HIV-infection status was collected when possible from neonatal admission records, maternal delivery notes, or from the mother herself. HIV exposure was determined by abstracting antenatal HIV test results of mothers and supplemented by HIV ELISA results from maternal or infant blood tests conducted by attending physicians. The HIV infection status of HIV-exposed infants was

determined using qualitative HIV PCR tests at the discretion of the attending

physician. Gestational age assessment was based on the available obstetric details (last menstrual period date, ultrasound, serial symphysis- fundal height) or neonatal assessment by the attending physicians.

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Data were entered into a customised Microsoft access database. Data on live births in Soweto were obtained from CHBAH and community clinics, and antenatal survey HIV-prevalence data were utilised to estimate denominators for HIV-infected and HIV-uninfected women.

Data were analysed using STATA/ IC 13.0 (Statacorp, College Station, TX, USA), and details of analyses are included in the methods sections of following chapters.

2.2.4. Ethics

The surveillance study was approved by the University of the Witwatersrand Human Research Ethics Committee (HREC) on 2nd November 2003 (HREC references M03- 10-07 and M100367, appendix 2) and the institutional review board of the Centers for Disease Control and Prevention, Atlanta, USA (protocol # 4128).

Written informed consent was obtained from a parent (usually mother) of infants enrolled prospectively into the surveillance study. If a patient was only identified by study staff after demise or discharge, the HREC waivered consent requirement, and data and GBS isolate (if relevant) were collected retrospectively.

2.2.5. Funding

The surveillance study was funded by the Centers for Disease Control and

Prevention (Cooperative Agreement number: U50/CCU02196 and U01 CI000318)) and the Bill and Melinda Gates Foundation (Grant number 39415).

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