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Towards High Negative Predictive Value Meningitis Testing in Neonates Abelian 1 A, Mund 2 T, Pritchard 3 I, Pelham 2 H, Dear 4 PH . 1Department of Paediatrics, 3Department of Pathology, Maelor Hospital, BCUHB, Wrexham, UK; 2Laboratory of Molecular Biology, Medical Research Council Centre, Cambridge, UK; 4Mote Research Ltd., Cambridge, UK Funding: UK Medical Research Council Problem with neonatal CSF culture: 1072 CSF specimens (Abelian A, Pritchard I. J Ped Inf Dis. 2011;6:227-9 and unpublished data) No growth No growth Very low negative predictive value (NPV) ------------------------------------------------------------------------------Solution: Adapt 16S RNA PCR to achieve very high NPV and hence No growth accurate exclusion of meningitis. Method: (a) CSF samples; (b) Intact bacteria are pelleted along with patient cells and debris and (potentially) some free bacterial DNA; (c) The pellet is resuspended in the assay mixture (PCR reagents, ethidium azide, buffer); (d) light activates the ethidium azide, destroying exposed DNA (from dead bacteria or from contaminants in the reagents) but leaving intact bacteria unaffected; during PCR (e), heat causes the bacteria to lyse, making their DNA available for amplification. (f) amplification products are then detected by a simple fluorometric assay (melting-curve analysis) using a widely-available real-time PCR machine. The results shown in the graph (f) are from five samples of neonatal CSF which were “spiked” with E. coli at an average concentration of ~0.5 bacteria per sample (hence, about half the samples would be expected to contain one bacterium). Total assay time is approximately 2 . hours. Total reagent costs (excluding plastics) is <<£1. Examples of the results to date: . A – exposure to light and elimination of contaminating bacterial DNA. B – E coli detection in the concentration between 1.5 to five colony-forming units. C – E coli and Listeria innocua are still detected after a series of fivefold dilutions. -----------------------------------------------------------------------------Future: an assay adapted for NHS laboratories, tested in case-control and cross-sectional studies and providing accurate, fast and inexpensive exclusion of meningitis, and helping to reduce hospital stay and antibiotic use.