Identification of bacterial pathogens in paediatric and neonatal bloodstream Infection by blood culture is associated with significant delays. Rapid species level diagnosis in bloodstream infection may allow early introduction of targeted antibiotic therapy, and improve outcomes. Molecular tests can detect bacterial DNA directly from blood and are rapid, but not sufficiently sensitive for adoption as routine tests. Aims: The aim of the project was to test the hypothesis that a shortened culture step prior to molecular testing would improve the sensitivity of molecular testing, and reduce the time to species-level diagnosis in neonatal and paediatric bloodstream infection. Methods: A panel of real time polymerase chain reaction assays was assembled, specifically targeting the common bacterial pathogens in neonatal sepsis and neutropenic sepsis. A novel method incorporating whole genome analysis was developed to identify target sequences for bacterial speciation, and an assay detecting Enterobacter cloacae designed and validated. A suitable DNA extraction protocol was identified. In a group of 88 episodes of suspected sepsis, in preterm infants and neutropenic children, clinical samples were cultured in blood culture media for 6 hours before DNA extraction and molecular testing. The results were compared with matched blood culture samples. Results: 11 out of 88 blood culture samples were positive by blood culture, and the qPCR panel detected five of these. The remaining six isolates were coagulase-negative staphylococci. The culture-molecular approach adopted here had an overall sensitivity of 45% (17 -76%) and a specificity of 97% (91- 99%). The processing time for the molecular test was 9 hours in total. Conclusion : The incorporation of a culture step into a molecular testing strategy did not improve sensitivity of molecular tests in children, but the panel developed was highly specific. Further refinement of this approach may impact favourably on diagnostic accuracy.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:680059 |
| Date | January 2014 |
| Creators | Moriarty, P. D. |
| Publisher | Queen's University Belfast |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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