Tool For Point-of-care Testing Research Articles

A novel extraction-free dual HiFi-LAMP assay for detection of methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a leading cause of bacteremia and blood stream infections. Methicillin-resistant S. aureus (MRSA) that first appeared in 1961 often caused hospital-acquired infections (HAIs) and community-acquired infections (CAIs) and was associated with high mortality rate. Accurate and rapid point-of-care testing (POCT) of MRSA is crucial for clinical management and treatment of MRSA infections, as well as the prevention and control of HAIs and CAIs. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of methicillin-susceptible S. aureus and MRSA. The dual HiFi-LAMP assay can detect 30 copies/reaction of nuc and mecA genes with detection limits of 147 and 158 copies per 25 µL reaction, respectively. A retrospective clinical evaluation with 107 clinical S. aureus isolates showed both sensitivity and specificity of 100%. A prospective clinical evaluation with 35 clinical samples revealed a specificity of 100% and a sensitivity of 92.3%. The dual HiFi-LAMP assay can detect almost all S. aureus samples (141/142; 99.3%) within 20 min, implying that the entire HiFi-LAMP assay (including sample process) can be completed within 40 min, extremely significantly shorter than 3-5 days by the traditional clinical microbial culture and antibiotic susceptibility testing. The novel extraction-free dual HiFi-LAMP assay can be used as a robust POCT tool to promote precise diagnosis and treatment of MRSA infections in hospitals and to facilitate surveillance of MRSA at hospital and community settings.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) was associated with high mortality rate and listed as a "priority pathogen" by the World Health Organization. Accurate and rapid point-of-care testing (POCT) of MRSA is critically required for clinical management and treatment of MRSA infections. Some previous LAMP-based POCT assays for MRSA might be questionable due to their low specificity and the lack of appropriate evaluation directly using clinical samples. Furthermore, they are relatively tedious and time-consuming because they require DNA extraction and lack multiplex detection capacity. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of MRSA and methicillin-susceptible S. aureus. The assay has high specificity and sensitivity and can be completed within 40 min. Clinical evaluation with real clinical samples and clinical isolates showed excellent performance with 100% specificity and 92.3%-100% sensitivity. The novel extraction-free assay may be a robust POCT tool to promote precise diagnosis of MRSA infections and facilitate surveillance of MRSA at hospital and community settings.

Multifunction nanoenzyme-assisted ion-selective and oxidation catalysis SERS biosensors for point-of-care nitrite testing

Point-of-care testing (POCT) for nitrite has significant implications for the diagnosis, treatment, and prognosis of various diseases. However, the low polarization and Raman cross section of nitrite present obstacles to monitoring nitrite molecules using the surface-enhanced Raman scattering (SERS) method. In this study, a bionic enzyme (cyanocobalamin (VB12))-modified matrix of metal-organic frameworks (ZIF-67) is chosen as a carrier for loading Au colloid complexes and subsequently utilized as ion-selective SERS biosensors (ADA@ZIF-67 bionic enzyme biosensors). By leveraging the mixed-state of the cobalt (Co) element, the as-prepared biosensors have demonstrated dual-mode nanoenzyme properties, providing increased peroxide activity for nitrite oxidation. Simultaneously, the presence of the distinct bionic state Co in the biosensors enables the ion-selective capture of nitrite. When utilized as a POCT reagent, the biosensors demonstrate high sensitivity, with a detection limit of 1.67 nM for nitrite and a wide linear response range of nitrite spanning from 1 M to 100 nM. Real salivary samples, encompassing varied activities such as working, eating, exercising, and sleeping, are evaluated using the proposed method and commercially available nitrite kits. The SERS biosensors demonstrate superior performance and hold promise as a potential tool for POCT, detection, and healthcare applications.

Automated Microfluidic Nucleic Acid Detection Platform-Integrated RPA-T7-Cas13a for Pathogen Diagnosis

There is a growing urgent need for point-of-care testing (POCT) devices that integrate sample pretreatment and nucleic acid detection in a rapid, economical, and non-labor-intensive way. Here, we have developed an automated, portable nucleic acid detection system employing microfluidic chips integrating rotary valve-assisted sample pretreatment and recombinase polymerase amplification (RPA)-T7-Cas13a into one-step nucleic acid detection. The RPA and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a were integrated into a single-chamber reaction. As a validation model, we used this method to detect Group B streptococci (GBS) DNA and achieved a detection sensitivity of 8 copies/reaction, which is 6 times more sensitive than gold-standard polymerase chain reactions (PCRs). Dual specific recognition of RPA with CRISPR/Cas13a makes our method ultraspecific, with correct detection of Group B streptococci from 8 kinds of pathogenic bacteria. For the 16 positive and 24 negative clinical GBS samples, our assay achieved 100% accuracy compared to the PCR technique. The whole procedure can be automatically completed within 30 min, providing a more robust, sensitive, and accurate molecular diagnostic tool for POCT.

Rapid visualization of Clostridioides difficile toxins A and B by multiplex RPA combined with CRISPR-Cas12a.

Clostridioides difficile (C. difficile) infection is the most common cause of nosocomial infection, which is a severe challenge in modern medical care. Currently, many laboratory diagnostic methods for C. difficile are available, such as PCR, culture-based tests, and antigen-based tests. However, these methods are not suitable for rapid point-of-care testing (POCT). Therefore, it is of great significance to develop a rapid, sensitive, and cost-effective method to detect C. difficile toxin genes. Recently, the development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for rapid POCT. In this study, we developed a rapid and specific detection platform for dual C. difficile toxins by combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a. The platform includes multiplex RPA-cas12a-fluorescence assay and multiplex RPA-cas12a-LFS (Lateral flow strip) assay, through which the detection limit for tcdA and tcdB was 10 copies/μL and 1 copy/μL, respectively. The results can be more clearly distinguished using a violet flashlight, which realized a portable visual readout. The platform can be tested within 50 min. Furthermore, our method did not cross-react with other pathogens that cause intestinal diarrhea. The results of 10 clinical samples using our method was 100% consistent with those from real-time PCR detection. In conclusion, the CRISPR-based double toxin gene detection platform for C. difficile is an effective, specific, and sensitive detection method, which can be used as a powerful on-site detection tool for POCT in the future.

Development of chemiluminescent lab-on-fiber immunosensors for rapid point-of-care testing of anti-SARS-CoV-2 antibodies and evaluation of longitudinal immune response kinetics following three-dose inactivation virus vaccination.

Developing reliable, rapid, and quantitative point‐of‐care testing (POCT) technology of SARS‐CoV‐2 specific antibodies and understanding longitudinal vaccination response kinetics are highly required to restrain the ongoing Coronavirus Disease 2019 (COVID‐19) pandemic. We demonstrate a novel portable, sensitive, and rapid chemiluminescent lab‐on‐fiber detection platform for detection of anti‐SARS‐CoV‐2 antibodies: the c‐LOFI. Using SARS‐CoV‐2 Spike S1 RBD protein functionalized fiber bio‐probe, the c‐LOFI can detect anti‐SARS‐CoV‐2 IgG and IgM antibodies with high sensitivity based on their respective HRP labeled secondary antibodies. The limits of detection of anti‐SARS‐CoV‐2 IgG and IgM antibodies were 0.6 ng/mL and 0.3 ng/mL, respectively. The c‐LOFI was successfully applied for direct detection of anti‐SARS‐CoV‐2 antibodies in whole blood samples with simple dilution, which can serve as a finger prick test to rapidly detect antibodies. Furthermore, the longitudinal immune response (>12 months) kinetics following three‐dose inactivated virus vaccines was evaluated based on anti‐SARS‐CoV‐2 IgG detection results, which can provide importance significance for understanding the immune mechanism against COVID‐19 and identify individuals who may benefit from the vaccination and booster vaccination. The c‐LOFI has great potential to become a sensitive, low‐cost, rapid, high‐frequency POCT tool for the detection of both SARS‐CoV‐2 specific antibodies and other biomarkers.This article is protected by copyright. All rights reserved.

Validity test of POCT (Point of Care Testing) method on blood glucose examination using whole blood samples, serum, and EDTA plasma

Medical laboratory services can support disease diagnosis or monitor patient recovery. The reality of the POCT method has been widely used by clinical laboratories. The tool is not only used for screening but is also used to check the diagnosis of diabetes mellitus. This research aims to determine the validity of the POCT method on whole blood, serum, and plasma EDTA samples on blood glucose tests. The research method of this study used the descriptive-analytical method using the POCT method on blood glucose tests using three different types of samples, whole blood, serum, and plasma EDTA. Precision tests were accepted on normal and pathological serum samples with CV% of 2.02% and 2.27%. The accuracy test was accepted on a normal and pathological serum with TE% values ​​of 8.54% and 6.03%. The linearity test is accepted on serum–plasma EDTA samples with an r2 value of 0.998. The sigma values ​​are in the unacceptable area. The use of the POCT tool for blood glucose examination has a valid performance value. The deviation of the examination results is influenced by pre-analytical errors such as sampling and processing samples so that the total error obtained is higher than the total allowed error. The POCT tool can be used for all types of samples.

Visual Detection of Adenosine Triphosphate by Taylor Rising: A Simple Point-of-Care Testing Method Based on Rolling Circle Amplification.

Rapid and sensitive point-of-care testing (POCT) is an extremely critical mission in practical applications, especially for rigorous military medicine, home health care, and in the third world. Here, we report a visual POCT method for adenosine triphosphate (ATP) detection based on Taylor rising in the corner of quadratic geometries between two rod surfaces. We discuss the principle of Taylor rising, demonstrating that it is significantly influenced by contact angle, surface tension, and density of the sample, which are controlled by ATP-dependent rolling circle amplification (RCA). In the presence of ATP, RCA reaction effectively suppresses Taylor-rising behavior, due to the increased contact angle, density, and decreased surface tension. Without addition of ATP, untriggered RCA reaction is favorable for Taylor rising, resulting in a significant height. With this proposed method, visual sensitive detection of ATP without the aid of other instruments is realized with only a 5 μL droplet, which has good selectivity and a low detection limit (17 nM). Importantly, this visual method provides a promising POCT tool for user-friendly molecular diagnostics.

EVALUASI ANALITIK POCT METODE GLUCOSE DEHYDROGENASE PARAMETER GLUKOSA PADA SPESEIMEN SERUM DAN PLASMA EDTA

Diabetes is a non-communicable disease that can be prevented and detected as early as possible by routine glucose checks. Point Of Care Testing (POCT) as a diabetes test screening tool is widely used by health care institutions and is even used independently by the wider community. Blood glucose can be measured by serum, plasma, and whole blood specimens. The standard specimen for glucose measurement using POCT is whole blood. Based on the author's survey, the use of the POCT tool in several health care institutions was not following the insert of the tool kit, one of which was the type of specimen used. Many laboratories use EDTA serum and plasma specimens for glucose testing using the POCT device. There are many methods used in the POCT tool, one of these methods is the glucose dehydrogenase method. Therefore analytic evaluation is necessary to ensure the accuracy and accuracy of laboratory examination results in glucose examination. This type of research is the analytical method with a comparative design. The data was collected using the total sampling method. The data is reported using Microsoft Excel and SPSS applications. The number of samples for analytic evaluation was 40 samples. Based on the results of tests performed in duplicate, there were clinical differences in glucose values using serum and plasma EDTA samples so that the use of these samples was not suitable for use for diagnostic purposes using the POCT glucose dehydrogenase method. There was a statistical difference in serum glucose and plasma EDTA against whole blood that was examined using the POCT glucose dehydrogenase method with a correlation value (R) of 0.9722 and 0.9695.

Abstract 17060: Machine Learning and Video Recognition for Automated Detection of Fluid Status in Heart Failure Patients

Introduction: There is a need for innovative solutions to better screen and diagnose the 7 million patients with chronic heart failure. A key component of assessing these patients is monitoring fluid status by evaluating for the presence and height of jugular venous distension (JVD). We hypothesize that video analysis of a patient’s neck using machine learning algorithms and image recognition can identify the amount of JVD. We propose the use of high fidelity video recordings taken using a mobile device camera to determine the presence or absence of JVD, which we will use to develop a point of care testing tool for early detection of acute exacerbation of heart failure. Methods: In this feasibility study, patients in the Duke cardiac catheterization lab undergoing right heart catheterization were enrolled. RGB and infrared videos were captured of the patient’s neck to detect JVD and correlated with right atrial pressure on the heart catheterization. We designed an adaptive filter based on biological priors that enhances spatially consistent frequency anomalies and detects jugular vein distention, with implementation done on Python. Results: We captured and analyzed footage for six patients using our model. Four of these six patients shared a similar strong signal outliner within the frequency band of 95bpm – 200bpm when using a conservative threshold, indicating the presence of JVD. We did not use statistical analysis given the small nature of our cohort, but in those we detected a positive JVD signal the RA mean was 20.25 mmHg and PCWP mean was 24.3 mmHg. Conclusions: We have demonstrated the ability to evaluate for JVD via infrared video and found a relationship with RHC values. Our project is innovative because it uses video recognition and allows for novel patient interactions using a non-invasive screening technique for heart failure. This tool can become a non-invasive standard to both screen for and help manage heart failure patients.

Vertical Flow Assay for Inflammatory Biomarkers Based on Nanofluidic Channel Array and SERS Nanotags.

There is a great demand for the development of detection assays for inflammation infection diagnosis with high throughput and ultrasensitivity. Herein, a vertical flow assay system with functionalized nanoporous anodic aluminum oxide (AAO) as sensing membrane, and encoded core-shell surface enhanced Raman scattering (SERS) nanotags as labels for multiple inflammatory biomarkers detection is presented. A 2 × 2 test array on the porous AAO is developed and modified with multiple capture antibodies to capture inflammatory biomarkers from samples. Due to the high surface area to volume ratio of the AAO membrane, and its influence on plasmonic coupling, the electromagnetic field of the encoded core-shell SERS nanotags is enhanced. Detection limits of 53.4, 4.72, 48.3, and 7.53 fg mL-1 are realized for C reactive protein, interleukin-6, serum amyloid A, and procalcitonin, respectively, with a linear dynamic range spanning at least five orders of magnitude. In addition, the proposed method also shows acceptable accuracy and repeatability for the detection of clinical samples. Therefore, this approach is expected to be a powerful point of care testing tool for disease diagnosis in facility limited areas.

DNA Tetrahedra-Cross-linked Hydrogel Functionalized Paper for Onsite Analysis of DNA Methyltransferase Activity Using a Personal Glucose Meter.

Despite the recent developments on the construction of point-of-care testing (POCT) devices, it is still a big challenge to build portable POCT tools for simple, sensitive, selective, and quantitative determination of disease-related biomarkers. With this in mind, we developed a simple and user-friendly POCT tool for onsite analysis of DNA adenine methyltransferase (Dam) activity by using DNA tetrahedra-based hydrogel to trap glucose-producing enzymes for target recognition and signal transduction. The enzyme-encapsulated DNA hydrogel and the substrate of enzyme were separately modified on papers and then combined onto a commercial glucose test strip for the sensitive evaluation of Dam activity via using a personal glucose meter (PGM) for quantitative signal readout. Taking advantage of the great amount of enzyme entrapped in DNA hydrogel and the high signal amplification ability of enzyme, this POCT tool is capable of highly sensitive and selective determination of Dam activity with a direct detection limit down to 0.001 U/mL, which is superior to that of most previously reported biosensors. Furthermore, this device can also be applied to screen inhibitor and analyze Dam activity in spiked serum samples, indicating the great potential for clinical practice and diagnostic applications. Additionally, all the reactions for Dam assay are performed on paper, which is simple and deliverable to end-users for medical diagnostics at home or in-field.

Development of point-of-care testing tool using immunochromatography for rapid diagnosis of human paragonimiasis

Paragonimiasis, an important food-borne zoonosis, is caused by lung flukes of the genus Paragonimus. Several million people are actually infected or at risk. Paragonimiasis is a re-emerging disease in developing countries. Diagnosis of pulmonary paragonimiasis is made by finding eggs in sputa and/or fecal samples. Eggs are typically not found in ectopic paragonimiasis cases, so diagnosis depends on supportive information, such as a history of eating fresh water crabs or crayfishes, radiographic findings and immunological tests. Here, a new point-of-care-testing (POCT) tool is presented. It uses immunochromatography for serodiagnosis of human paragonimiasis using excretory-secretory antigen from Paragonimus heterotremus. It proved effective in diagnosing infections due to P. heterotremus, and was also successfully diagnosed with sera from infections with P. westermani and P. miyazakii. The diagnostic sensitivity, specificity, positive and negative predictive values and accuracy were 97.9%, 87.6%, 78%, 98.9%, and 90.8%, respectively. The developed POCT tool is rapid and simple to use not only for clinical diagnosis of paragonimiasis at the bedside or at well-equipped laboratories, but also at local and remote hospitals with limited facilities. Moreover, the POCT tool could be applied for epidemiological surveys of paragonimaisis in Asia where P. heterotremus, P. westermani and P. miyazakii are endemic.

Tests in the point of care – glucose meter as a POCT tool. Implement a quality system control

Introduction: Point of care testing (POCT) are tests carried out near to the patient, outside the laboratory. There is a continuing increase interest in POCT, however the issue of quality of those tests rises concerns. Glucose concentration measurements are carried out using glucometers, which makes those analyzers a POCT tool. All devices outside the laboratory should be included in internal quality control system according to ISO 22870, so it is necessary to control glucometers as well.<br>Aim: To perform the quality control of glucometers as a POCT tools in University Hospital in Wroclaw, evaluate the correct use of glucometers, conduct replacement of devices, and implement constant internal quality control.<br>Materials and methods: The approximate count of blood glucose measurements in the hospital was determined on the basis of laboratory data and glucometers strips orders. Quality control was performed using MediSense certified control materials at two concentrations levels. A total of 272 control measurements were used for interpretation, which were made on 25 FreeStyle Optium H glucometers. The results were registered in quality control sheet.<br>Results: Laboratory determinations of glucose concentration represented 24.6% of all glucose measurements, POCT tests 75,4%. Among control measurements 80.88% were within ± 15% error recommended by Polish Diabetology Association and 19.12% exceeded it, 7.35% exceeded ± 20%. However none measurement exceeded the error recommended by glucometer manufacturer.<br>Conclusions: Based on our results several glucometers were replaced and the systematic internal quality control was introduced. Numerous of glucometer training courses for medical staff were conducted.

Hemoglobin A1C determination by point-of-care testing: Its correlation to standard method

The hemoglobin A1C (HbA1C) is the useful test for following up of glycemic control of the diabetic patient. Due to the present advance in point-of-care testing (POCT) technology, the new POCT for determination of HbA1C is available. Here, the author performs a laboratory comparative study between the new POCT tool (DCA) and classical tool (COBAS) for analysis of HbA1C level. This study is the second confirmation study in Thailand performed in the new setting adding to the previous author's report. The results show that there is a good correlation between two techniques and the error of the POCT tool is within acceptable limit.

Point of care testing for pediatric following up of hemoglobin A1C: Concern on product's property

Point of care testing (POCT) is a new concept in laboratory medicine. The POCT tool helps perform the laboratory test at site giving fast result and requires only small amount of sample. It is presently and increasingly used in pediatrics. In pediatric diabetology, the use of POCT for following up of hemoglobin A1C is a new concept. At present, there are several POCT tools for this purpose. In this specific article, the author compares on important aspects (turnaround time and required amount of specimen) of 5 available POCT tools for measurement of hemoglobin A1C level. The test that is the fastest and requires the least amount of blood sample, which means the least harm to the pediatric patient is the most preferable. Here, it can be seen that there is no ideal POCT that has both properties. However, if it is standardized to compare the amount of required blood sample, the special concern in the pediatric patients, the product from multiplication shows that Siemen DCA 2000+ and Siemen DCA Vantage are the two most preferable POCT tools for hemoglobin A1C determination in pediatric diabetes mellitus management.

Development of a point of care testing tool to classify peritoneal effusion as exudate and transudate

BackgroundRecently we developed a bedside test to classify pleural effusion into exudate and transudate but point of care classification of peritoneal effusion is yet not published. MethodsWe analyzed the Boyer's criteria parameters from bloodless peritoneal fluid and classified the biofluid as exudate or transudate and also estimated some parameters of oxidative stress in the biofluid by established spectrophotometric procedure. Two hundred microliters of sample was used and 10μl of 30% hydrogen peroxide was added, followed by inspection of the sample for the appearance of bubbles. ResultsAll exudative ascitic fluids (n=50) have shown the appearance of profuse bubbles within 1min addition of hydrogen peroxide along with significantly more catalase activity compared to transudate. All transudative ascitic fluids (n=50) have not shown bubble formation within 1min after the addition of hydrogen peroxide. The exudate does not show bubble formation if added with catalase inhibitors prior to the addition of hydrogen peroxide. Blood mixed transudate have shown profuse bubble formation after the addition of hydrogen peroxide. ConclusionThe hydrogen peroxide bubbling reaction has the potential to be developed as a point of care test to classify peritoneal fluid as exudate or transudate.