Point-of-Care Testing in Primary Care

Point-of-care testing (POCT) in pediatric primary care is essential for clinicians to make a timely and accurate diagnosis. The COVID-19 pandemic has highlighted the importance of timely and accurate testing strategies to correctly identify the etiology of upper and lower respiratory infections. Additionally, pediatric POCT continues to be important in rural and underserved communities where access to hospital laboratories may be less available. This chapter will focus on seven rapid tests: Group A streptococcus (GAS), influenza A & B, SARS-CoV-2 (COVID-19), human immunodeficiency virus (HIV), C-reactive protein (CRP), human chorionic gonadotropin (hCG), and hemoglobin A1c (HbA1c). © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022.

Point-of-care testing of SARS -CoV-2 and influenza A and B at hospital emergency room during Covid-19 pandemic among patients with acute respiratory diseases

Introduction. The most common causes of acute respiratory diseases (ARD) with significant morbidity and mortality include, for a long time, the influenza virus and, in recent years, also the SARS-Cov-2 virus. Patients with various clinical symptoms are triaged in emergency rooms of hospitals, and therefore their rapid and reliable diagnostics is essential in order to prevent the spread of ARD. Molecular genetic point-of-care testing (POCT) at the point of care represents a significant advance in clinical diagnostics. Materials and methods. Diagnostics of viral agents of ARD took place from December 2021 to February 2022. 1046 nasopharynx swabs samples were collected in the emergency room of I. Internal Clinic of the Faculty of Medicine at Comenius University and the University Hospital in Bratislava. SARSCov-2 and influenza were detected from the same sample using the cobas® SARS-Cov-2 & Influenza A/B test on the cobas® Liat® system. Results. From the total number of biological material collected, the SARS-Cov-2 virus was detected in 135 samples (12.9 %), while the highest incidence of positive samples was in February 2022 - 86 (20.9 % positivity), followed by December 2021 - 31 (23.0 % positivity) and January 2022 - 18 (13.3% positivity). Influenza type A virus was detected in two samples (0.2%) and influenza type B virus was not detected. Conclusion. POCT made it possible to significantly improve the screening of patients and minimize the risk of nosocomial transmission of respiratory infections in the hospital thanks to the rapid and accurate diagnosis of the SARS-CoV-2 virus and influenza A/B (Fig. 3, Ref. 32). Text v PDF www.lekarsky.herba. sk. © 2023, Lekarsky Obzor. All Rights Reserved.

[CRISPR-based molecular diagnostics: a review].

Rapid and accurate detection technologies are crucial for disease prevention and control. In particular, the COVID-19 pandemic has posed a great threat to our society, highlighting the importance of rapid and highly sensitive detection techniques. In recent years, CRISPR/Cas-based gene editing technique has brought revolutionary advances in biotechnology. Due to its fast, accurate, sensitive, and cost-effective characteristics, the CRISPR-based nucleic acid detection technology is revolutionizing molecular diagnosis. CRISPR-based diagnostics has been applied in many fields, such as detection of infectious diseases, genetic diseases, cancer mutation, and food safety. This review summarized the advances in CRISPR-based nucleic acid detection systems and its applications. Perspectives on intelligent diagnostics with CRISPR-based nucleic acid detection and artificial intelligence were also provided.

An IoT-based smart optical platform for colorimetric analyzing multiple samples of biomarkers

In this work, we have designed, implemented, and characterized an IoT-enabled optical platform that serves as a highly efficient colorimetric analyzer for disposable rapid diagnostic test kits. We have taken color images of the test chip using an external camera module connected to an IoT-enabled single-board mini-computer, which are then analyzed using an image processing algorithm. To prevent image burning and to reduce the impact of ambient lighting, we have proposed a unique light-diffusing model and have maintained a strategic distance within the white acrylic imaging enclosure of the platform. In order to ensure even and uniform distribution of light on the sample within the platform, we have carried out a study with the incorporation of a highly bright single LED, an LED array, and attaching a PDMS-made light diffuser on the LED array at varying light incident angles on custom-made single-channel test kits containing blood hematocrit. Finally, based on the optimized lighting conditions, we have successfully applied the proposed platform for the detection of blood hematocrit, β-hCG, and SARS-CoV-2 antigen, and were able to identify their different levels of concentration. Since the device is portable in size and cost-effective to implement, it can also be used for analyzing other biomarkers in resource-limited communities for point-of-care (POC) applications.

Prospective population-level validation of the Abbott ID NOW severe acute respiratory syndrome coronavirus 2 device implemented in multiple settings for testing asymptomatic and symptomatic individuals.

OBJECTIVE: Diagnostic evaluation of the ID NOW coronavirus disease 2019 (COVID-19) assay in various real-world settings among symptomatic and asymptomatic individuals. METHODS: Depending on the setting, the ID NOW testing was performed using oropharyngeal swabs (OPSs) taken from patients with symptoms suggestive of COVID-19, asymptomatic close contacts, or asymptomatic individuals as part of outbreak point prevalence screening. From January to April 2021, a select number of sites switched from using OPS to combined oropharyngeal and nasal swab (O + NS) for ID NOW testing. For every individual tested, two swabs were collected by a health care worker: one swab (OPS or O + NS) for ID NOW testing and a separate swab (OPS or nasopharyngeal swab) for RT-PCR. RESULTS: A total of 129 112 paired samples were analysed (16 061 RT-PCR positive). Of these, 81 697 samples were from 42 COVID-19 community collection sites, 16 924 samples were from 69 rural hospitals, 1927 samples were from nine emergency shelters and addiction treatment facilities, 23 802 samples were from six mobile units that responded to 356 community outbreaks, and 4762 O + NS swabs were collected from three community collection sites and one emergency shelter. The ID NOW assay sensitivity was the highest among symptomatic individuals presenting to community collection sites (92.5%; 95% CI, 92.0-93.0%) and the lowest for asymptomatic individuals associated with community outbreaks (73.9%; 95% CI, 69.8-77.7%). Specificity was >99% in all populations tested. DISCUSSION: The sensitivity of ID NOW severe acute respiratory syndrome coronavirus 2 testing is the highest when used in symptomatic community populations not seeking medical care. Sensitivity and positive predictive value drop by approximately 10% when tested on asymptomatic populations. Using combined oropharyngeal and nasal swabs did not improve the performance of ID NOW assay.

CRISPR-based point-of-care plant disease diagnostics.

The coupling of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas RNA-programmable nucleases with nucleic acid detection platforms has brought radical changes to the field of disease diagnosis. Recently, Sánchez et al. developed a simple, rapid, highly sensitive, precise, and in-field deployable point-of-care (POC) and point-of-need (PON) molecular disease detection tool that can be used in diverse agricultural applications.

Current Advances in Paper-Based Biosensor Technologies for Rapid COVID-19 Diagnosis.

The global coronavirus disease 2019 (COVID-19) pandemic has had significant economic and social impacts on billions of people worldwide since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China, in November 2019. Although polymerase chain reaction (PCR)-based technology serves as a robust test to detect SARS-CoV-2 in patients with COVID-19, there is a high demand for cost-effective, rapid, comfortable, and accurate point-of-care diagnostic tests in medical facilities. This review introduces the SARS-CoV-2 viral structure and diagnostic biomarkers derived from viral components. A comprehensive introduction of a paper-based diagnostic platform, including detection mechanisms for various target biomarkers and a COVID-19 commercial kit is presented. Intrinsic limitations related to the poor performance of currently developed paper-based devices and unresolved issues are discussed. Furthermore, we provide insight into novel paper-based diagnostic platforms integrated with advanced technologies such as nanotechnology, aptamers, surface-enhanced Raman spectroscopy (SERS), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. Finally, we discuss the prospects for the development of highly sensitive, accurate, cost-effective, and easy-to-use point-of-care COVID-19 diagnostic methods.

A novel linear displacement isothermal amplification with strand displacement probes (LDIA-SD) in a pocket-size device for point-of-care testing of infectious diseases.

Nucleic acid amplification is crucial for disease diagnosis, especially lethal infectious diseases such as COVID-19. Compared with PCR, isothermal amplification methods are advantageous for point-of-care testing (POCT). However, complicated primer design limits their application in detecting some short targets or sequences with abnormal GC content. Herein, we developed a novel linear displacement isothermal amplification (LDIA) method using two pairs of conventional primers and Bacillus stearothermophilus (Bst) DNA polymerase, and reactions could be accelerated by adding an extra primer. Pseudorabies virus gE (high GC content) and Salmonella fimW (low GC content) genes were used to evaluate the LDIA assay. Using strand displacement (SD) probes, a LDIA-SD method was developed to realize probe-based specific detection. Additionally, we incorporated a nucleic acid-free extraction step and a pocket-sized device to realize POCT applications of the LDIA-SD method. The LDIA-SD method has advantages including facile primer design, high sensitivity and specificity, and applicability for POCT, especially for amplification of complex sequences and detection of infectious diseases.

Electrochemical Gene Amplification Signal Detection of Disposable Biochips Using Electrodes.

Real-time Polymerase Chain Reaction (RT-PCR), a molecular diagnostic technology, is spotlighted as one of the quickest and fastest diagnostic methods for the actual coronavirus (SARS-CoV-2). However, the fluorescent label-based technology of the RT-PCR technique requires expensive equipment and a sample pretreatment process for analysis. Therefore, this paper proposes a biochip based on Electrochemical Impedance Spectroscopy (EIS). In this paper, it was possible to see the change according to the concentration by measuring the impedance with a chip made of two electrodes with different shapes of sample DNA.

Establishment of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of swine acute diarrhea syndrome coronavirus (SADS-CoV).

BACKGROUND: Swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute vomiting and diarrhea in piglets, leading to significant financial losses for the pig industry. Recombinase polymerase amplification (RPA) is a rapid nucleic acid amplification technology used under constant temperature conditions. The study established a real-time reverse transcription (RT)-RPA assay for early diagnosis of SADS-CoV.  RESULTS: The detection limit of the real-time RT-RPA was 74 copies/µL of SADS-CoV genomic standard recombinant plasmid in 95% of cases. The assay was performed in less than 30 min and no cross-reactions were observed with eight other common viruses that affect swine, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudo rabies virus (PRV), swine influenza virus (SIV), seneca valley virus (SVA), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV). The coefficient of variation (C.V.) values of the two standards dilutions and three positive clinical sample ranged from 2.95% to 4.71%. A total of 72 clinical fecal samples from swine with diarrheal symptoms were analyzed with the developed RT-RPA and quantitative RT-PCR. There was 98.61% agreement between the RT-RPA and the quantitative real-time PCR results. CONCLUSIONS: These results indicated that the developed RT-RPA assay had good specificity, sensitivity, stability and repeatability. The study successfully established a broadly reactive RT-RPA assay for SADS-CoV detection.

Optimizing Public Health Preparedness for Highly Infectious Diseases in Central Vietnam.

Our primary objectives were (a) to determine the need for and the availability of point-of-care testing (POCT) for infectious diseases and (b) to recommend point-of-care testing strategies and Spatial Care PathsTM (SCPs) that enhance public health preparedness in the regional districts of Thua Thien Hue Province (TTHP), Central Vietnam, where we conducted field surveys. Medical professionals in seven community health centers (CHCs), seven district hospitals (DHs) and one provincial hospital (PH) participated. Survey questions (English and Vietnamese) determined the status of diagnostic testing capabilities for infectious diseases and other acute medical challenges in TTHP. Infectious disease testing was limited: six of seven CHCs (86%) lacked infectious disease tests. One CHC (14%, 1/7) had two forms of diagnostic tests available for the detection of malaria. All CHCs lacked adequate microbiology laboratories. District hospitals had few diagnostic tests for infectious diseases (tuberculosis and syphilis), blood culture (29%, 2/7), and pathogen culture (57%, 4/7) available. The PH had broader diagnostic testing capabilities but lacked preparedness for highly infectious disease threats (e.g., Ebola, MERS-CoV, SARS, Zika, and monkeypox). All sites reported having COVID-19 rapid antigen tests; COVID-19 RT-PCR tests were limited to higher-tier hospitals. We conclude that infectious disease diagnostic testing should be improved and POC tests must be supplied near patients' homes and in primary care settings for the early detection of infected individuals and the mitigation of the spread of new COVID-19 variants and other highly infectious diseases.

CRISPR-Cas12a-Empowered Electrochemical Biosensor for Rapid and Ultrasensitive Detection of SARS-CoV-2 Delta Variant.

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The gold standard method for the diagnosis of SARS-CoV-2 depends on quantitative reverse transcription-polymerase chain reaction till now, which is time-consuming and requires expensive instrumentation, and the confirmation of variants relies on further sequencing techniques. Herein, we first proposed a robust technique-methodology of electrochemical CRISPR sensing with the advantages of rapid, highly sensitivity and specificity for the detection of SARS-CoV-2 variant. To enhance the sensing capability, gold electrodes are uniformly decorated with electro-deposited gold nanoparticles. Using DNA template identical to SARS-CoV-2 Delta spike gene sequence as model, our biosensor exhibits excellent analytical detection limit (50 fM) and high linearity (R2 = 0.987) over six orders of magnitude dynamic range from 100 fM to 10 nM without any nucleic-acid-amplification assays. The detection can be completed within 1 h with high stability and specificity which benefits from the CRISPR-Cas system. Furthermore, based on the wireless micro-electrochemical platform, the proposed biosensor reveals promising application ability in point-of-care testing.

Planning education for best practices in point-of-care testing

The increasing availability and use of POCT are influenced by many factors, including the industry trend toward patient-centered care and decentralization of health care;the increasing prevalence of infectious diseases, which includes the current use of rapid SARS-CoV-2 testing;the increasing incidence of lifestyle diseases such as diabetes, heart disease, and hypertension;and advances in in-vitro medical diagnostics. The use of POCT can increase the efficiency of services and improve patient outcomes. However, the variability of the testing environment and conditions, as well as the competency of the staff performing the tests, can have a significant impact on the quality and accuracy of POCT results. Most personnel performing POCT are not trained laboratory staff and may not be as familiar with the processes associated with testing, such as patient preparation, specimen collection, management of equipment and supplies, calibration and maintenance of equipment, the performance of the test, quality control, interpretation of results, and reporting/documentation of results related to the particular patient. Therefore, personnel performing POCT must have the appropriate training and experience to ensure that test results are accurate and reliable. This review outlines the specific personnel training requirements based on international standards that must be considered to ensure the quality of test results and describe the competency criteria required for POCT compliance. [ FROM AUTHOR] Copyright of Turkish Journal of Biochemistry / Turk Biyokimya Dergisi is the property of De Gruyter and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) and lateral flow strip based on CRISPR/Cas13a for sensitive and visual detection of SARS-CoV-2.

COVID-19 has erupted and quickly swept across the globe, causing huge losses to human health and wealth. It is of great value to develop a quick, accurate, visual, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we developed a biosensor based on CRISPR/Cas13a combined with recombinase polymerase amplification (RPA) to detect S and Orf1ab genes of SARS-CoV-2 within 30 min. Most important of all, we developed an automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) with a 3D-printed microfluidic chip for sensitively detecting SARS-CoV-2, which addressed aerosol contamination issue and provided a more accurate and high-throughput detection during the on-site detection process. The detection limits of S gene and Orf1ab gene were as low as 0.68 fM and 4.16 fM. Furthermore, we used the lateral flow strip to realize visualization and point of care testing (POCT) of SARS-CoV-2. Therefore, profit from the efficient amplification of RPA and the high specificity of CRISPR/Cas13a, APHF-analyzer and the lateral flow strip to simultaneous detection of S gene and Orf1ab gene would be applied as a promising tool in the field of SARS-CoV-2 detection.

The Coronavirus Disease 2019 Spatial Care Path: Home, Community, and Emergency Diagnostic Portals.

This research uses mathematically derived visual logistics to interpret COVID-19 molecular and rapid antigen test (RAgT) performance, determine prevalence boundaries where risk exceeds expectations, and evaluate benefits of recursive testing along home, community, and emergency spatial care paths. Mathematica and open access software helped graph relationships, compare performance patterns, and perform recursive computations. Tiered sensitivity/specificity comprise: (T1) 90%/95%; (T2) 95%/97.5%; and (T3) 100%/≥99%, respectively. In emergency medicine, median RAgT performance peaks at 13.2% prevalence, then falls below T1, generating risky prevalence boundaries. RAgTs in pediatric ERs/EDs parallel this pattern with asymptomatic worse than symptomatic performance. In communities, RAgTs display large uncertainty with median prevalence boundary of 14.8% for 1/20 missed diagnoses, and at prevalence > 33.3-36.9% risk 10% false omissions for symptomatic subjects. Recursive testing improves home RAgT performance. Home molecular tests elevate performance above T1 but lack adequate validation. Widespread RAgT availability encourages self-testing. Asymptomatic RAgT and PCR-based saliva testing present the highest chance of missed diagnoses. Home testing twice, once just before mingling, and molecular-based self-testing, help avoid false omissions. Community and ER/ED RAgTs can identify contagiousness in low prevalence. Real-world trials of performance, cost-effectiveness, and public health impact could identify home molecular diagnostics as an optimal diagnostic portal.

A Review of Apta-POF-Sensors: The Successful Coupling between Aptamers and Plastic Optical Fibers for Biosensing Applications

Aptamers represent the next frontier as biorecognition elements in biosensors thanks to a smaller size and lower molecular weight with respect to antibodies, more structural flexibility with the possibility to be regenerated, reduced batch-to-batch variation, and a potentially lower cost. Their high specificity and small size are particularly interesting for their application in optical biosensors since the perturbation of the evanescent field are low. Apart from the conventional plasmonic optical sensors, platforms based on silica and plastic optical fibers represent an interesting class of devices for point-of-care testing (POCT) in different applications. The first example of the coupling between aptamers and silica optical fibers was reported by Pollet in 2009 for the detection of IgE molecules. Six years later, the first example was published using a plastic optical fiber (POF) for the detection of Vascular Endothelial Growth Factor (VEGF). The excellent flexibility, great numerical aperture, and the large diameter make POFs extremely promising to be coupled to aptamers for the development of a sensitive platform easily integrable in portable, small-size, and simple devices. Starting from silica fiber-based surface plasmon resonance devices, here, a focus on significant biological applications based on aptamers, combined with plasmonic-POF probes, is reported.

[Isothermal amplification technology based on microfluidic chip].

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification in molecular diagnostics. The PCR includes multiple reaction stages (denaturation, annealing, and extension), and a complicated thermalcycler is required to repetitively provide different temperatures for different stages for 30-40 cycles within at least 1-2 hours. Due to the complicated devices and the long amplification time, it is difficult to adopt conventional PCR in point-of-care testing (POCT). Comparing to conventional PCR, isothermal amplification is able to provide a much faster and more convenient nucleic acid detection because of highly efficient amplification at a constant reaction temperature provided by a simple heating device. When isothermal amplification is combined with microfluidics, a more competent platform for POCT can be established. For example, various diagnosis devices based on isothermal amplification have been used to rapidly and conveniently detect SARS-CoV-2 viruses. This review summarized the recent development and applications of the microfluidics-based isothermal amplification. First, different typical isothermal amplification methods and related detection methods have been introduced. Subsequently, different types of microfluidic systems with isothermal amplification were discussed based on their characteristics, for example, functionality, system structure, flow control, and operation principles. Furthermore, detection of pathogens (e.g. SARS-CoV-2 viruses) based on isothermal amplification was introduced. Finally, the combination of isothermal amplification with other new technologies, e.g. CRISPR, has been introduced as well.

Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe.

Viral evolution impacts diagnostic test performance through the emergence of variants with sequences affecting the efficiency of primer binding. Such variants that evade detection by nucleic acid-based tests are subject to selective pressure, enabling them to spread more efficiently. Here, we report a variant-tolerant diagnostic test for SARS-CoV-2 using a loop-mediated isothermal nucleic acid-based amplification (LAMP) assay containing high-fidelity DNA polymerase and a high-fidelity DNA polymerase-medicated probe (HFman probe). In addition to demonstrating a high tolerance to variable SARS-CoV-2 viral sequences, the mechanism also overcomes frequently observed limitations of LAMP assays arising from non-specific amplification within multiplexed reactions performed in a single "pot". Results showed excellent clinical performance (sensitivity 94.5%, specificity 100%, n = 190) when compared directly to a commercial gold standard reverse transcription quantitative polymerase chain reaction assay for the extracted RNA from nasopharyngeal samples and the capability of detecting a wide range of sequences containing at least alpha and delta variants. To further validate the test with no sample processing, directly from nasopharyngeal swabs, we also detected SARS-CoV-2 in positive clinical samples (n = 49), opening up the possibility for the assay's use in decentralized testing.

Microfluidic technology and its application in the point-of-care testing field.

Since the outbreak of the coronavirus disease 2019 (COVID-19), countries around the world have suffered heavy losses of life and property. The global pandemic poses a challenge to the global public health system, and public health organizations around the world are actively looking for ways to quickly and efficiently screen for viruses. Point-of-care testing (POCT), as a fast, portable, and instant detection method, is of great significance in infectious disease detection, disease screening, pre-disease prevention, postoperative treatment, and other fields. Microfluidic technology is a comprehensive technology that involves various interdisciplinary disciplines. It is also known as a lab-on-a-chip (LOC), and can concentrate biological and chemical experiments in traditional laboratories on a chip of several square centimeters with high integration. Therefore, microfluidic devices have become the primary implementation platform of POCT technology. POCT devices based on microfluidic technology combine the advantages of both POCT and microfluids, and are expected to shine in the biomedical field. This review introduces microfluidic technology and its applications in combination with other technologies.