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Towards high accuracy meningitis testing in neonates

Presented as a poster at the Neonatal Society 2016 Summer Meeting.

Abelian A1, Mund T2, Pelham H2, Ogilvy-Stuart A3, Curran M4, Dear PH5

1 Maelor Hospital, Wrexham, UK
2 Laboratory of Molecular Biology, MRC Centre, Cambridge, UK
3 Rosie Maternity Hospital, Cambridge, UK
4 Molecular Diagnostics Laboratory, Addenbrooke’s Hospital, Cambridge, UK
5 Mote Research Ltd., Cambridge, UK

Background: The negative predictive value of bacterial culture is low and often inadequate to confidently rule out neonatal bacterial meningitis (NBM). This results in over-testing and over-treatment with prolonged hospitalisation and antibiotic use. Molecular techniques have the potential to augment and even replace bacterial culture.

Methods: “PCRctic” – novel assay based on 16S rDNA PCR technology – utilises ethidium monoazide to eliminate false positive results due to dead bacteria and/or contaminating bacterial DNA, and a single-step closed-tube nested PCR format to achieve sensitivity of single bacterium per 200 microlitres of CSF (5 drops). It offers fast turn-around time (under 2 hours) and minimal per assay cost (under £1). In-built internal control enables to identify false-negative results. It has been optimised to work on the platform of real-time PCR equipment – widely available in NHS molecular diagnostics laboratories.

Results: “PCRctic” detected a single colony-forming unit of the common causes of NBM: E coli, Streptococcus agalactiae (Group B Streptococcus), Streptococcus pneumoniae, and Listeria monocytogenes, whilst avoiding false positive results from contaminating bacterial DNA. Sequence analysis of the rarer bacteria implicated in NBM – Serratia marcescens, Staphylococcus aureus, Salmonella berta and agama, Ureaplasma parvum, Pasteurella multocida, Enterobacter sakazakii (Cronobacter), Sneathia species, Chryseobacterium meningosepticum – showed full-to-good match with PCRctic primers and therefore high probability of successful detection.

Conclusion: PCRctic has been adapted for NHS molecular diagnostics laboratory and is entering a feasibility trial with a tertiary neonatal unit with a multi-centre cross-sectional study to follow. Successful translation of advances in molecular diagnosis into neonatal practice will require concerted efforts of neonatologists, public health specialists, molecular biologists, and biotech

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