Transcript the poster - Meningitis Research Foundation

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and unpublished data)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.