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Bridging the gap between detection and confirmation of B. anthracis in blood cultures

Student thesis: Doctoral Thesis

The spore forming bacterium, Bacillus anthracis is the aetiological agent of
anthrax. The 2001 US anthrax letter attacks and the 2009‐2010 outbreak of
injectional anthrax in the UK highlighted the importance of early detection and
confirmation of this agent, both for patient outcome and forensic investigations.

A reliable and consistent method was used in this study to safely simulate
blood cultures with B. anthracis and used to determine the time to positive
detection. This was performed with different strains and with varying
concentrations of inoculum. An inverse linear relationship was observed with
all strains and used to estimate the bacterial blood concentration of anthrax
patients based on data gathered from the literature and front‐line laboratories
in the UK.

The study explored a method to potentially reduce the turnaround times for the
confirmation of B. anthracis at the national reference laboratory. Serum
separator tubes were used to concentrate the bacteria from simulated blood
cultures. A simple wash step was performed prior to performing confirmatory
phenotypic tests and inactivation for rapid molecular detection. A comparison
of test results with and without serum separator tube processing was made for
B. anthracis and bacterial isolates referred during the outbreak of injectional
anthrax. Simulated mixed blood cultures of B. anthracis and possible common
contaminants were also tested. Compared to routine methods, confirmatory
phenotypic test results were achieved 24 hours sooner using the method. The
simple wash step and inactivation was sufficient to provide nucleic acid for
molecular confirmatory assays and genotyping. A new ‘sample to answer’
platform, the Biofire Filmarray® was also trialled and correctly identified B.
anthracis directly from simulated blood culture and provided results within one
hour.

Aspects relating to potential biosafety concerns for processing B. anthracis blood
cultures were explored. The data generated suggests the aerosol risk is low for
B. anthracis. Viability of material on microscopy slides was examined and the
data supports the recommended use of alcohol fixation for slide preparation.
There has been no previous evidence reported for sporulation occurring in
blood culture bottles and the study findings suggest this is possible five days
post positive detection.

Interactive e‐learning modules have been produced to disseminate the study
outcome. The e‐learning is intended for front‐line laboratories to raise
awareness for the safe handling and laboratory identification of B. anthracis.
Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award dateFeb 2015

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