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Feasibility study of a novel assay for detection of bacteria in neonatal

Presented at the Neonatal Society 2017 Autumn Meeting.

Abelian A1, Mund T2, Curran M3, Charan C4, Mitra N4, Ogilvy-Stuart A4, Pelham H2, Dear PH5

1 Maelor Hospital, Wrexham, UK
2 MRC Laboratory of Molecular Biology, Cambridge, UK
3 Addenbrookes Hospital, Cambridge, UK
4 Rosie Maternity Hospital, Cambridge, UK
5 Mote Research Ltd., Cambridge, UK

Background: An assay based on 16S rDNA PCR technology has been designed to detect a single intact bacterium whilst eliminating free DNA from dead bacteria, thus offering unprecedented sensitivity and scope to the analysis of bacterial carriage in clinical specimens. We hypothesised that application of such an assay to neonatal CSF will enable accurate, fast and inexpensive discrimination of bacteria-free specimens, and will have a small but clinically acceptable false-positive rate.

Methods: Design of PCRctic – a novel assay based on 16S rDNA PCR technology utilising ethidium azide for elimination of free bacterial DNA and optimised for neonatal CSF – was presented to this Society in summer 2016. In this prospective study lasting 12 months, the feasibility of PCRctic was investigated in CSF specimens obtained from newborn babies tested for meningitis. Following midway interim analysis, sterile snap-top tubes (EppendorfTM) replaced standard universal containers for collection of CSF, and ChloraPrepTM replaced Unisept as the choice of antiseptic. Study received National REC and HRA approvals and was funded by the MRC.

Results: Forty specimens of CSF were tested before the interim analysis (1st phase) and 33 after (2nd phase). In phase 1, the assay detected bacteria in 15 specimens (38%) and sequencing revealed several organisms of Flavobacteriaceae family (Cloacibacterium, Flavobacterium, Hymenobacter), as well as Ochrobactrum (Brucellaceae), Sneathia amnii (Leptotrichiaceae), Pseudomonas spp, Acinetobacter, Sphingomonadaceae, Oscillatoriales (Cyanobacteria), Ureaplasma urealyticum, Staphylococcus auricularis, Streptococcus spp, Bdellovibrio, and Aerococcus christensenii. In phase 2, bacteria were detected in six specimens (18%) and sequencing revealed Cloacibacterium, Methylobacterium, Pedobacter (Sphingomonadaceae), Geobacter and Staphylococcus. No clinical cases of neonatal bacterial meningitis occurred during the study. A positive signal was detected in only one out of 23 negative controls designed to test for environmental contamination (4%).

Conclusion: Neither the origin nor significance of the detected bacteria is clear at this stage. The diversity of organisms as well as the performance of negative controls argues against environmental contamination. The possibility that bacteria originated from either blood or CSF is worth exploring further.

Corresponding author: abelartur@doctors.org.uk

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