Molecular Detection of Bloodstream Pathogens in Critical Illness

  • Huda Al_Griw

    Student thesis: Phd


    Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved.Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identification of bacterial and fungal DNA in blood.Methods: A range of commercial and "in-house" PCR-based assays for detection of bacterial and fungal DNA were developed and/or optimised for use in clinical blood samples. These included LightCycler SeptiFast, a CE-marked multi-pathogen assay for common bloodstream pathogens, BactScreen and GramScreen, broad spectrum bacterial assays based on 16S rRNA gene and real-time PCR assays developed to detect a range of clinically important fungal pathogens. Novel approaches to speciation of pathogen DNA using melting temperature (Tm) profiling and high resolution melting analysis (HRMA) were developed. Clinical evaluation of assays was either on blinded clinical isolates or blood samples from critically ill patients with clinical suspicion of bloodstream infection against conventional microbiological culture. Several techniques aimed at improving extraction of pathogen DNA from blood were also investigated. Results: The CE-marked commercial assay SeptiFast showed analytical sensitivity and specificity of 79% and 83% respectively. Concordance with positive culture results was good but high levels of "false positives" were detected possibly attributed to detection of free pathogen DNA not associated with viable pathogens. The predictive value of a negative SeptiFast test was 98% suggesting that absence of pathogen DNA is a strong indicator of absence of infection. Further studies were aimed at detailed optimisation and validation of 16S rRNA gene real-time PCR assays for bacterial DNA. BactScreen and GramScreen were able to detect a broad range of clinically important bacteria down to
    Date of Award1 Aug 2012
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorPaul Dark (Supervisor) & Geoffrey Warhurst (Supervisor)


    • High resolution melting curve analysis (HRMA)
    • Melting peak analysis
    • Species identification
    • SeptiFast
    • Sepsis
    • Bacteria, Candidemia
    • 16S rRNA
    • Universal primers
    • Real-time PCR
    • Molecular diagnosis
    • Bloodstream infection

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