RESUMO
Rapid, simple, inexpensive, accurate, and sensitive point-of-care (POC) detection of viral pathogens in bodily fluids is a vital component of controlling the spread of infectious diseases. The predominant laboratory-based methods for sample processing and nucleic acid detection face limitations that prevent them from gaining wide adoption for POC applications in low-resource settings and self-testing scenarios. Here, we report the design and characterization of an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a 2-stage microfluidic cartridge for sample processing and nucleic acid amplification, and a clip-on detection instrument that interfaces with the image sensor of a smartphone. The cartridge is designed to release viral RNA from Zika virus in whole blood using chemical lysis, followed by mixing with the assay buffer for performing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) reactions in six parallel microfluidic compartments. The battery-powered handheld detection instrument uniformly heats the compartments from below, and an array of LEDs illuminates from above, while the generation of fluorescent reporters in the compartments is kinetically monitored by collecting a series of smartphone images. We characterize the assay time and detection limits for detecting Zika RNA and gamma ray-deactivated Zika virus spiked into buffer and whole blood and compare the performance of the same assay when conducted in conventional PCR tubes. Our approach for kinetic monitoring of the fluorescence-generating process in the microfluidic compartments enables spatial analysis of early fluorescent "bloom" events for positive samples, in an approach called "Spatial LAMP" (S-LAMP). We show that S-LAMP image analysis reduces the time required to designate an assay as a positive test, compared to conventional analysis of the average fluorescent intensity of the entire compartment. S-LAMP enables the RT-LAMP process to be as short as 22 minutes, resulting in a total sample-to-answer time in the range of 17-32 minutes to distinguish positive from negative samples, while demonstrating a viral RNA detection as low as 2.70 × 102 copies per µl, and a gamma-irradiated virus of 103 virus particles in a single 12.5 µl droplet blood sample.
Assuntos
Infecção por Zika virus , Zika virus , Humanos , Microfluídica , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , Sensibilidade e Especificidade , Smartphone , Instrumentos Cirúrgicos , Zika virus/genética , Infecção por Zika virus/diagnósticoRESUMO
Understanding the efficacy of digital biomarkers in vision-based human motion analysis is essential, not only for interpreting the computer-aided exam results but also for advancing the next generation of digital health tool solutions. This study extensively analyzes digitized neurological examination (DNE) biomarkers for detecting and documenting exam features of Parkinson's disease (PD) and other neurological disorders (OD). Collected over 113 participants, DNE-113, a multi-test DNE database of finger tapping, finger to finger, forearm roll, stand-up and walk, and facial activation tests, covering a broader range of neurological abnormalities beyond PD is first proposed. Subsequently, DNE-113 is integrated into pyDNE - a convenient open-source toolbox, streamlining the creation and assessment of digital biomarkers. This toolbox empowers us to assess the quality of DNE biomarkers across diverse classification tasks. We showcase the discriminative potency of DNE biomarkers, successfully characterizing abnormal signals in neurological patients. Our findings highlight not only the potential use cases but also the persisting challenges in constructing digital biomarkers for computer-aided movement analysis on PD and OD patients.
RESUMO
The COVID-19 pandemic, in addition to the co-occurrence of influenza virus and respiratory syncytial virus (RSV), has emphasized the requirement for efficient and reliable multiplex diagnostic methods for respiratory infections. While existing multiplex detection techniques are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) and extraction and purification kits, the need for complex instrumentation and elevated cost limit their scalability and availability. In this study, we have developed a point-of-care (POC) device based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that can simultaneously detect four respiratory viruses (SARS-CoV-2, Influenza A, Influenza B, and RSV) and perform two controls in less than 30 min, while avoiding the use of the RNA extraction kit. The system includes a disposable microfluidic cartridge with mechanical components that automate sample processing, with a low-cost and portable optical reader and a smartphone app to record and analyze fluorescent images. The application as a real point-of-care platform was validated using swabs spiked with virus particles in nasal fluid. Our portable diagnostic system accurately detects viral RNA specific to respiratory pathogens, enabling deconvolution of coinfection information. The detection limits for each virus were determined using virus particles spiked in chemical lysis buffer. Our POC device has the potential to be adapted for the detection of new pathogens and a wide range of viruses by modifying the primer sequences. This work highlights an alternative approach for multiple respiratory virus diagnostics that is well-suited for healthcare systems in resource-limited settings or at home.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , SARS-CoV-2 , Humanos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/genética , COVID-19/diagnóstico , COVID-19/virologia , Vírus da Influenza B/isolamento & purificação , Vírus da Influenza B/genética , RNA Viral/análise , RNA Viral/isolamento & purificação , RNA Viral/genética , Técnicas de Diagnóstico Molecular/métodos , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Vírus Sinciciais Respiratórios/isolamento & purificação , Vírus Sinciciais Respiratórios/genéticaRESUMO
Neuroaxonal dystrophy (NAD) is a neurodegenerative disease characterized by spheroid (swollen axon) formation in the nervous system. In the present study, we focused on a newly established autosomal recessive mutant strain of F344-kk/kk rats with hind limb gait abnormalities and ataxia from a young age. Histopathologically, a number of axonal spheroids were observed throughout the central nervous system, including the spinal cord (mainly in the dorsal cord), brain stem, and cerebellum in F344-kk/kk rats. Transmission electron microscopic observation of the spinal cord revealed accumulation of electron-dense bodies, degenerated abnormal mitochondria, as well as membranous or tubular structures in the axonal spheroids. Based on these neuropathological findings, F344-kk/kk rats were diagnosed with NAD. By a positional cloning approach, we identified a missense mutation (V95E) in the Hspa8 (heat shock protein family A (Hsp70) member 8) gene located on chromosome 8 of the F344-kk/kk rat genome. Furthermore, we developed the Hspa8 knock-in (KI) rats with the V95E mutation using the CRISPR-Cas system. Homozygous Hspa8-KI rats exhibited ataxia and axonal spheroids similar to those of F344-kk/kk rats. The V95E mutant HSC70 protein exhibited the significant but modest decrease in the maximum hydrolysis rate of ATPase when stimulated by co-chaperons DnaJB4 and BAG1 in vitro, which suggests the functional deficit in the V95E HSC70. Together, our findings provide the first evidence that the genetic alteration of the Hspa8 gene caused NAD in mammals.
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The ability to use digitally recorded and quantified neurological exam information is important to help healthcare systems deliver better care, in-person and via telehealth, as they compensate for a growing shortage of neurologists. Current neurological digital biomarker pipelines, however, are narrowed down to a specific neurological exam component or applied for assessing specific conditions. In this paper, we propose an accessible vision-based exam and documentation solution called Digitized Neurological Examination (DNE) to expand exam biomarker recording options and clinical applications using a smartphone/tablet. Through our DNE software, healthcare providers in clinical settings and people at home are enabled to video capture an examination while performing instructed neurological tests, including finger tapping, finger to finger, forearm roll, and stand-up and walk. Our modular design of the DNE software supports integrations of additional tests. The DNE extracts from the recorded examinations the 2D/3D human-body pose and quantifies kinematic and spatio-temporal features. The features are clinically relevant and allow clinicians to document and observe the quantified movements and the changes of these metrics over time. A web server and a user interface for recordings viewing and feature visualizations are available. DNE was evaluated on a collected dataset of 21 subjects containing normal and simulated-impaired movements. The overall accuracy of DNE is demonstrated by classifying the recorded movements using various machine learning models. Our tests show an accuracy beyond 90% for upper-limb tests and 80% for the stand-up and walk tests.
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Smartphone , Software , Dedos , Humanos , Aprendizado de Máquina , Exame NeurológicoRESUMO
The RIß subunit of cAMP-dependent protein kinase (PKA), encoded by Prkar1b, is a neuronal isoform of the type I regulatory subunit of PKA. Mice lacking the RIß subunit exhibit normal long-term potentiation (LTP) in the Schaffer collateral pathway of the hippocampus and normal behavior in the open-field and fear conditioning tests. Here, we combined genetic, electrophysiological, and behavioral approaches to demonstrate that the RIß subunit was involved in body tremor, LTP in the Schaffer collateral pathway, and fear conditioning memory in rats. Genetic analysis of WTC-furue, a mutant strain with spontaneous tremors, revealed a deletion in the Prkar1b gene of the WTC-furue genome. Prkar1b-deficient rats created by the CRISPR/Cas9 system exhibited body tremor. Hippocampal slices from mutant rats showed deficient LTP in the Schaffer collateral-CA1 synapse. Mutant rats also exhibited decreased freezing time following contextual and cued fear conditioning, as well as increased exploratory behavior in the open field. These findings indicate the roles of the RIß subunit in tremor pathogenesis and contextual and cued fear memory, and suggest that the hippocampal and amygdala roles of this subunit differ between mice and rats and that rats are therefore beneficial for exploring RIß function.
Assuntos
Subunidade RIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Hipocampo/metabolismo , Transtornos da Memória/genética , Tremor/genética , Animais , Comportamento Animal/fisiologia , Sistemas CRISPR-Cas/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Modelos Animais de Doenças , Medo/fisiologia , Hipocampo/patologia , Humanos , Memória/fisiologia , Transtornos da Memória/fisiopatologia , Camundongos , Mutação/genética , Plasticidade Neuronal/genética , Neurônios/metabolismo , Neurônios/patologia , Ratos , Tremor/fisiopatologiaRESUMO
Gastrointestinal (GI) endoscopy has been an active field of research motivated by the large number of highly lethal GI cancers. Early GI cancer precursors are often missed during the endoscopic surveillance. The high missed rate of such abnormalities during endoscopy is thus a critical bottleneck. Lack of attentiveness due to tiring procedures, and requirement of training are few contributing factors. An automatic GI disease classification system can help reduce such risks by flagging suspicious frames and lesions. GI endoscopy consists of several multi-organ surveillance, therefore, there is need to develop methods that can generalize to various endoscopic findings. In this realm, we present a comprehensive analysis of the Medico GI challenges: Medical Multimedia Task at MediaEval 2017, Medico Multimedia Task at MediaEval 2018, and BioMedia ACM MM Grand Challenge 2019. These challenges are initiative to set-up a benchmark for different computer vision methods applied to the multi-class endoscopic images and promote to build new approaches that could reliably be used in clinics. We report the performance of 21 participating teams over a period of three consecutive years and provide a detailed analysis of the methods used by the participants, highlighting the challenges and shortcomings of the current approaches and dissect their credibility for the use in clinical settings. Our analysis revealed that the participants achieved an improvement on maximum Mathew correlation coefficient (MCC) from 82.68% in 2017 to 93.98% in 2018 and 95.20% in 2019 challenges, and a significant increase in computational speed over consecutive years.