Development and integration of simplified real-world to chip interfaces for use in the detection of infectious diseases

Bacterial-based infectious disease, such as sexually transmitted infections and hospital-acquired infections, present a worldwide burden on healthcare issues. To control the spread of infection and to inform clinical treatment, rapid point of care (POC) diagnosis is required. Although some are currently available, these are commonly limited by a requirement for sample processing prior to analysis and a requirement for user intervention. Novel real world-to-chip interfaces are required which can receive a sample with little or no pre-processing and should be manufactured with minimal cost. In addition, manufacturing protocols should ideally be developed to allow easy adjustment of design for customising to process a wide variety of sample types and volumes.Here, focussing on bacterial-based POC diagnostics, a microfluidic platform has been developed which holds the potential to receive a variety of sample types for the detection of infectious organisms by implementing multiple sample-to-chip interfaces. The platform consists of a glass microfluidic device which is incorporated in to a custom-made integrated genetic analyser (IGA) for sample processing.A series of interfacing substrates were investigated using two types of porous silica and the biopolymer chitosan (α(1→4)-linked 2-amino-2-deoxy-β-D-glucopyranose) as contributing materials. For analysis of urine samples, a porous silica monolith, synthesised from tetramethyl orthosilicate, was developed, capable of receiving and processing human urine samples (≈ 150 μl) for DNA capture and purification. Due to the nature of synthesis, these monoliths hold the potential for resizing and shaping, dependent on the sample volume and for integration to downstream steps, such as polymerase chain reaction (PCR) amplification. The monolith was first optimised structurally using flow systems made from monoliths encased in heat shrink wrap and was incorporated in to a microfluidic device by way of disk. The latter was achieved by sealing the monolith in place with a sondary porous silica phase synthesised from potassium silicate, creating a dual porous silica (DPS) real world interface. The DNA extraction efficiencies of monolithic flow systems and the DPS system were 51 % and 44 % respectively. The DPS was shown to provide DNA of sufficient quality and integrity to support PCR amplification for both Chlamydia trachomatis and Neisseria gonorrhoea target sequences. The system did however lack sensitivity (1.3 x 10¯³ ng DNA μl¯¹ urine), when compared with systems of similar applications in the literature, likely due to large elution volumes (> 20 μl) and/or ethanol carryover. In addition, chitosan was introduced to the silica surface of the monolith as an alternative methodology for DNA extraction by anion exchange. The system provided DNA extraction efficiencies of 40 % and DNA was subsequently amplified by PCR.Using an alternative application, an investigation was also carried out in to the analysis of small volume blood samples (≤ 6 μl) for use in the same system. This was achieved by implementing a Phusion® blood direct kit in to a glass microfluidic device to amplify gDNA of bacterial target, methicillin resistant Staphylococcus aureus (MRSA). The system was shown to amplify DNA in 6 μl blood (0.083 ng μl¯¹) off-chip. It was then demonstrated to work on-chip in a 12.5 μl glass chamber, using a Peltier system for thermal cycling.For use in conjunction with all interfaces, an IGA with a built in DNA separation/ detection system, based on plug injection, capillary electrophoresis separation and fluorescence detection has been shown to reproducibly report the presence of target DNA sequences, against that of a custom-made size ladder. The detection of a 107 bp generic Staphylococcus aureus marker and 532 bp sequence from the mecA gene unique to MRSA were detected in 20 and 25 min, respectively. Importantly, the detection system is designed to integrate directly to upstream steps and has a Peltier element fitted for thermal cycling.The work described here contributes towards a platform which offers the opportunity to tackle a number of diagnostic applications in one fit-for-all instrument

Tracking of motor vehicles from aerial video imagery using the OT-MACH correlation filter

Accurately tracking moving targets in a complex scene involving moving cameras, occlusions and targets embedded in noise is a very active research area in computer vision. In this paper, an optimal trade-off maximum correlation height (OT-MACH) filter has been designed and implemented as a robust tracker. The algorithm allows selection of different objects as a target, based on the operator’s requirements. The user interface is designed so as to allow the selection of a different target for tracking at any time. The filter is updated, at a frequency selected by the user, which makes the filter more resistant to progressive changes in the object’s orientation and scale. The tracker has been tested on both colour visible band as well as infra-red band video sequences acquired from the air by the Sussex County police helicopter. Initial testing has demonstrated the ability of the filter to maintain a stable track on vehicles despite changes of scale, orientation and lighting and the ability to re-acquire the track after short losses due to the vehicle passing behind occlusions

Reality Check of Laboratory Service Effectiveness during Pandemic (H1N1) 2009, Victoria, Australia

TOC summary: The greatest challenges were insufficient staff and test reagents

What change in body mass index is needed to improve metabolic health status in childhood obesity:protocol for a systematic review

PRISMA-P (Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols) 2015 checklist: recommended items to address in a systematic review protocol*. PRISMA is an evidence-based minimum set of items for reporting in systematic reviews and meta-analyses. (DOC 80.5 kb

Phenotypic analysis of extracellular vesicles:a review on the applications of fluorescence

Extracellular vesicles (EVs) have numerous potential applications in the field of healthcare and diagnostics, and research into their biological functions is rapidly increasing. Mainly because of their small size and heterogeneity, there are significant challenges associated with their analysis and despite overt evidence of the potential of EVs in clinical diagnostic practice, guidelines for analytical procedures have not yet been properly established. Here, we present an overview of the main methods for studying the properties of EVs based on the principles of fluorescence. Setting aside the isolation, purification and physicochemical characterization strategies which answer questions about the size, surface charge and stability of EVs (reviewed elsewhere), we focus on available optical tools that enable the direct analysis of phenotype and mechanisms of interaction with tissues. In brief, the topics on which we elaborate range from the most popular approaches such as nanoparticle tracking analysis and flow cytometry, to less commonly used techniques such as fluorescence depolarization and microarrays as well as emerging areas such as fast fluorescence lifetime imaging microscopy (FLIM). We highlight that understanding the strengths and limitations of each method is essential for choosing the most appropriate combination of analytical tools. Finally, future directions of this rapidly developing area of medical diagnostics are discussed

Real time unmanned aerial vehicle tracking of fast moving small target on ground

In order to solve problems of occlusion and fast motion of small targets in UAV (Unmanned Aerial Vehicle) target tracking , an adaptive algorithm which fuses the improved color histogram tracking response and the correlation filter tracking response based on multi-channel HOG features is proposed to realize small target tracking with high accuracy. The state judgment index is used to determine whether the target is in a fast motion or an occlusion state. In the fast motion state, the search area is enlarged, and the color optimal model which suppresses the suspected area is used for rough detection. Then, re-detection in the location of multiple peaks in the rough detection response is carried out using the correlation filter to accurately locate the target. In an occlusion state, the model stops updating, the search area is expanded, and the current color model is used for rough detection. Then, re-detection in the place of multiple peaks in the rough detection response is carried out using the correlation filter to accurately locate the target. Experimental results show that the proposed method can track small targets accurately. The frame rate of the proposed method is 40.23 frames/second, indicating usable real-time performance