Microfluidic methods for the diagnosis of acute respiratory tract infections.

Acute respiratory tract infections (ARTIs) are caused by sporadic or pandemic outbreaks of viral or bacterial pathogens, and continue to be a considerable socioeconomic burden for both developing and industrialized countries alike. Diagnostic methods and technologies serving as the cornerstone for disease management, epidemiological tracking, and public health interventions are evolving continuously to keep up with the demand for higher sensitivity, specificity and analytical throughput. Microfluidics is becoming a key technology in these developments as it allows for integrating, miniaturizing and automating bioanalytical assays at an unprecedented scale, reducing sample and reagent consumption and improving diagnostic performance in terms of sensitivity, throughput and response time. In this article, we describe relevant ARTIs-pneumonia, influenza, severe acute respiratory syndrome, and coronavirus disease 2019-along with their pathogenesis. We provide a summary of established methods for disease diagnosis, involving nucleic acid amplification techniques, antigen detection, serological testing as well as microbial culture. This is followed by a short introduction to microfluidics and how flow is governed at low volume and reduced scale using centrifugation, pneumatic pumping, electrowetting, capillary action, and propagation in porous media through wicking, for each of these principles impacts the design, functioning and performance of diagnostic tools in a particular way. We briefly cover commercial instruments that employ microfluidics for use in both laboratory and point-of-care settings. The main part of the article is dedicated to emerging methods deriving from the use of miniaturized, microfluidic systems for ARTI diagnosis. Finally, we share our thoughts on future perspectives and the challenges associated with validation, approval, and adaptation of microfluidic-based systems.

Detection of renal biomarkers in chronic kidney disease using microfluidics: progress, challenges and opportunities.

Chronic kidney disease (CKD) typically evolves over many years in a latent period without clinical signs, posing key challenges to detection at relatively early stages of the disease. Accurate and timely diagnosis of CKD enable effective management of the disease and may prevent further progression. However, long turn-around times of current testing methods combined with their relatively high cost due to the need for established laboratory infrastructure, specialized instrumentation and trained personnel are drawbacks for efficient assessment and monitoring of CKD, especially in underserved and resource-poor locations. Among the emerging clinical laboratory approaches, microfluidic technology has gained increasing attention over the last two decades due to the possibility of miniaturizing bioanalytical assays and instrumentation, thus potentially improving diagnostic performance. In this article, we review current developments related to the detection of CKD biomarkers using microfluidics. A general trend in this emerging area is the search for novel, sensitive biomarkers for early detection of CKD using technology that is improved by means of microfluidics. It is anticipated that these innovative approaches will be soon adopted and utilized in both clinical and point-of-care settings, leading to improvements in life quality of patients.

Quantitative nonlinear optical assessment of atherosclerosis progression in rabbits.

Quantification of atherosclerosis has been a challenging task owing to its complex pathology. In this study, we validated a quantitative approach for assessing atherosclerosis progression in a rabbit model using a numerical matrix, optical index for plaque burden, derived directly from the nonlinear optical microscopic images captured on the atherosclerosis-affected blood vessel. A positive correlation between this optical index and the severity of atherosclerotic lesions, represented by the age of the rabbits, was established based on data collected from 21 myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits with age ranging between new-born and 27 months old. The same optical index also accurately identified high-risk locations for atherosclerotic plaque formation along the entire aorta, which was validated by immunohistochemical fluorescence imaging.

Original article: novelty of Canadian manufacture nasopharyngeal swabs for collection of samples being tested for SARS-CoV-2 in a pandemic setting.

Objectives: The COVID-19 pandemic caused a global shortage of nasopharyngeal (NP) swabs, required for RT-PCR testing. Canadian manufacturers were contacted to share NP swab innovations. The primary objective was to determine whether novel NP test swabs were comparable to commercially available swabs regarding user characteristics, ability to collect a specimen, and diagnostic performance using RT-PCR testing. Methods: Participants were randomized by swab (test/control) and nostril (left/right). A calculated positive percent agreement ≥90% was considered successful. Mean Ct values of viral genes and housekeeping gene (RNase P) were considered similar if a Ct difference ≤ 2 between control and test group was obtained. There also was a qualitative assessment of swabs usability. Results: 647 participants were enrolled from Huaycan Hospital in Lima, Peru, distributed over 8 NP swabs brands. Seven brands agreed to share their results. There were no statistically significant differences between the test swabs of these 7 brands and control swabs. Conclusion: All the seven brands are comparable to the commercially available flocked swabs used for SARS-CoV-2 regarding test results agreement, ability to collect a specimen, and user characteristics.

Photodynamic therapy of Porphyromonas gingivalis via liposome-encapsulated sensitizers.

Photodynamic therapy exploits the light-activation of a photosensitizer to cause cytotoxicity. Liposomes can be used to deliver hydrophobic photosensitizers to bacteria. Positively charged dioleoyltrimethylammoniumpropane:palmitoyloleoylphosphatidylcholine (1:1) liposomes bound quantitatively to the periodontal pathogen, Porphyromonas gingivalis. Following illumination, free and liposomal zinc phthalocyanine reduced the colony-forming unit (CFU) to 65 percent and 23 percent of controls, respectively. Thus, localization of the photosensitizer at the surface of bacteria via liposome binding enhanced the photodynamic cytotoxicity of zinc phthalocyanine.

Inflammatory Cytokines Induce Sustained CTLA-4 Cell Surface Expression on Human MAIT Cells.

Mucosal-associated invariant T (MAIT) cells acquire effector function in response to proinflammatory signals, which synergize with TCR-mediated signals. We asked if cell-intrinsic regulatory mechanisms exist to curtail MAIT cell effector function akin to the activation-induced expression of inhibitory receptors by conventional T cells. We examined human MAIT cells from blood and oral mucosal tissues by RNA sequencing and found differential expression of immunoregulatory genes, including CTLA-4, by MAIT cells isolated from tissue. Using an ex vivo experimental setup, we demonstrate that inflammatory cytokines were sufficient to induce CTLA-4 expression on the MAIT cell surface in the absence of TCR signals. Even brief exposure to the cytokines IL-12, IL-15, and IL-18 was sufficient for sustained CTLA-4 expression by MAIT cells. These data suggest that control of CTLA-4 expression is fundamentally different between MAIT cells and conventional T cells. We propose that this mechanism serves to limit MAIT cell-mediated tissue damage.

Fractal dimension and directional analysis of elastic and collagen fiber arrangement in unsectioned arterial tissues affected by atherosclerosis and aging.

Structural proteins like collagen and elastin are major constituents of the extracellular matrix (ECM). ECM degradation and remodeling in diseases significantly impact the microorganization of these structural proteins. Therefore, tracking the changes of collagen and elastin fiber morphological features within ECM impacted by disease progression could provide valuable insight into pathological processes such as tissue fibrosis and atherosclerosis. Benefiting from its intrinsic high-resolution imaging power and superior biochemical specificity, nonlinear optical microscopy (NLOM) is capable of providing information critical to the understanding of ECM remodeling. In this study, alterations of structural fibrillar proteins such as collagen and elastin in arteries excised from atherosclerotic rabbits were assessed by the combination of NLOM images and textural analysis methods such as fractal dimension (FD) and directional analysis (DA). FD and DA were tested for their performance in tracking the changes of extracellular elastin and fibrillar collagen remodeling resulting from atherosclerosis progression/aging. Although other methods of image analysis to study the organization of elastin and collagen structures have been reported, the simplified calculations of FD and DA presented in this work prove that they are viable strategies for extracting and analyzing fiber-related morphology from disease-impacted tissues. Furthermore, this study also demonstrates the potential utility of FD and DA in studying ECM remodeling caused by other pathological processes such as respiratory diseases, several skin conditions, or even cancer. NEW & NOTEWORTHY Textural analyses such as fractal dimension (FD) and directional analysis (DA) are straightforward and computationally viable strategies to extract fiber-related morphological data from optical images. Therefore, objective, quantitative, and automated characterization of protein fiber morphology in extracellular matrix can be realized by using these methods in combination with digital imaging techniques such as nonlinear optical microscopy (NLOM), a highly effective visualization tool for fibrillar collagen and elastic network. Combining FD and DA with NLOM is an innovative approach to track alterations of structural fibrillar proteins. The results illustrated in this study not only prove the effectiveness of FD and DA methods in extracellular protein characterization but also demonstrate their potential value in clinical and basic biomedical research where protein microstructure characterization is critical.

Early dental caries detection using a fibre-optic coupled polarization-resolved Raman spectroscopic system.

A new fibre-optic coupled polarization-resolved Raman spectroscopic system was developed for simultaneous collection of orthogonally polarized Raman spectra in a single measurement. An application of detecting incipient dental caries based on changes observed in Raman polarization anisotropy was also demonstrated using the developed fibre-optic Raman spectroscopic system. The predicted reduction of polarization anisotropy in the Raman spectra of caries lesions was observed and the results were consistent with those reported previously with Raman microspectroscopy. The capability of simultaneous collection of parallel- and cross-polarized Raman spectra of tooth enamel in a single measurement and the improved laser excitation delivery through fibre-optics demonstrated in this new design illustrates its future clinical potential.

Detection of early dental caries using polarized Raman spectroscopy.

A new technique based on polarized Raman spectroscopy is demonstrated for detecting early dental caries on extracted human teeth. Sound tooth enamel exhibited strong Raman polarization anisotropy whereas early caries consistently showed a lower degree of Raman polarization anisotropy. In particular, for sound enamel, the Raman peak arising from the symmetric nu1 vibration of PO(4) (3-) at 959 cm(-1) is strongly polarized. This is in contrast to the spectra of carious lesions that displayed weaker polarization dependence at 959 cm(-1). Such difference in the degree of Raman polarization anisotropy allows for discrimination between early dental caries and sound enamel.

Semi-parametric estimation in the compositional modeling of multicomponent systems from Raman spectroscopic data.

Identification and quantification of molecular species are central applications of molecular spectroscopy. In complex multicomponent systems like tissue samples, linear parametric models are often used to estimate the relative concentrations of the biochemical components of the sample. In situations where not all of the components of the sample are known or modeled, such parametric models can suffer from omitted variable bias and result in skewed estimates of component concentrations. We propose a semi-parametric approach that tries to avoid this omitted variable bias by effectively including unknown covariates as a non-parametric term in the regression equation. Constituent concentrations estimated with such partial linear models should outperform strict parametric linear models when the user has limited information on the composition of a multi-constituent system.

Review of near-infrared methods for wound assessment.

Wound management is a challenging and costly problem that is growing in importance as people are living longer. Instrumental methods are increasingly being relied upon to provide objective measures of wound assessment to help guide management. Technologies that employ near-infrared (NIR) light form a prominent contingent among the existing and emerging technologies. We review some of these technologies. Some are already established, such as indocyanine green fluorescence angiography, while we also speculate on others that have the potential to be clinically relevant to wound monitoring and assessment. These various NIR-based technologies address clinical wound management needs along the entire healing trajectory of a wound.

Ex vivo detection and characterization of early dental caries by optical coherence tomography and Raman spectroscopy.

Early dental caries detection will facilitate implementation of nonsurgical methods for arresting caries progression and promoting tooth remineralization. We present a method that combines optical coherence tomography (OCT) and Raman spectroscopy to provide morphological information and biochemical specificity for detecting and characterizing incipient carious lesions found in extracted human teeth. OCT imaging of tooth samples demonstrated increased light backscattering intensity at sites of carious lesions as compared to the sound enamel. The observed lesion depth on an OCT image was approximately 290 microm matching those previously documented for incipient caries. Using Raman microspectroscopy and fiber-optic-based Raman spectroscopy to characterize the caries further, spectral changes were observed in PO4 (3-) vibrations arising from hydroxyapatite of mineralized tooth tissue. Examination of various ratios of PO4 (3-) nu2, nu3, nu4 vibrations against the nu1 vibration showed consistent increases in carious lesions compared to sound enamel. The changes were attributed to demineralization-induced alterations of enamel crystallite morphology and/or orientation. OCT imaging is useful for screening carious sites and determining lesion depth, with Raman spectroscopy providing biochemical confirmation of caries. The combination has potential for development into a new fiber-optic diagnostic tool enabling dentists to identify early caries lesions with greater sensitivity and specificity.

Light- and pH-activated intracellular drug release from polymeric mesoporous silica nanoparticles.

Surface modified mesoporous silica nanoparticles (MSNs) with reduced toxicity were prepared for light and pH dual triggerable drug delivery system. Both 413 nm light and acidic environment can activate the drug release process, improving the pharmacological action. By applying rhodamine B (RhB) as a model drug, the accumulative RhB release is as high as 95% in pH 5.0 and in irradiation of 413 nm light, compared to only 55% in pH 7.4 and in dark. The anti-cancer drug camptothecin (CPT) loaded nanoparticles can kill cancer cells with IC50 value of 0.02 µg mL(-1) in exposure of 413 nm light, which is much lower than free CPT (about 0.1 µg mL(-1)). Multimodal nonlinear optical imaging microscopy (NLOM) was employed to acquire in vitro coherent anti-Stokes Raman (CARS) and two-photon excited fluorescence (TPEF) images of live MCF-7 cells and showed that the nanoparticles can be taken up by breast tumor cell MCF-7 with high efficiency, indicating its great potential for anti-cancer drug delivery system.

Investigation of white matter structure in velocardiofacial syndrome: a diffusion tensor imaging study.

OBJECTIVE: Velocardiofacial syndrome, caused by a deletion on chromosome 22q11.2, is often accompanied by cognitive, behavioral, and psychiatric impairments. Specifically, velocardiofacial syndrome has been proposed as a disease model for a genetically mediated subtype of schizophrenia. Velocardiofacial syndrome is also known to affect brain structure. The most prominent structural findings in velocardiofacial syndrome are reduced white matter volumes. However, the structure of white matter and extent of specific regional involvement in this syndrome have never been investigated. The current study used diffusion tensor imaging to investigate white matter structure in children and young adults with velocardiofacial syndrome. METHOD: Nineteen participants with velocardiofacial syndrome and 19 age- and gender-matched comparison subjects underwent diffusion-weighted magnetic resonance imaging scans. Whole brain voxel-by-voxel analyses were conducted to investigate white matter fractional anisotropy differences between the groups. RESULTS: Relative to the comparison group, the velocardiofacial syndrome group had reduced white matter anisotropy in the frontal, parietal, and temporal regions as well as in tracts connecting the frontal and temporal lobes. CONCLUSIONS: This study demonstrates that alterations of white matter tract structure occur in velocardiofacial syndrome. Reduced white matter anisotropy was observed in individuals with velocardiofacial syndrome in areas previously implicated in the neurocognitive phenotype of velocardiofacial syndrome. The finding of aberrant parietal white matter tracts as well as aberrant frontotemporal connectivity in velocardiofacial syndrome and in previous schizophrenia studies may be associated with increased vulnerability for development of psychotic symptoms.

Reducible polyamidoamine-magnetic iron oxide self-assembled nanoparticles for doxorubicin delivery.

We report a reducible copolymer self-assembled with superparamagnetic iron oxide nanoparticles (SPIONs) to deliver doxorubicin (DOX) for cancer therapy. The copolymer of reducible polyamidoamine (rPAA) with poly(ethylene glycol)(PEG)/dodecyl amine graft was synthesized by Michael addition. rPAA@SPIONs were formed by the alkyl grafts of reducible copolymers intercalated with the oleic acid layer capped on the surface of magnetite nanocrystals. The intercalating area formed a reservoir for hydrophobic anti-cancer drug (DOX), whilst the PEG moiety in the copolymers helped the nanoparticle well-dispersible in aqueous solution. We employed two-photon excited fluorescence (TPEF) and coherent anti-Stokes Raman (CARS) to investigate drug delivery in intra-cellular structures of live cells, and used Vivaview(®) technique to show real-time inhibition efficacy of nanoparticles in live cells. rPAA@SPIONs present efficiently drug loading with reducible responsibility in vitro tests. Finally, rPAA@SPIONs were tested in mice with xenograft MDA-MB-231 breast tumor though i.v. injection and inhibited tumor growth efficiently. MRI was used to monitor nanoparticles aggregation in tumor site. Histology and Prussian blue on kidney, liver, and heart in mice indicated that DOX/rPAA@SPIONs showed no significant toxicity for mice organs after 24 days treatment.

NIR initiated and pH sensitive single-wall carbon nanotubes for doxorubicin intracellular delivery.

In this work, we synthesized a pH sensitive poly(ethylene glycol)-doxorubicin (PEG-DOX) on single-wall carbon nanotubes (SWNTs). Using multimodal nonlinear optical imaging microscopy, we found that low power (1 mW cm-2) near-infrared radiation can initiate prodrug burst release from the carbon nanotubes in seconds. The successful SWNTs capping with PEG-DOX (denoted PEG-DOX@SWNT) was determined by transmission electron microscopy and FTIR results. The in vitro release of DOX from the PEG-DOX@SWNT was evaluated upon changes of pH values and NIR treating time. The cytotoxicity of the PEG-DOX@SWNT was also evaluated. This dual-sensitive delivery system based on SWNTs provides a facile approach to promote drug release and kill cancer cells.

Collagen morphology and texture analysis: from statistics to classification.

In this study we present an image analysis methodology capable of quantifying morphological changes in tissue collagen fibril organization caused by pathological conditions. Texture analysis based on first-order statistics (FOS) and second-order statistics such as gray level co-occurrence matrix (GLCM) was explored to extract second-harmonic generation (SHG) image features that are associated with the structural and biochemical changes of tissue collagen networks. Based on these extracted quantitative parameters, multi-group classification of SHG images was performed. With combined FOS and GLCM texture values, we achieved reliable classification of SHG collagen images acquired from atherosclerosis arteries with >90% accuracy, sensitivity and specificity. The proposed methodology can be applied to a wide range of conditions involving collagen re-modeling, such as in skin disorders, different types of fibrosis and muscular-skeletal diseases affecting ligaments and cartilage.

Nonlinear optical microscopy in decoding arterial diseases.

Pathological understanding of arterial diseases is mainly attributable to histological observations based on conventional tissue staining protocols. The emerging development of nonlinear optical microscopy (NLOM), particularly in second-harmonic generation, two-photon excited fluorescence and coherent Raman scattering, provides a new venue to visualize pathological changes in the extracellular matrix caused by atherosclerosis progression. These techniques in general require minimal tissue preparation and offer rapid three-dimensional imaging. The capability of label-free microscopic imaging enables disease impact to be studied directly on the bulk artery tissue, thus minimally perturbing the sample. In this review, we look at recent progress in applications related to arterial disease imaging using various forms of NLOM.

Evaluation of texture parameters for the quantitative description of multimodal nonlinear optical images from atherosclerotic rabbit arteries.

The composition and structure of atherosclerotic lesions can be directly related to the risk they pose to the patient. Multimodal nonlinear optical (NLO) microscopy provides a powerful means to visualize the major extracellular components of the plaque that critically determine its structure. Textural features extracted from NLO images were investigated for their utility in providing quantitative descriptors of structural and compositional changes associated with plaque development. Ten texture parameters derived from the image histogram and gray level co-occurrence matrix were examined that highlight specific structural and compositional motifs that distinguish early and late stage plaques. Tonal-texture parameters could be linked to key histological features that characterize vulnerable plaque: the thickness and density of the fibrous cap, size of the atheroma, and the level of inflammation indicated through lipid deposition. Tonal and texture parameters from NLO images provide objective metrics that correspond to structural and biochemical changes that occur within the vessel wall in early and late stage atherosclerosis.

Differentiating atherosclerotic plaque burden in arterial tissues using femtosecond CARS-based multimodal nonlinear optical imaging.

A femtosecond CARS-based nonlinear optical microscope was used to simultaneously image extracellular structural proteins and lipid-rich structures within intact aortic tissue obtained from myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits (WHHLMI). Clear differences in the NLO microscopic images were observed between healthy arterial tissue and regions dominated by atherosclerotic lesions. In the current ex-vivo study, we present a single parameter based on intensity changes derived from multi-channel NLO image to classify plaque burden within the vessel. Using this parameter we were able to differentiate between healthy regions of the vessel and regions with plaque, as well as distinguish plaques relative to the age of the WHHLMI rabbit.