Feasibility of a point-of-care ultrasound protocol for cardiorespiratory evaluation of horses in different clinical settings.

BACKGROUND: A point-of-care ultrasound (POCUS) protocol for evaluation of the cardiac and respiratory systems in horses does not exist. OBJECTIVES: (a) Describe the windows of a POCUS protocol for cardiorespiratory assessment of horses (CRASH); (b) Estimate the number of acoustic windows that can be acquired by a sonographer-in-training; (c) Estimate the time required to complete the protocol for specific groups of horses; (d) Describe the sonographic abnormalities detected in horses presented with cardiovascular, respiratory, or systemic disease. ANIMALS: Twenty-seven healthy horses, 14 horses competing in athletic events, and 120 horses with clinical disease. METHOD: A pocket-sized ultrasound device was used to acquire 7 sonographic cardiorespiratory windows in various clinical scenarios. The duration of the examination was timed, and images were evaluated for diagnostic quality. Abnormalities in horses with clinical disease were determined by an expert sonographer. RESULTS: The CRASH protocol could be performed in healthy and diseased horses in hospital, barn, and competition settings between 5.5 ± 0.9 (athletic horses) and 6.9 ± 1.9 min (horses with clinical disease). Thoracic windows were obtained most consistently, followed by right parasternal long-axis echocardiographic windows. Frequently detected abnormalities were pleural fluid, lung consolidation, B-lines, and moderate-to-severe left-sided heart disease. CONCLUSIONS: The CRASH protocol was feasible using a pocket-sized ultrasound device in various groups of horses, could be completed rapidly in a variety of settings, and frequently identified sonographic abnormalities when evaluated by an expert sonographer. The diagnostic accuracy, observer agreement, and utility of the CRASH protocol merit further evaluation.

Current regulatory landscape for viral point-of-care testing in the United States.

Historically, the diagnosis of viral infections has been accomplished using a combination of laboratory-based methods, including culture, serology, antigen-based tests, and molecular (e.g., real-time PCR) assays. Although these methods provide an accurate way to detect viral pathogens, testing in a centralized laboratory may delay results, which could impact patient diagnosis and management. Point-of-care tests, including antigen- and molecular-based assays, have been developed to assist with the timely diagnosis of several viral infections, such as influenza, respiratory syncytial virus, and COVID-19. Despite the ability of point-of-care tests to provide rapid results (i.e., <30 min), there are issues to consider prior to their routine use, including test performance and specific regulatory requirements. This review will provide a summary of the regulatory landscape of point-of-care tests for viral infections in the United States, and address important considerations such as site certification, training and inspection readiness.

CRISPR Cas12a-enabled biosensors coupled with commercial pregnancy test strips for the visible point-of-care testing of SARS-CoV-2.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has promoted the development of nucleic acid diagnosis technology. Several platforms with isothermal amplification methods have achieved sensitive and specific detection of SARS-CoV-2. However, they still suffer from complicated operations, delicate instruments, and unintuitive signal output modes. Here, a system consisting of CRISPR Cas12a-based biosensors and commercial pregnancy test strips (CRISPR-PTS) was established for the point-of-care testing of SARS-CoV-2. The target viral nucleic acids were finally reflected on the test strips through four steps, namely sample pretreatment, RT-RAA amplification, CRISPR Cas12a reaction, and separation-free hCG detection. This CRISPR-PTS assay possessed an outstanding sensitivity of as low as 1 copy per µL for SARS-CoV-2 detection and showed an excellent specificity in distinguishing the SARS-CoV-2 pseudovirus as well as other SARS-like viral clinical samples. In addition, the CRISPR-PTS assay performed well in practical applications, with 96.3% agreement versus RT-qPCR in spiked samples. With the advantages of low reagent cost, simple operation procedure, and visible signal output, CRISPR-PTS assay was expected to provide a strong supplement in the prevention and early diagnosis of infectious diseases in resource-limited situations.

Ten Influential Point-of-Care Ultrasound Papers: 2022 in Review.

Keeping up with the latest developments in the point-of-care ultrasound (POCUS) literature is challenging, as with any area of medicine. Our group of POCUS experts has selected 10 influential papers from the past 12 months and provided a short summary of each. We hope to provide emergency physicians, intensivists, and other acute care providers with a succinct update concerning some key areas of ultrasound interest.

Nucleic Acid Point-of-Care Testing to Improve Diagnostic Preparedness.

Testing programs for severe acute respiratory syndrome coronavirus 2 have relied on high-throughput polymerase chain reaction laboratory tests and rapid antigen assays to meet diagnostic needs. Both technologies are essential; however, issues of cost, accessibility, manufacturing delays, and performance have limited their use in low-resource settings and contributed to the global inequity in coronavirus disease 2019 testing. Emerging low-cost, multidisease point-of-care nucleic acid tests may address these limitations and strengthen pandemic preparedness, especially within primary healthcare where most cases of disease first present. Widespread deployment of these novel technologies will also help close long-standing test access gaps for other diseases, including tuberculosis, human immunodeficiency virus, cervical cancer, viral hepatitis, and sexually transmitted infections. We propose a more optimized testing framework based on greater use of point-of-care nucleic acid tests together with rapid immunologic assays and high-throughput laboratory molecular tests to improve the diagnosis of priority endemic and epidemic diseases, as well as strengthen the overall delivery of primary healthcare services.

A universal CRISPR/Cas12a-powered intelligent point-of-care testing platform for multiple small molecules in the healthcare, environment, and food.

Growing studies focusing on nuclear acid detection via the emerging CRISPR technique demonstrate its promising application. However, limited works solve the identification of non-nucleic acid targets, especially multiple small molecules. To address challenges for point-of-care testing (POCT) in complex matrices for healthcare, environment, and food safety, we developed CRISPR Cas12a-powered highly sensitive, high throughput, intelligent POCT (iPOCT) for multiple small molecules based on a smartphone-controlled reader. As a proof of concept, aflatoxin B1 (AFB1), benzo[a]pyrene (BaP), and capsaicin (CAP) were chosen as multiple targets. First, three antigens were preloaded in independent microwells. Then, the antibody/antigen-induced fluorescent signals were consecutively transferred from the biotin-streptavidin to CRISPR/Cas12a system. Third, the fluorescent signals were recorded by a smartphone-controlled handheld dark-box readout. Under optimization, detection limits in AFB1, BaP, and CAP were 0.00257, 4.971, and 794.6 fg/mL with wide linear ranges up to four orders of magnitude. Using urine, water, soybean oil, wheat, and peanuts as the complex matrix, we recorded high selectivity, considerable recovery, repeatability, and high consistency comparison to HPLC-MS/MS methods. This work promises a practical intelligent POCT platform for multiple targets in lipid-soluble and water-soluble matrices and could be extensively applied for healthcare, environment, and food safety.

Trends of respiratory virus detection in point-of-care testing: A review.

In resource-limited conditions such as the COVID-19 pandemic, on-site detection of diseases using the Point-of-care testing (POCT) technique is becoming a key factor in overcoming crises and saving lives. For practical POCT in the field, affordable, sensitive, and rapid medical testing should be performed on simple and portable platforms, instead of laboratory facilities. In this review, we introduce recent approaches to the detection of respiratory virus targets, analysis trends, and prospects. Respiratory viruses occur everywhere and are one of the most common and widely spreading infectious diseases in the human global society. Seasonal influenza, avian influenza, coronavirus, and COVID-19 are examples of such diseases. On-site detection and POCT for respiratory viruses are state-of-the-art technologies in this field and are commercially valuable global healthcare topics. Cutting-edge POCT techniques have focused on the detection of respiratory viruses for early diagnosis, prevention, and monitoring to protect against the spread of COVID-19. In particular, we highlight the application of sensing techniques to each platform to reveal the challenges of the development stage. Recent POCT approaches have been summarized in terms of principle, sensitivity, analysis time, and convenience for field applications. Based on the analysis of current states, we also suggest the remaining challenges and prospects for the use of the POCT technique for respiratory virus detection to improve our protection ability and prevent the next pandemic.

Advancements in CRISPR-Based Biosensing for Next-Gen Point of Care Diagnostic Application.

With the move of molecular tests from diagnostic labs to on-site testing becoming more common, there is a sudden rise in demand for nucleic acid-based diagnostic tools that are selective, sensitive, flexible to terrain changes, and cost-effective to assist in point-of-care systems for large-scale screening and to be used in remote locations in cases of outbreaks and pandemics. CRISPR-based biosensors comprise a promising new approach to nucleic acid detection, which uses Cas effector proteins (Cas9, Cas12, and Cas13) as extremely specialized identification components that may be used in conjunction with a variety of readout approaches (such as fluorescence, colorimetry, potentiometry, lateral flow assay, etc.) for onsite analysis. In this review, we cover some technical aspects of integrating the CRISPR Cas system with traditional biosensing readout methods and amplification technologies such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA) and continue to elaborate on the prospects of the developed biosensor in the detection of some major viral and bacterial diseases. Within the scope of this article, we also discuss the recent COVID pandemic and the numerous CRISPR biosensors that have undergone development since its advent. Finally, we discuss some challenges and future prospects of CRISPR Cas systems in point-of-care testing.

Point-of-care laboratory testing in primary care: utilization, limitations and perspectives of general practitioners in Germany.

BACKGROUND: Due to their fast turnaround time and user-friendliness, point-of-care tests (POCTs) possess a great potential in primary care. The purpose of the study was to assess general practitioners' (GPs) perspectives on POCT use in German primary care, including utilization, limitations and requirements. METHODS: We conducted a cross-sectional survey study among GPs in Germany (federal states of Thuringia, Bremen and Bavaria (Lower Franconia), study period: 04/22-06/2022). RESULTS: From 2,014 GPs reached, 292 participated in our study (response rate: 14.5%). The median number of POCTs used per GP was 7.0 (IQR: 5.0-8.0). Six POCTs are used by the majority of surveyed GPs (> 50%): urine dipstick tests (99%), glucose (urine [91%] and plasma [69%]), SARS-CoV-2 (80%), urine microalbumin (77%), troponin I/T (74%) and prothrombin time / international normalized ratio (65%). The number of utilized POCTs did not differ between GP practice type (p = 0.307) and population size of GP practice location (p = 0.099). The great majority of participating German GPs (93%) rated POCTs as useful diagnostic tools in the GP practice. GPs ranked immediate decisions on patient management and the increase in diagnostic certainty as the most important reasons for performing POCTs. The most frequently reported limitations of POCT use in the GP practice were economic aspects (high costs and inadequate reimbursement), concerns regarding diagnostic accuracy, and difficulties to integrate POCT-testing into practice routines (e.g. time and personnel expenses). CONCLUSION: Although participating German GPs generally perceive POCTs as useful diagnostic tools and numerous POCTs are available, several test-related and contextual factors contribute to the relatively low utilization of POCTs in primary care.

The critical role of engineering in the rapid development of COVID-19 diagnostics: Lessons from the RADx Tech Test Verification Core.

Engineering plays a critical role in the development of medical devices, and this has been magnified since 2020 as severe acute respiratory syndrome coronavirus 2 swept over the globe. In response to the coronavirus disease 2019, the National Institutes of Health launched the Rapid Acceleration of Diagnostics (RADx) initiative to help meet the testing needs of the United States and effectively manage the pandemic. As the Engineering and Human Factors team for the RADx Tech Test Verification Core, we directly assessed more than 30 technologies that ultimately contributed to an increase of the country's total testing capacity by 1.7 billion tests to date. In this review, we present central lessons learned from this "apples-to-apples" comparison of novel, rapidly developed diagnostic devices. Overall, the evaluation framework and lessons learned presented in this review may serve as a blueprint for engineers developing point-of-care diagnostics, leaving us better prepared to respond to the next global public health crisis rapidly and effectively.

The application of nanoparticles in point-of-care testing (POCT) immunoassays.

The Covid-19 pandemic has led to greater recognition of the importance of the fast and timely detection of pathogens. Recent advances in point-of-care testing (POCT) technology have shown promising results for rapid diagnosis. Immunoassays are among the most extensive POCT assays, in which specific labels are used to indicate and amplify the immune signal. Nanoparticles (NPs) are above the rest because of their versatile properties. Much work has been devoted to NPs to find more efficient immunoassays. Herein, we comprehensively describe NP-based immunoassays with a focus on particle species and their specific applications. This review describes immunoassays along with key concepts surrounding their preparation and bioconjugation to show their defining role in immunosensors. The specific mechanisms, microfluidic immunoassays, electrochemical immunoassays (ELCAs), immunochromatographic assays (ICAs), enzyme-linked immunosorbent assays (ELISA), and microarrays are covered herein. For each mechanism, a working explanation of the appropriate background theory and formalism is articulated before examining the biosensing and related point-of-care (POC) utility. Given their maturity, some specific applications using different nanomaterials are discussed in more detail. Finally, we outline future challenges and perspectives to give a brief guideline for the development of appropriate platforms.

Specific lateral flow detection of isothermal nucleic acid amplicons for accurate point-of-care testing.

For point-of-care testing (POCT), coupling isothermal nucleic acid amplification schemes (e.g., recombinase polymerase amplification, RPA) with lateral flow assay (LFA) readout is an ideal platform, since such integration offers both high sensitivity and deployability. However, isothermal schemes typically suffers from non-specific amplification, which is difficult to be differentiated by LFA and thus results in false-positives. Here, we proposed an accurate POCT platform by specific recognition of target amplicons with peptide nucleic acid (PNA, assisted by T7 Exonuclease), which could be directly plugged into the existing RPA kits and commercial LFA test strips. With SARS-CoV-2 as the model, the proposed method (RPA-TeaPNA-LFA) efficiently eliminated the false-positives, exhibiting a lowest detection concentration of 6.7 copies/µL of RNA and 90 copies/µL of virus. Using dual-gene (orf1ab and N genes of SARS-CoV-2) as the targets, RPA-TeaPNA-LFA offered a high specificity (100%) and sensitivity (RT-PCR Ct < 31, 100%; Ct < 40, 71.4%), and is valuable for on-site screening or self-testing during isolation. In addition, the dual test lines in the test strips were successfully explored for simultaneous detection of SARS-CoV-2 and H1N1, showing great potential in response to future pathogen-based pandemics.

Recent Advances in Novel Lateral Flow Technologies for Detection of COVID-19.

The development of reliable and robust diagnostic tests is one of the most efficient methods to limit the spread of coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, most laboratory diagnostics for COVID-19, such as enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR), are expensive, time-consuming, and require highly trained professional operators. On the other hand, the lateral flow immunoassay (LFIA) is a simpler, cheaper device that can be operated by unskilled personnel easily. Unfortunately, the current technique has some limitations, mainly inaccuracy in detection. This review article aims to highlight recent advances in novel lateral flow technologies for detecting SARS-CoV-2 as well as innovative approaches to achieve highly sensitive and specific point-of-care testing. Lastly, we discuss future perspectives on how smartphones and Artificial Intelligence (AI) can be integrated to revolutionize disease detection as well as disease control and surveillance.

Microfluidics for COVID-19: From Current Work to Future Perspective.

Spread of coronavirus disease 2019 (COVID-19) has significantly impacted the public health and economic sectors. It is urgently necessary to develop rapid, convenient, and cost-effective point-of-care testing (POCT) technologies for the early diagnosis and control of the plague's transmission. Developing POCT methods and related devices is critical for achieving point-of-care diagnosis. With the advantages of miniaturization, high throughput, small sample requirements, and low actual consumption, microfluidics is an essential technology for the development of POCT devices. In this review, according to the different driving forces of the fluid, we introduce the common POCT devices based on microfluidic technology on the market, including paper-based microfluidic, centrifugal microfluidic, optical fluid, and digital microfluidic platforms. Furthermore, various microfluidic-based assays for diagnosing COVID-19 are summarized, including immunoassays, such as ELISA, and molecular assays, such as PCR. Finally, the challenges of and future perspectives on microfluidic device design and development are presented. The ultimate goals of this paper are to provide new insights and directions for the development of microfluidic diagnostics while expecting to contribute to the control of COVID-19.

Rapid PCR kit: lateral flow paper strip with Joule heater for SARS-CoV-2 detection.

Polymerase chain reaction (PCR)-based diagnostic kits for point-of-care (POC) testing are highly desirable to prevent the spread of infectious diseases. Here, we demonstrate a rapid PCR testing kit that involves integrating a lateral flow paper strip with a nichrome-based thin film heater. The use of a paper membrane as a PCR-solution container results in fast thermocycling without a cooler because the membrane can contain the solution with a high specific surface area where Joule heating is applied. After PCR, amplified products are simultaneously detected at the lateral flow paper strip with the naked eye. Severe acute respiratory syndrome ß-coronavirus RNA can be detected within 30 min after PCR solution injection. This work reveals that the paper membrane can act as not only a capillary flow channel but also as a promising platform for fast PCR and detection.

Gargle sample is an effective option in a novel fully automated molecular point-of-care test for influenza: a multicenter study.

BACKGROUND: We conducted a multicenter study to evaluate the performance of a novel fully automated molecular point-of-care test using transcription-reverse transcription concerted reaction that can detect influenza A and B within 15 min in nasopharyngeal swabs and gargle samples (TRCsatFLU). METHODS: Patients who visited or were hospitalized at eight clinics and hospitals with influenza-like illnesses between December 2019 and March 2020 participated in this study. We collected nasopharyngeal swabs from all patients and gargle samples from patients whom the physician judged fit to perform gargling. The result of TRCsatFLU was compared to a conventional reverse transcription-polymerase chain reaction (RT-PCR). If the results of TRCsatFLU and conventional RT-PCR were different, the samples were analyzed by sequencing. RESULTS: We evaluated 233 nasopharyngeal swabs and 213 gargle samples from 244 patients. The average age of the patients was 39.3 ± 21.2. Of the patients, 68.9% visited a hospital within 24 h of symptom onset. The most common symptoms were fever (93.0%), fatigue (79.5%), and nasal discharge (64.8%). All patients in whom the gargle sample was not collected were children. Influenza A or B was detected in 98 and 99 patients in nasopharyngeal swabs and gargle samples using TRCsatFLU, respectively. Four and five patients in nasopharyngeal swabs and gargle samples, respectively, with different TRCsatFLU and conventional RT-PCR results. Influenza A or B was detected using sequencing in all samples with different results. Based on the combined conventional RT-PCR and sequencing results, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of TRCsatFLU for influenza detection in nasopharyngeal swabs were 0.990, 1.000, 1.000, and 0.993, respectively. In the gargle samples, the sensitivity, specificity, PPV, and NPV of the TRCsatFLU for detecting influenza were 0.971, 1.000, 1.000, and 0.974, respectively. CONCLUSIONS: The TRCsatFLU showed great sensitivity and specificity for the detection of influenza in nasopharyngeal swabs and gargle samples. TRIAL REGISTRATION: This study was registered in the UMIN Clinical Trials Registry (reference number: UMIN000038276) on October 11, 2019. Before sample collection, written informed consent for the participation and publication of this study was obtained from all participants.

Assessment of Urinary Biomarkers for Infectious Diseases Using Lateral Flow Assays: A Comprehensive Overview.

Screening of biomarkers is a powerful approach for providing a holistic view of the disease spectrum and facilitating the diagnosis and prognosis of the state of infectious diseases. Unaffected by the homeostasis mechanism in the human body, urine accommodates systemic changes and reflects the pathophysiological condition of an individual. Easy availability in large volumes and non-invasive sample collection have rendered urine an ideal source of biomarkers for various diseases. Infectious diseases may be communicable, and therefore early diagnosis and treatment are of immense importance. Current diagnostic approaches preclude the timely identification of clinical conditions and also lack portability. Point-of-care (POC) testing solutions have gained attention as alternative diagnostic measures due to their ability to provide rapid and on-site results. Lateral flow assays (LFAs) are the mainstay in POC device development and have attracted interest owing to their potential to provide instantaneous results in resource-limited settings. The discovery and optimization of a definitive biomarker can render POC testing an excellent platform, thus impacting unwarranted antibiotic administration and preventing the spread of infectious diseases. This Review summarizes the importance of urine as an emerging biological fluid in infectious disease research and diagnosis in clinical settings. We review the academic research related to LFAs. Further, we also describe commercial POC devices based on the identification of urinary biomarkers as diagnostic targets for infectious diseases. We also discuss the future use of LFAs in developing more effective POC tests for urinary biomarkers of various infections.