Label-free and amplification-free viral RNA quantification from primate biofluids using a trapping-assisted optofluidic nanopore platform.

Viral outbreaks can cause widespread disruption, creating the need for diagnostic tools that provide high performance and sample versatility at the point of use with moderate complexity. Current gold standards such as PCR and rapid antigen tests fall short in one or more of these aspects. Here, we report a label-free and amplification-free nanopore sensor platform that overcomes these challenges via direct detection and quantification of viral RNA in clinical samples from a variety of biological fluids. The assay uses an optofluidic chip that combines optical waveguides with a fluidic channel and integrates a solid-state nanopore for sensing of individual biomolecules upon translocation through the pore. High specificity and low limit of detection are ensured by capturing RNA targets on microbeads and collecting them by optical trapping at the nanopore location where targets are released and rapidly detected. We use this device for longitudinal studies of the viral load progression for Zika and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections in marmoset and baboon animal models, respectively. The up to million-fold trapping-based target concentration enhancement enables amplification-free RNA quantification across the clinically relevant concentration range down to the assay limit of RT-qPCR as well as cases in which PCR failed. The assay operates across all relevant biofluids, including semen, urine, and whole blood for Zika and nasopharyngeal and throat swab, rectal swab, and bronchoalveolar lavage for SARS-CoV-2. The versatility, performance, simplicity, and potential for full microfluidic integration of the amplification-free nanopore assay points toward a unique approach to molecular diagnostics for nucleic acids, proteins, and other targets.

Optofluidic multiplex detection of single SARS-CoV-2 and influenza A antigens using a novel bright fluorescent probe assay.

The urgency for the development of a sensitive, specific, and rapid point-of-care diagnostic test has deepened during the ongoing COVID-19 pandemic. Here, we introduce an ultrasensitive chip-based antigen test with single protein biomarker sensitivity for the differentiated detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A antigens in nasopharyngeal swab samples at diagnostically relevant concentrations. The single-antigen assay is enabled by synthesizing a brightly fluorescent reporter probe, which is incorporated into a bead-based solid-phase extraction assay centered on an antibody sandwich protocol for the capture of target antigens. After optimization of the probe release for detection using ultraviolet light, the full assay is validated with both SARS-CoV-2 and influenza A antigens from clinical nasopharyngeal swab samples (PCR-negative spiked with target antigens). Spectrally multiplexed detection of both targets is implemented by multispot excitation on a multimode interference waveguide platform, and detection at 30 ng/mL with single-antigen sensitivity is reported.

Optofluidic Flow-Through Biosensor Sensitivity - Model and Experiment.

We present a model and simulation for predicting the detected signal of a fluorescence-based optical biosensor built from optofluidic waveguides. Typical applications include flow experiments to determine pathogen concentrations in a biological sample after tagging relevant DNA or RNA sequences. An overview of the biosensor geometry and fabrication processes is presented. The basis for the predictive model is also outlined. The model is then compared to experimental results for three different biosensor designs. The model is shown to have similar signal statistics as physical tests, illustrating utility as a pre-fabrication design tool and as a predictor of detection sensitivity.

Greatly Enhanced Single Particle Fluorescence Detection Using High Refractive Index Liquid-Core Waveguides.

High sensitivity and easy integration with microfabrication techniques has made silicon photonics one of the leading technologies used to build biosensors for diagnostic applications. Here we introduce a new silicon dioxide based optofluidic platform having a planar solid-core (SC) waveguide orthogonally intersecting a liquid-core (LC) waveguide with high refractive index ZnI2 salt solution as core. This enables both more uniform collection of particle fluorescence by the core mode and its propagation to an off-chip detector. This approach results in ultra-high sensitivity performance, demonstrated by achieving 8X enhancement in signal-to-noise ratio, a 45x increase in detection efficiency, and a 100x lower detection limit of 80 aM of fluorescent nanobeads. This represents a key step towards an ultrasensitive biosensor system for analyzing pathogens at clinical concentrations.

Optofluidic Amplification-free Multiplex Detection of Viral Hemorrhagic Fevers.

Infectious disease outbreaks such as Ebola and other Viral Hemorrhagic Fevers (VHF) require low-complexity, specific, and differentiated diagnostics as illustrated by the recent outbreak in the Democratic Republic of Congo. Here, we describe amplification-free spectrally multiplex detection of four different VHF total RNA samples using multi-spot excitation on a multimode interference waveguide platform along with combinatorial fluorescence labeling of target nucleic acids. In these experiments, we observed an average of 8-fold greater fluorescence signal amplitudes for the Ebola total RNA sample compared to three other total RNA samples: Lake Victoria Marburg Virus, Ravn Marburg Virus, and Crimean-Congo Hemorrhagic Fever. We have attributed this amplitude amplification to an increased amount of RNA during synthesis of soluble glycoprotein in infection. This hypothesis is confirmed by single molecule detection of the total RNA sample after heat-activated release from the carrier microbeads. From these experiments, we observed at least a 5.3x higher RNA mass loading on the Ebola carrier microbeads compared to the Lake Victoria Marburg carrier microbeads, which is consistent with the known production of soluble glycoprotein during infection.

Free-Space Excitation of Optofluidic Devices for Pattern-Based Single Particle Detection.

Optofluidic sensors have enabled single molecule sensing using planar, waveguide dependent multi-spot fluorescence excitation. Here, we demonstrate a new approach to single-particle fluorescence sensing using free-space, top-down illumination of liquid-core antiresonant reflecting optical waveguide (ARROW) devices using two different multi-spot excitation techniques. First, the liquid core ARROW waveguide is excited with a focused beam through a slit pattern milled into an opaque aluminum film, showing comparable performance for single bead fluorescence detection as in-plane, multi-mode interference waveguide based excitation. The second top-down illumination technique images the spot pattern from a Y-splitter SiO2 waveguide chip directly onto the detection device for efficient power utilization and circumventing the need for an opaque cover, producing a further 2.7x improvement in signal-to-noise ratio. The two top-down approaches open up new possibilities for chip-based optical particle sensing with relaxed alignment tolerances.

Availability as key determinant in the palliative home care setting from the patients' and family caregivers' perspectives: A quantitative-qualitative-content analysis approach.

OBJECTIVE: A sense of security is important in palliative home care. Yet, knowledge about which components contribute most to feeling secure from the patients' and family caregivers' perspectives, especially since the introduction of specialist palliative home care, is sparse. The goal of the current study was to determine the key components contributing to a sense of security and how they relate to each other as experienced by patients and family caregivers in specialist and generalist palliative home care. METHODS: The current sub-study, as part of a larger study, was performed in different regions in Germany. Palliative care patients and family caregivers of at least 18 years of age, being cared for at home were interviewed using semi-structured interview guides following a three-factor model and analyzed by using a combined quantitative-qualitative-content approach. RESULTS: One hundred and ninty-seven patients and 10 carers completed interviews between December 2017 and April 2019. The majority of patients were diagnosed with an oncological disease. Sense of security was mentioned particularly often suggesting its high relevance. We identified nine subcategories that were all mentioned more frequently by specialist than generalist palliative home care recipients in the following order of priority and relation: (i) patient-centeredness: availability, provision of information/education, professional competence, patient empowerment, and trust (ii) organizational work: comprehensive responsibility, external collaboration, and internal cooperation, and (iii) direct communication. SIGNIFICANCE OF RESULTS: The work of specialist palliative home care services in particular was perceived as very effective and beneficial. Our findings confirm a previously developed three-factor model allowing for generalizability and revealed that availability was most important for improving the sense of security for effective palliative home care.

[Neurological complications of hepatitis C infections]. / Neurologische Komplikationen der Hepatitis-C-Infektion.

Chronic hepatitis C virus (HCV) infection is a highly prevalent systemic disease, which can cause a variety of neurological complications. The HCV-associated symptoms can be differentiated into central and peripheral nervous systems as well as the musculature. Important pathomechanisms are HCV-associated autoimmunity (e.g. mixed cryoglobulinemia with polyneuropathy) and direct neurotoxic effects of the virus (e.g. HCV-associated cognitive deficits). Distal symmetric polyneuropathies, small fiber neuropathies and cognitive deficits are the most prevalent neurological manifestations. Furthermore, HCV infection is a risk factor for ischemic and hemorrhagic stroke as well as Parkinson's disease. As HCV infection has become a permanently curable disease in >90% of patients, early identification and antiviral treatment of HCV positive patients is of utmost importance.

[Smarter Medicine in Headache Care - presentation and discussion of 5 recommendations]. / Smarter Medicine in Diagnose und Behandlung von Kopfschmerzen.

Smarter Medicine in Headache Care - presentation and discussion of 5 recommendations Abstract. An unequivocal headache diagnosis cannot always be made. The lack of diagnostic tests able to prove primary headaches often prompts physicians to perform unnecessary examinations to reduce their uncertainty. When setting out the therapeutic strategy, again, insecurity often leads to mendable choices. In this Delphi study, members of the therapy commission of the Swiss Headache Society collected, rated, and re-rated doubtful and questionable procedures. Five recommendations that resulted from this survey are presented and reviewed in this article. The recommendations are: (A) no repeated cerebral imaging in headaches with unchanged phenotype; (B) no computed tomography in the work-up of non-acute headaches; (C) no tooth extraction to treat persistent idiopathic facial pain, (D) no migraine surgery; (E) no removal of amalgam fillings to treat headache disorders.

Pneumatically tunable optofluidic DFB dye laser using corrugated sidewalls.

Polydimethylsiloxane-based optofluidics provides a powerful platform for a complete analytical lab-on-chip. Here, we report on a novel on-chip laser source that can be integrated with sample preparation and analysis functions. A corrugated sidewall structure is integrated into a microfluidic channel to form a distributed feedback (DFB) laser using rhodamine 6G dissolved in an ethylene glycol and water solution. Lasing is demonstrated with a threshold pump power of 87.9 µW, corresponding to a pump intensity of 52.7mW/cm2. Laser threshold and output power are optimized with respect to rhodamine 6G concentration and core index and found to be in good agreement with a rate equation model. Additionally, the laser can be switched on and off mechanically using a pneumatic cell inducing positive pressure on the grating.

Effects of Post-Etch Microstructures on the Optical Transmittance of Silica Ridge Waveguides.

Silica waveguides are often etched by reactive ion etch (RIE) processes. These processes can leave residual topography that can increase optical loss. We investigated the relation between optical loss and various RIE etch. A wet etch step meant to remove microstructures was also considered and compared. Ridge waveguides were fabricated in plasma enhanced chemical vapor deposited films by three different RIE processes, each with a different gas composition, pressure setting, and applied power setting. Half of each set of waveguides were also subjected to a hydrofluoric acid (HF) solution. The waveguides were tested for optical transmission via the cutback method. The transmission vs waveguide length measurements were plotted to fit an exponential curve and the optical loss and measurement uncertainty for each waveguide set was calculated. Clear distinctions in optical loss were found between the different RIE processes. The HF treatment also has an effect, significantly reducing optical loss for two processes and increasing it for the third. Of the tested RIE processes, one can be suggested for silica waveguides. It results in the lowest optical loss and coincidently has the fastest etch rate.

Solid-state membranes formed on natural menisci.

We present a method to create robust, nanoscale solid-state membranes using the natural shape of a liquid meniscus as a template. A narrow, open channel is etched into a silicon substrate and then a photoresist polymer is introduced into the channel through spontaneous capillary action. The natural concave meniscus formed by the polymer is then covered by a thin chemical vapor deposited membrane. The polymer is removed by sacrificial etching, leaving behind a suspended membrane. Membranes as large as 20 µm by 9 mm can be fabricated with a thickness as low as 50 nm.

Enhanced Detection of Single Viruses On-Chip via Hydrodynamic Focusing.

Planar optofluidics provide a powerful tool for facilitating chip-scale light-matter interactions. Silicon-based liquid core waveguides have been shown to offer single molecule sensitivity for efficient detection of bioparticles. Recently, a PDMS based planar optofluidic platform was introduced that opens the way to rapid development and prototyping of unique structures, taking advantage of the positive attributes of silicon dioxide-based optofluidics and PDMS based microfluidics. Here, hydrodynamic focusing is integrated into a PDMS based optofluidic chip to enhance the detection of single H1N1 viruses on-chip. Chip-plane focusing is provided by a system of microfluidic channels to force the particles towards a region of high optical collection efficiency. Focusing is demonstrated and enhanced detection is quantified using fluorescent polystyrene beads where the coefficient of variation is found to decrease by a factor of 4 with the addition of hydrodynamic focusing. The mean signal amplitude of fluorescently tagged single H1N1 viruses is found to increase with the addition of focusing by a factor of 1.64.

Voxel-wise correlation of functional imaging parameters in HNSCC patients receiving PET/MRI in an irradiation setup.

PURPOSE: The purpose of this study was to demonstrate the feasibility of voxel-wise multiparametric characterization of head and neck squamous cell carcinomas (HNSCC) using hybrid multiparametric magnetic resonance imaging and positron emission tomography with [18F]-fluorodesoxyglucose (FDG-PET/MRI) in a radiation treatment planning setup. METHODS: Ten patients with locally advanced HNSCC were examined with a combined FDG-PET/MRI in an irradiation planning setup. The multiparametric imaging protocol consisted of FDG-PET, T2-weighted transverse short tau inversion recovery sequence (STIR) and diffusion-weighted MRI (DWI). Primary tumours were manually segmented and quantitative imaging parameters were extracted. PET standardized uptake values (SUV) and DWI apparent diffusion coefficients (ADC) were correlated on a voxel-wise level. RESULTS: Images acquired in this specialised radiotherapy planning setup achieved good diagnostic quality. Median tumour volume was 4.9 [1.1-42.1] ml. Mean PET SUV and ADC of the primary tumours were 5 ± 2.5 and 1.2 ± 0.3 10-3 mm2/s, respectively. In voxel-wise correlation between ADC values and corresponding FDG SUV of the tumours, a significant negative correlation was observed (r = -0.31 ± 0.27, p < 0.05). CONCLUSION: Multiparametric voxel-wise characterization of HNSCC is feasible using combined PET/MRI in a radiation planning setup. This technique may provide novel insights into tumour biology with regard to radiation therapy in the future.

Multi-channel velocity multiplexing of single virus detection on an optofluidic chip.

Liquid-core waveguide-based optofluidic devices have proven to be valuable tools for analysis of biological samples in fluid. They have enabled single bioparticle sensitivity while maintaining in-plane detection via light-induced fluorescence. The incorporation of multi-spot excitation with multimode interference (MMI) waveguides has enabled spatially and spectrally multiplexed detection of single viruses on an oxide-based optofluidic platform. Here, we introduce a new way of MMI-based multiplexing where multiple analysis channels are placed within a single multi-spot pattern. This stacked channel design enables both velocity and spectral multiplexing of single particles. The principle is demonstrated with differentiated detection of single H3N2 and H1N1 viruses on a polydimethylsiloxane platform.

18F-FDG-PET detects complete response to PD1-therapy in melanoma patients two weeks after therapy start.

PURPOSE: The aim of the study was to evaluate if 18F-FDG-PET has the potential to detect complete responders to PD1-therapy in patients with unresectable metastasized melanoma two weeks after therapy initiation. METHODS: Between September 2014 and May 2016, ten patients (four females; 65 ± 12 y) received a whole-body 18F-FDG-PET/MRI examination at three time points: Before therapy start (t0, base-line), two weeks (t1, study examination) and three months after treatment initiation (t2, reference standard). Therapy response was assessed with PET response criteria in solid tumors (PERCIST). Time to progression and overall survival (OS) were obtained for all patients. RESULTS: Three patients with partial metabolic response in PET at t1 turned out to have complete response at t2. No tumor relapse was observed in those patients so far (observation period: 265, 511 and 728 days, respectively). At t2, progressive metabolic disease (PMD) was seen in six patients from whom four showed PMD and two showed stable metabolic disease (SMD) at t1. OS in patients with PMD at t2 varied between 148 and 814 days. SMD at both t1 and t2 was seen in one patient, tumor progress was observed after 308 days. CONCLUSION: Our study indicates that whole-body 18F-FDG-PET might be able to reliably identify complete responders to PD1-therapy as early as two weeks after therapy initiation in stage IV melanoma patients. This might help to shorten therapy regimes and avoid unnecessary side effects in the future.

Buried Rib SiO2 Multimode Interference Waveguides for Optofluidic Multiplexing.

Multimode interference (MMI) waveguides can be used to create wavelength-dependent spot patterns which enables simultaneous analyte detection on a single optofluidic chip, useful for disease diagnostics. The fidelity of such multi-spot patterns is important for high sensitivity and accurate target identification. Buried rib structures have been incorporated into these SiO2-based waveguides to improve environmental stability. Through experiments and simulation, this letter explores design parameters for a buried MMI rib waveguide based on anti-resonant reflecting optical waveguides in order to produce high-fidelity spot patterns. Optimal rib heights and widths are reported in the context of available microfabrication etch technology and performance for an optimized biosensor is shown.

Optimized ARROW-Based MMI Waveguides for High Fidelity Excitation Patterns for Optofluidic Multiplexing.

Multimode interference (MMI) waveguides can be used for multiplexing and de-multiplexing optical signals. High fidelity, wavelength dependent multi-spot patterns from MMI waveguides are useful for sensitive and simultaneous identification of multiple targets in multiplexed fluorescence optofluidic biosensors. Through experiments and simulation, this paper explores design parameters for an MMI rib anti-resonant reflecting optical waveguide (ARROW) in order to produce high fidelity spot patterns at the liquid core biomarker excitation region. Width and etch depth of the single excitation rib waveguide used to excite the MMI waveguide are especially critical because they determine the size of the input optical mode which is imaged at the MMI waveguide's output. To increase optical throughput into the MMI waveguide when light is coupled in from an optical fiber, tapers in the waveguide width can be used for better mode matching.

Cognitive impairment and quality of life of people with epilepsy and neurocysticercosis in Zambia.

Cognitive impairment and quality of life (Qol) are important to assess the burden of epilepsy and neurocysticercosis (NCC), which are common but neglected in Sub-Saharan Africa (SSA). The aims of this study were to assess cognitive performance and Qol of people with epilepsy (PWE) in Zambia and to explore differences in PWE with and without NCC. In this community based, cross-sectional case-control-study, 47 PWE and 50 healthy controls completed five neuropsychological tests (Mini Mental State Examination (MMSE), Digit Span, Selective Reminding Test (SRT), Spatial Recall Test (SPART), Test Battery of Attentional Performance (TAP)) and a World Health Organization (WHO) questionnaire of Qol. Comparisons were made between PWE (n=47) and healthy controls (n=50) and between PWE with NCC (n=28) and without NCC (n=19), respectively, using Analysis of Covariance (ANCOVA) and Linear Models (LMs) while correcting for confounders such as age, sex, and schooling years, and adjusting for multiplicity. Working memory, spatial memory, verbal memory, verbal learning, orientation, speech and language reception, visuoconstructive ability, and attentional performance were significantly reduced in PWE compared with healthy controls (ANCOVA and LM, p<0.05). Quality of life of PWE was significantly lower in three domains (psychological, social, environmental) and in overall Qol compared with healthy controls (ANCOVA, p<0.05). There were no significant differences between PWE with NCC and PWE without NCC detected by ANCOVA. Using LM, significant differences between the groups were detected in four tests, indicating worse performance of PWE without NCC in MMSE, Digit Span, SPART, and lower physical Qol. Epilepsy was found to be associated with cognitive impairment and reduced Qol. People with epilepsy due to NCC had similar cognitive impairment and Qol compared with PWE due to other causes. Further studies should investigate the role of different conditions of NCC and the role of seizures on cognition and Qol.

Needs of people with advanced dementia in their final phase of life: A multi-perspective qualitative study in nursing homes.

BACKGROUND: People with advanced dementia present an important target group for palliative care. They suffer a range of symptoms, and their verbal communication abilities are highly restricted. At present, little is known about their needs in the final phase of life. AIM: To identify the needs of people with advanced dementia in their final phase of life and to explore the aspects relevant to first recognize and then meet these needs. DESIGN: Multi-perspective qualitative study using grounded theory methodology conducting group discussions, individual interviews, and participant observation. SETTING/PARTICIPANTS: The study encompassed nursing homes and involved health professionals, relatives, and residents with advanced dementia. RESULTS: Data were collected in six nursing homes. Nine group discussions and three individual interviews were conducted comprising 42 health professionals and 14 relatives. Participant observations aided in giving the perspective of 30 residents with advanced dementia. Data analysis generated a total of 25 physical, psychosocial, and spiritual needs divided into 10 categories. Physical needs were classified as follows: "food intake," "physical well-being," and "physical activity and recovery." Categories of psychosocial needs were classified as follows: "adaptation of stimuli," "communication," "personal attention," "participation," "familiarity and safety," as well as "self-determination." Spiritual needs addressed "religion." The results revealed a multitude of key aspects for recognizing and meeting these needs, stressing the importance of personhood. CONCLUSION: People with advanced dementia in their final phase of life have a multitude of individual and complex needs. This evidence contributes to narrowing the current research gap, offering an orientation framework for research and practice.