Development of methods for the detection Staphylococcal Protein A in ICU patient urine

Varley, Chris and Lauder, Bob (2016) Development of methods for the detection Staphylococcal Protein A in ICU patient urine. Masters thesis, Lancaster University.

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Hospital acquired Staphylococcus aureus bacteraemia presents a serious health risk to inpatients, due to the high risk of transmission and high mortality rates, which is in part is due to the emergence of multiple drug resistant strains of S. aureus. Therefore, the prompt diagnosis and treatment of S. aureus bacteraemia is of paramount importance in the Intensive Care department. Staphylococcal Protein A (SpA) is an immunoglobulin-binding surface protein, expressed by more than 98.9% of S. aureus strains, with a primary function of aiding S. aureus in the colonisation of the host. While many Staphylococcal toxins are excreted by the kidneys, the method of SpA processing and removal by the body has not been confirmed. If SpA is present in urine, it could provide a key marker of S. aureus infection. The aim of this research was to develop techniques for the detection of SpA in samples, and evaluate the surface protein as a target for S. aureus urinary antigen testing. This was to be assessed in a sample population of 45 Intensive Care patients, each providing up to 8 samples over a 48 hour inpatient stay. An effective and specific Western Blot was developed for the detection of commercial SpA in both buffer and control urine; however, this method failed to detect any SpA bands in patient urine samples. Subsequently, an optimised ELISA displayed increased sensitivity, being able to detect lower levels of SpA down to 0.78ng/ml. Using this technique, we detected an increased absorbance in 25% of patient samples tested, implying the presence of SpA. However, these samples did not display the same characteristics as commercial SpA, lacking the heat-resistance of the purified protein when ELISA samples were subject to boiling. Additionally, the application of mass spectrometry to analysing the SpA ELISA positive samples did not identify SpA. Furthermore, positive ELISA results were significantly associated with renal failure, but not with markers of infection. This research represents a comprehensive analysis of immunoblotting and immunoassay methods for detecting SpA in urine samples. Further work is required to fully assess the route of excretion of SpA, and the techniques developed could prove useful in the testing of other clinical samples, such as serum, for SpA.

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Thesis (Masters)
?? protein astaphylococcus aureusurinary antigen testing ??
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Deposited On:
04 Oct 2016 09:04
Last Modified:
31 Dec 2023 00:06