Introducing a New Algorithm for Classification of Etiology in Studies on Pediatric Pneumonia: Protocol for the Trial of Respiratory Infections in Children for Enhanced Diagnostics Study

Background: There is a need to better distinguish viral infections from antibiotic-requiring bacterial infections in children presenting with clinical community-acquired pneumonia (CAP) to assist health care workers in decision making and to improve the rational use of antibiotics.Objective: The overall aim of the Trial of Respiratory infections in children for ENhanced Diagnostics (TREND) study is to improve the differential diagnosis of bacterial and viral etiologies in children aged below 5 years with clinical CAP, by evaluating myxovims resistance protein A (MxA) as a biomarker for viral CAP and by evaluating an existing (multianalyte point-of-care antigen detection test system [mariPOC respi] ArcDia International Oy Ltd.) and a potential future point-of-care test for respiratory pathogens.Methods: Children aged 1 to 59 months with clinical CAP as well as healthy, hospital-based, asymptomatic controls will be included at a pediatric emergency hospital in Stockholm, Sweden. Blood (analyzed for MxA and C-reactive protein) and nasopharyngeal samples (analyzed with real-time polymerase chain reaction as the gold standard and antigen-based mariPOC respi test as well as saved for future analyses of a novel recombinase polymerase amplification-based point-of-care test for respiratory pathogens) will be collected. A newly developed algorithm for the classification of CAP etiology will be used as the reference standard.Results: A pilot study was performed from June to August 2017. The enrollment of study subjects started in November 2017. Results are expected by the end of 2019.Conclusions: The findings from the TREND study can be an important step to improve the management of children with clinical CAP

Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level

Microfluidic anoikis-chip for screening of cytostatic drugs for cander treatment

Background: Anoikis (“homeless”) refers to the process of detachment of cells from their matrix which causes cell death. For detailed drug-screening, microfluidics is ideal for studying the balance between cell survival and cell death, as cells can be studied in real-time at a single cell level in the presence of different (dosages of) drugs. Methods: MCF-7 breast cancer cells were incubated with a mixture of TNF-α and cycloheximide (CHX) or staurosporine (SSP) to induce apoptosis. The process of anoikis was measured conventional with a DELFIA assay and analyzed in real-time on chip at a single cell level using Annexin V and propidium iodide (PI). Results: TNF-α/CHX and SSP both activated the apoptotic cascade, confirmed with light microscopy, and demonstrated an increase in time in the Annexin V-Europium fluorescent intensity in the adherent cell fraction and the fraction with floating cells and apoptotic bodies. For detailed cellular-based experiments, MCF-7 cells were successfully cultured in a microfluidic device. Continuous administration of 50μM SSP showed 100% positive for Annexin V and PI, though cells did not detach. Incubation over longer periods with TNF-α/CHX demonstrated all the characteristics of the apoptotic process (shrinkage, fragmentation, membrane blebbing) though cells hardly detach. Differences in material (pyrex glass vs. polystyrene) and the way of administration (single vs. continuous administration) might account for the effects seen and therefore further analysis is necessary. Conclusions: Microfluidics has the potential for effective drugscreening and when using patient’s own cells obtained via biopsy optimal selection of cytotoxic treatment can be made

Micro-droplet based directed evolution outperforms conventional laboratory evolution

We present droplet adaptive laboratory evolution (DrALE), a directed evolution method used to improve industrial enzyme producing microorganisms for e.g. feedstock digestion. DrALE is based linking a desired phenotype to growth rate allowing only desired cells to proliferate. Single cells are confined in microfluidic droplets to prevent the phenotype, e.g. secreted enzymes, from leaking between cells. The method was benchmarked against and found to significantly outperform conventional adaptive laboratory evolution (ALE) in enriching enzyme producing cells. It was furthermore applied to enrich a whole-genome mutated library of yeast cells for α-amylase activity

Simultaneous impedance and fluorescence detection of proteins in a cyclo olefin polymer chip containing a column with an ordered pillar array with integrated gold microelectrodes

In this work, we report the detection of proteins by means of simultaneous fluorescence and impedance measurements in a cyclo olefin polymer (COP) chip containing an ordered pillar array column, used for reversed-phase liquid chromatography, with integrated microband gold electrodes at the end of the channel

Microfluidic single-cell array cytometry for the analysis of tumour apoptosis

Limitations imposed by conventional analytical technologies for cell biology, such as flow cytometry or microplate imaging, are often prohibitive for the kinetic analysis of single-cell responses to therapeutic compounds. In this paper, we describe the application of a microfluidic array to the real-time screening of anticancer drugs against arrays of single cells. The microfluidic platform comprises an array of micromechanical traps, designed to passively corral individual nonadherent cells. This platform, fabricated in the biologically compatible elastomer poly(dimethylsiloxane), PDMS, enables hydrodynamic trapping of cells in low shear stress zones, enabling time-lapse studies of nonadherent hematopoietic cells. Results indicate that these live-cell, microfluidic microarrays can be readily applied to kinetic analysis of investigational anticancer agents in hematopoietic cancer cells, providing new opportunities for automated microarray cytometry and higher-throughput screening. We also demonstrate the ability to quantify on-chip the anticancer drug induced apoptosis. Specifically, we show that with small numbers of trapped cells (∼300) under careful serial observation we can achieve results with only slightly greater statistical spread than can be obtained with single-pass flow cytometer measurements of 15 000-30 000 cells