A TWO-DIMENSIONAL MICROFLUIDIC SEPARATION PLATFORM FOR HIGH-THROUGHPUT GENETIC MUTATIONAL ANALYSIS

A principal challenge to upgrading genetic-based disease prevention and management is the establishment of new mutation detection strategies that surpass the performance and capabilities of traditional gel-based approaches. The need to screen individuals for the presence of mutations across a large number of genes requires the development of technology that can more rapidly and more cost-effectively identify DNA sequence heterogeneity. In this work, we demonstrate the feasibility of microfluidic separation platforms to transform traditional genetic screening methods by implementing these techniques in miniaturized systems to achieve far superior throughput than what is realized in contemporary clinical laboratories. Furthermore, to maximize the cost-effectiveness of these microfluidic systems, we have explored the use of inexpensive polymer materials as the substrates for these systems. In the initial part of this work, fabrication methods for polymeric microfluidic separation platforms were developed. The initial system consisting of a hybrid polydimthylsiloxane-silicon structure was used to demonstrate the capability of microfluidic platforms to perform field-effect electroosmotic flow control at enhanced efficiencies. Polycarbonate microfluidic devices were developed to demonstrate temperature gradient gel electrophoresis (TGGE) for the first time in a miniaturized platform. TGGE analyses of model mutant DNA fragments, each containing a single base substitution, were achieved using both single and 10-channel parallel measurements in a microfluidic platform. When compared to conventional capillary systems, the polymer microfluidic TGGE system described here demonstrated similar separation efficiency at one tenth of the analysis time, thus displaying the potential of this technology to dramatically increase the throughput of TGGE analyses. Finally the conception and realization of a parallel two-dimensional microfluidic platform for genetic mutation screening is described here. The system provides for online coupling of an initial size-based gel electrophoresis separation with a secondary parallel sequence-based TGGE separation. Such a separation strategy allows for the highthrouput analysis of complex genomic samples such as a mixture of multiplexed polymerase chain reaction (PCR) products. The comprehensive two-dimensional mutation analysis performed using this microfluidic system demonstrated the capability to detect mutations in DNA fragments at speeds approximately one to two orders of magnitude higher than is possible with conventional gel-based gene scanning methods

Vector-borne pathogens of zoonotic concern in hunting dogs of southern Italy

Dogs are commonly exposed to vector-borne pathogens (VBPs), yet few data are available on hunting dogs, which are often at high risk of infection due to their involvement in field activities. To investigate the occurrence of VBPs and evaluate the relative performance of different diagnostic tools, blood and serum samples were collected from hunting dogs (n = 1,433) in rural areas of southern Italy. All samples were tested by Knott's technique for filarioids, serologically (SNAP® 4Dx® Plus) for Anaplasma spp., Borrelia burgdorferi sensu lato, Dirofilaria immitis and Ehrlichia spp. and molecularly (qPCR) for all except B. burgdorferi of the above pathogens plus Babesia spp. and Leishmania infantum. Logistic regression was run to evaluate the statistical associations between the risk of VBP infection and independent variables (such as geographic area of provenience, age class and sex) and K-Cohen formula for assessing the concordance among diagnostic tests. Overall, out of 321 dogs (22.4%) positive to at least one VBP, 28 (1.9%) were infected by filarial species at the Knott's technique. In particular, Acanthocheilonema reconditum was the most prevalent (1.6%), followed by D. immitis (0.2%) and Dirofilaria repens (0.1%). One hundred forty (9.8%) and 231 (16.1%) dogs scored positive to VBPs by serological and molecular methods, respectively. The most prevalent pathogens detected were Ehrlichia spp. (7.3%) with SNAP® 4Dx® Plus, and A. reconditum (7.7%) by qPCR. Statistics revealed a significant association (p < 0.001) between A. reconditum infestation and both Ehrlichia spp. seropositivity and geographical origin of dogs. An agreement of 99.9%, 94.0% and 95.7% for Knott - SNAP® 4Dx® Plus, Knott - qPCR and SNAP® 4Dx® Plus - qPCR for D. immitis was found, respectively. Data demonstrate a high prevalence of VBPs in hunting dogs, indicating that this group of animals is largely exposed to several arthropod vector species and suggesting the transmission risk of pathogens to humans in rural areas of southern Italy. A multi-diagnostic approach and a deeper cooperation among healthcare and stakeholders are required to prevent VBP infections to animals and humans

Association of acute Babesia canis infection and serum lipid, lipoprotein, and apoprotein concentrations in dogs

Background Babesia canis infection induces a marked acute phase response (APR) that might be associated with alteration in lipid and lipoprotein metabolism and disease prognosis. Hypothesis Dogs with B. canis-induced APR develop dyslipidemia with altered lipoprotein concentration and morphology. Animals Twenty-nine client-owned dogs with acute B. canis infection and 10 clinically healthy control dogs. Methods Observational cross-sectional study. Serum amyloid A (SAA) was measured using ELISA. Cholesterol, phospholipids, and triglycerides were determined biochemically. Lipoproteins were separated using agarose gel electrophoresis. Lipoprotein diameter was assessed by polyacrylamide gradient gel electrophoresis; correlation with ApoA-1 (radioimmunoassay) and SAA was determined. Results Dogs with B. canis infection had a marked APR (median SAA, 168.3 mu g/mL; range, 98.1-716.2 mu g/mL) compared with controls (3.2 mu g/mL, 2.0-4.2 mu g/mL) (P < .001). Dogs with B. canis infection had significantly lower median cholesterol (4.79 mmol/L, 1.89-7.64 mmol/L versus 6.15 mmol/L, 4.2-7.4 mmol/L) (P = .02), phospholipid (4.64 mmol/L, 2.6-6.6 mmol/L versus 5.72 mmol/L, 4.68-7.0 mmol/L) (P = .02), and alpha-lipoproteins (77.5%, 27.7%-93.5% versus 89.2%, 75.1%-93.5%) (P = .04), and higher ApoA-1 (1.36 U, 0.8-2.56 U versus 0.95 U, 0.73-1.54 U) concentrations (P = .02). Serum amyloid A correlated with high-density lipoproteins (HDLs) diameter (rho = .43; P = .03) and ApoA-1 (rho = .63, P < .001). Conclusions and Clinical Importance Major changes associated with B. canis-induced APR in dogs are related to concentration, composition, and morphology of HDL particles pointing to an altered reverse cholesterol transport. Parallel ApoA-1 and SAA concentration increase is a unique still unexplained pathophysiological finding

Associations between clinical canine leishmaniosis and multiple vector-borne co-infections: a case-control serological study

Dogs that have clinical leishmaniosis (ClinL), caused by the parasite Leishmania infantum, are commonly co-infected with other pathogens, especially vector-borne pathogens (VBP). A recent PCR-based study found that ClinL dogs are more likely to be additionally infected with the rickettsial bacteria Ehrlichia canis. Further information on co-infections in ClinL cases with VBP, as assessed by serology, is required. The research described in this report determined if dogs with ClinL are at higher risk of exposure to VBP than healthy control dogs using a case-control serology study

Federated Learning for Breast Density Classification: A Real-World Implementation

Building robust deep learning-based models requires large quantities of diverse training data. In this study, we investigate the use of federated learning (FL) to build medical imaging classification models in a real-world collaborative setting. Seven clinical institutions from across the world joined this FL effort to train a model for breast density classification based on Breast Imaging, Reporting & Data System (BI-RADS). We show that despite substantial differences among the datasets from all sites (mammography system, class distribution, and data set size) and without centralizing data, we can successfully train AI models in federation. The results show that models trained using FL perform 6.3% on average better than their counterparts trained on an institute's local data alone. Furthermore, we show a 45.8% relative improvement in the models' generalizability when evaluated on the other participating sites' testing data.Comment: Accepted at the 1st MICCAI Workshop on "Distributed And Collaborative Learning"; add citation to Fig. 1 & 2 and update Fig.

Seropositivity of main vector-borne pathogens in dogs across Europe

Background Canine vector-borne disease (CVBD) has been an area of increasing interest in Europe over the last few decades, and there have been changes in the prevalence and distribution of many of these diseases. Monitoring CVBD infections in Europe is often done by individual countries, but aggregated data for the European countries are helpful to understand the distribution of CVBDs. Methods We used an extensive retrospective database of results from point-of-care rapid enzyme-linked immunosorbent assay (ELISA) tests on dogs across Europe to identify distribution and seropositivity in animals tested for selected CVBDs (Anaplasma spp., Ehrlichia spp., Borrelia burgdorferi, Leishmania spp., and Dirofilaria immitis) from 2016 through 2020. Geographic distribution of positive tests and relative percent positive values were mapped by the Nomenclature of Territorial Units for Statistics classification for regions with sufficient test results for reporting. Results A total of 404,617 samples corresponding to 1,134,648 canine results were available from dogs tested in 35 countries over the 5-year study period. Over this period the number of test results per year increased whereas test positivity decreased. Leishmania spp. had the largest increase in total test results from 25,000 results in 2016 to over 60,000 results in 2020. Test positivity for Leishmania spp. fell from 13.9% in 2016 to 9.4% in 2020. Test positivity fell for Anaplasma spp. (7.3 to 5.3%), Ehrlichia spp. (4.3 to 3.4%), and Borrelia burgdorferi (3.3 to 2.4%). Dirofilaria immitis test positivity trended down with a high of 2.7% in 2016 and low of 1.8% in 2018. Leishmania spp. test positivity was highest in endemic areas and in several non-endemic countries with low numbers of test results. Co-positivity rates were significantly higher than expected for all pathogen test positive pairs except for Ehrlichia spp. with Borrelia burgdorferi and D. immitis with Borrelia burgdorferi. Conclusions This study represents the largest data set on CVBD seropositivity in Europe to date. The increase in the number of test results and decreasing test positivity over the study period may reflect changes in testing behavior and increased screening of healthy animals. The Europe-wide mapping of CVBD provides expected test positivity that can help inform veterinarians’ decisions on screening and improve prevention and identification of these important, sometimes zoonotic, diseases

Integration of Isoelectric Focusing with Parallel Sodium Dodecyl Sulfate Gel Electrophoresis for Multidimensional Protein Separations in a Plastic Microfludic Network,” Anal.

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of ∼1700 using a chip with planar dimensions of as small as 2 cm × 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension