Rapid Detection of Bacteria in Blood

Author/Creator ORCID




Mechanical Engineering


Engineering, Mechanical

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Sepsis is the leading cause of death in U.S. hospitals. It is usually caused by an acute infection in the bloodstream, and treatment requires the timely prescription of antibiotics. Rapid detection of bacterial presence in blood, in combination with antibiotic susceptibility, is essential in the treatment of bloodstream bacterial infections. Unfortunately, the current procedure (blood cultures) takes 1 to 3 days to complete, which is time many patients do not have. Regardless of the method utilized, however, it is important to isolate bacteria from blood samples. A novel method to separate bacteria from analytic volumes of blood samples has been developed. The process takes 35 minutes to extract the bacteria-containing plasma. This device relies on the fact that blood cells sediment faster than bacteria. After the blood cells are sedimented, the plasma is extracted by pushing a fluid into the system. The difference in density between the plasma and red blood cells creates a velocity gradient across the sample in the channel. This velocity gradient extracts the plasma out of the tube while the red blood cells lag. The study optimized the method parameters. These parameters are the sedimentation time, the channel height, and the extraction velocity. The method was developed using a TygonTM tube taped to a bench. This made it challenging to provide reproducibility for separation of plasma. To increase the repeatability of the bacterial isolation process, a fluidic device was developed where the process takes place. The devices were fabricated using Polymethymethacrylate (PMMA) sheets that were bonded in a novel method. This method integrated solvent and thermal bonding. The bonding method was validated using Mode I and Mode II fracture toughness analysis. This method was also modified to bond expanded polytetrafluoroethylene (ePTFE) to ensure effective degassing in the system. To determine the optimum channel depth of the device, the sedimentation of red blood cells was studied. Experiments were carried out in Polymethymethacrylate (PMMA) blocks, these blocks contained an observation window where a camera recorded the sedimentation rate. The experimental results were compared to Stoke'sequation and the difference was reported. The resulting samples were paired with a previously developed fluorescence-based bacterial detection device. The device automates alamarBlue cell viability assay. This assay utilizes resazurin dye which acts as an indicator. The rate of the fluorescence increase is proportional to the number of viable cells in the sample. The fluorometer is portable. The method has been validated using infected mouse blood. The device resulted in 82% bacterial recovery when tested with non-typhoidal Salmonella and Methicillin-resistant Staphylococcus aureus (MRSA) with plasma purity of less than 1% (percentage of hemoglobin). Infected mice are used to validate the device with bacterial concentrations as low as 10 CFU/mL. All results from the devices were validated using absorbance and standard bacterial plating methods.