External Quality Assessment (EQA) for SARS-CoV-2 RNA Point-of-Care Testing in Primary Healthcare Services: Analytical Performance over Seven EQA Cycles.

In April 2020, the Aboriginal and Torres Strait Islander COVID-19 Point-of-Care (POC) Testing Program was initiated to improve access to rapid molecular-based SARS-CoV-2 detection in First Nations communities. At capacity, the program reached 105 health services across Australia. An external review estimated the program contributed to averting between 23,000 and 122,000 COVID-19 infections within 40 days of the first infection in a remote community, equating to cost savings of between AU$337 million and AU$1.8 billion. Essential to the quality management of this program, a customised External Quality Assessment (EQA) program was developed with the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP). From July 2020 to May 2022, SARS-CoV-2 EQA participation ranged from 93 to 100%. Overall concordance of valid EQA results was high (98%), with improved performance following the first survey. These results are consistent with those reported by 12 Australian and 4 New Zealand laboratories for three SARS-CoV-2 RNA EQA surveys in March 2020, demonstrating that SARS-CoV-2 RNA POC testing in primary care settings can be performed to an equivalent laboratory analytical standard. More broadly, this study highlights the value of quality management practices in real-world testing environments and the benefits of ongoing EQA program participation.

Photothermal biosensing integrated with microfluidic paper-based analytical device for sensitive quantification of sarcosine.

This article presents the development of a photothermal biosensing integrated with microfluidic paper-based analytical device (PT-µPAD) as a quantitative biosensor method for monitoring sarcosine in human control urine, plasma, and serum samples. The device utilizes gold nanoparticles (AuNPs) as both a peroxidase-like nanozyme and a photothermal substrate to enable sarcosine detection. In our PT-µPAD, hydrogen peroxide (H2O2) is generated through the oxidation of sarcosine by a sarcosine oxidase (SOx) enzyme. Subsequently, the H2O2 flows through the paper microchannels to the detection zone, where it etches the pre-deposited AuNPs, inducing a temperature change upon exposure by a 532 nm laser. The temperature variation is then measured using a portable and inexpensive infrared thermometer. Under optimized conditions, we obtained a linear range between 10.0 and 40.0 nmol L-1 (R2 = 0.9954) and a detection limit (LOD) of 32.0 pmol L-1. These values fall within the clinical range for sarcosine monitoring in prostate cancer diagnostics in humans. Moreover, our approach exhibits high selectivity without interfering effects. Recovery studies in various human control samples demonstrated a range of 99.05-102.11 % with the highest RSD of 2.25 %. The PT-µPAD was further validated for sarcosine determination in human control urine and compared with a commercial ELISA assay, revealing no significant difference between these two methods at a 95 % confidence level. Overall, our proposed sarcosine biosensor is well-suited for prostate cancer monitoring, given its affordability, sensitivity, and user-friendliness, even for unskilled individuals. Moreover, this strategy has promising prospects for broader applications, potentially detecting various biomarkers as a point-of-care (POC) diagnostic tool.

Impact of point-of-care HIV viral load and targeted drug resistance mutation testing on viral suppression among Kenyan pregnant and postpartum women: results from a prospective cohort study (Opt4Mamas).

INTRODUCTION: Lack of viral suppression (VS) among pregnant and breastfeeding women living with HIV poses challenges for maternal and infant health, and viral load (VL) monitoring via centralized laboratory systems faces many barriers. We aimed to determine the impact of point-of-care (POC) VL and targeted drug resistance mutation (DRM) testing in improving VS among pregnant and postpartum women on antiretroviral therapy. METHODS: We conducted a pre/post-intervention prospective cohort study among 820 pregnant women accessing HIV care at five public-sector facilities in western Kenya from 2019 to 2022. The pre-intervention or "control" group consisted of standard-of-care (SOC) centralized VL testing every 6 months and the post-intervention or "intervention" group consisted of a combined strategy of POC VL every 3 months, targeted DRM testing, and clinical management support. The primary outcome was VS (VL ≤1000 copies/ml) at 6 months postpartum; secondary outcomes included uptake and turnaround times for VL testing and sustained VS. RESULTS: At 6 months postpartum, 321/328 (98%) of participants in the intervention group and 339/347 (98%) in the control group achieved VS (aRR 1.00, 95% confidence interval [CI] 0.98, 1.02). When assessing VS using a threshold of <40 copies/ml, VS proportions were lower overall (90-91%) but remained similar between groups. Among women with viraemia (VL>1000 copies/ml) who underwent successful DRM testing in the intervention group, all (46/46, 100%) had some DRMs and 20 (43%) had major DRMs (of which 80% were nucleos(t)ide reverse transcriptase inhibitor mutations). POC VL testing uptake was high (>89%) throughout pregnancy, delivery, and postpartum periods, with a median turnaround time of 1 day (IQR 1, 4) for POC VL in the intervention group and 7 days (IQR 5, 9) for SOC VL in the control group. Sustained VS throughout follow-up was similar between groups with either POC or SOC VL testing (90-91% for <1000 copies/ml, 62-70% for <40 copies/ml). CONCLUSIONS: Our combined strategy markedly decreased turnaround time but did not increase VS rates, which were already very high, or sustained VS among pregnant and postpartum women living with HIV. Further research on how best to utilize POC VL and DRM testing is needed to optimize sustained VS among this population.

Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine.

The Rapid Visual CRISPR (RAVI-CRISPR) assay employs Cas12a and Cas13a enzymes for precise gene detection in a sample. However, RAVI-CRISPR is limited in single-tube multiplex detection applications due to the lack of specific single-strand (ss) DNA-fluorescently quenched (ssDNA-FQ) and RNA-fluorescently quenched (ssRNA-FQ) reporter cleavage mechanisms. We report the development of a sensitive and specific dual-gene Cas12a and Cas13a diagnostic system. To optimize the application for field testing, we designed a portable multiplex fluorescence imaging assay that could distinguish test results with the naked eye. Herein, dual gene amplified products from multiplex recombinase polymerase amplification (RPA) were simultaneously detected in a single tube using Cas12a and Cas13a enzymes. The resulting orthogonal DNA and RNA collateral cleavage specifically distinguishes individual and mixed ssDNA-FQ and ssRNA-FQ reporters using the green-red-yellow, fluorescent signal conversion reaction system, detectable with portable blue and ultraviolet (UV) light transilluminators. As a proof-of-concept, reliable multiplex RAVI-CRISPR detection of genome-edited pigs was demonstrated, exhibiting 100% sensitivity and specificity for the analysis of CD163 knockout, lactoferrin (LF) knock-in, and wild-type pig samples. This portable naked-eye multiplex RAVI-CRISPR detection platform can provide accurate point-of-care screening of genetically modified animals and infectious diseases in resource-limited settings.

A fully-enclosed prototype 'pen' for rapid detection of SARS-CoV-2 based on RT-RPA with dipstick assay at point-of-care testing.

A fully-enclosed prototype 'pen' for rapid detection of SARS-CoV-2 based on reverse transcriptase isothermal recombinase polymerase amplification (RT-RPA) with dipstick assay was developed. The integrated handheld device, consisting of amplification, detection and sealing modules, was developed to perform rapid nucleic acid amplification and detection under a fully enclosed condition. After RT-RPA amplification with a metal bath or a normal PCR instrument, the amplicons were mixed with dilution buffer prior to being detected on a lateral flow strip. To avoid aerosol contamination causing false-positive, from amplification to final detection, the detection 'pen' had been enclosed to isolate from the environment. With colloidal gold strip-based detection, the detection results could be directly observed by eyes. By cooperating with other inexpensive and rapid methods for POC nucleic acid extraction, the developed 'pen' could detect COVID-19 or other infectious diseases in a convenient, simple and reliable way.

A microfluidic system for rapid nucleic acid analysis based on real-time convective PCR at point-of-care testing.

A microfluidic system for rapid nucleic acid analysis based on real-time convective PCR is developed. To perform 'sample-in, answer-out' nucleic acid analysis, a microfluidic chip is developed to efficiently extract nucleic acid, and meanwhile convective PCR (CPCR) is applied for rapid nucleic acid amplification. With an integrated microfluidic chip consisting of reagent pre-storage chambers, a lysis & wash chamber, an elution chamber and a waste chamber, nucleic acid extraction based on magnetic beads can be automatically performed for a large size of test sample within a limited time. Based on an easy-to-operate strategy, different pre-stored reagents can be conveniently released for consecutive reaction at different steps. To achieve efficient mixing, a portable companion device is developed to introduce properly controlled 3-D actuation to magnetic beads in nucleic acid extraction. In CPCR amplification, PCR reagent can be spontaneously and repeatedly circulated between hot and cool zones of the reactor for space-domain thermal cycling based on pseudo-isothermal heating. A handheld real-time CPCR device is developed to perform nucleic acid amplification and in-situ detection. To extend the detection throughput, multiple handheld real-time CPCR devices can be grouped together by a common control system. It is demonstrated that influenza A (H1N1) viruses with the reasonable concentration down to 1.0 TCID50/ml can be successfully detected with the microfluidic system.

Smartphone-Based Multiplexed Biosensing Tools for Health Monitoring.

Many emerging technologies have the potential to improve health care by providing more personalized approaches or early diagnostic methods. In this review, we cover smartphone-based multiplexed sensors as affordable and portable sensing platforms for point-of-care devices. Multiplexing has been gaining attention recently for clinical diagnosis considering certain diseases require analysis of complex biological networks instead of single-marker analysis. Smartphones offer tremendous possibilities for on-site detection analysis due to their portability, high accessibility, fast sample processing, and robust imaging capabilities. Straightforward digital analysis and convenient user interfaces support networked health care systems and individualized health monitoring. Detailed biomarker profiling provides fast and accurate analysis for disease diagnosis for limited sample volume collection. Here, multiplexed smartphone-based assays with optical and electrochemical components are covered. Possible wireless or wired communication actuators and portable and wearable sensing integration for various sensing applications are discussed. The crucial features and the weaknesses of these devices are critically evaluated.

Reliability of point-of-care circulating cathodic antigen assay for diagnosing schistosomiasis mansoni in urine samples from an endemic area of Brazil after one year of storage at -20 degrees Celsius

ABSTRACT Background The World Health Organization recommends reliable point-of-care (POC) diagnostic testing to eliminate schistosomiasis. Lateral flow immunoassay that detects schistosome circulating cathodic antigen (CCA) in urine to establish prevalence thresholds for intervention in endemic areas is recommended. Stored urine may be useful if surveying at-risk populations is delayed or interrupted by unforeseen circumstances, such as the current COVID-19 pandemic. This study evaluated the manufacturer's claim that Schistosoma mansoni infection can be reliably diagnosed in urine samples stored at -20°C for one year. Methods Two-hundred-forty-two subjects from an endemic site in Brazil provided one urine sample each for testing with URINE CCA (SCHISTO) ECO TESTE® (POC-ECO) and one stool sample each for testing with Kato-Katz (KK) and Helmintex® (HTX) as a robust reference standard for infection status. At least 2 ml of urine from each participant was stored at -20°C; after one year, 76 samples were randomly selected for POC-ECO retesting. Results: The POC-ECO agreement between freshly collected and stored urine was inadequate considering trace results as positive (Cohen's kappa coefficient κ = 0.08) and negative (κ = 0.36). POC-ECO accuracy was not significantly greater than that of routine KK (54%; 95% confidence interval: 42.1%-65.5%). Conclusions The precision and accuracy of POC-ECO have to be optimized in both freshly collected and stored urine before it can be recommended for use in control programs in Brazil.

Optimizing viral load suppression in Kenyan children on antiretroviral therapy (Opt4Kids).

BACKGROUND: As many as 40% of the 1 million children living with HIV (CLHIV) receiving antiretroviral treatment (ART) in resource limited settings have not achieved viral suppression (VS). Kenya has a large burden of pediatric HIV with nearly 140,000 CLHIV. Feasible, scalable, and cost-effective approaches to ensure VS in CLHIV are urgently needed. The goal of this study is to determine the feasibility and impact of point-of-care (POC) viral load (VL) and targeted drug resistance mutation (DRM) testing to improve VS in children on ART in Kenya. METHODS: We are conducting a randomized controlled study to evaluate the use of POC VL and targeted DRM testing among 704 children aged 1-14 years on ART at health facilities in western Kenya. Children are randomized 1:1 to intervention (higher frequency POC VL and targeted DRM testing) vs. control (standard-of-care) arms and followed for 12 months. Our primary outcome is VS (VL < 1000 copies/mL) 12 months after enrollment by study arm. Secondary outcomes include time to VS and the impact of targeted DRM testing on VS. In addition, key informant interviews with patients and providers will generate an understanding of how the POC VL intervention functions. Finally, we will model the cost-effectiveness of POC VL combined with targeted DRM testing. DISCUSSION: This study will provide critical information on the impact of POC VL and DRM testing on VS among CLHIV on ART in a resource-limited setting and directly address the need to find approaches that maximize VS among children on ART. TRIALS REGISTRATION: NCT03820323.

An aptamer-based magnetic flow cytometer using matched filtering.

Facing unprecedented population-ageing, the management of noncommunicable diseases (NCDs) urgently needs a point-of-care (PoC) testing infrastructure. Magnetic flow cytometers are one such solution for rapid cancer cellular detection in a PoC setting. In this work, we report a giant magnetoresistive spin-valve (GMR SV) biosensor array with a multi-stripe sensor geometry and matched filtering to improve detection accuracy without compromising throughput. The carefully designed sensor geometry generates a characteristic signature when cells labeled with magnetic nanoparticles (MNPs) pass by thus enabling multi-parametric measurement like optical flow cytometers (FCMs). Enumeration and multi-parametric information were successfully measured across two decades of throughput (37 - 2730 cells/min). 10-µm polymer microspheres were used as a biomimetic model where MNPs and MNP-decorated polymer conjugates were flown over the GMR SV sensor array and detected with a signal-to-noise ratio (SNR) as low as 2.5 dB due to the processing gain afforded by the matched filtering. The performance was compared against optical observation, exhibiting a 92% detection efficiency. The system achieved a 95% counting accuracy for biomimetic models and 98% for aptamer-based pancreatic cancer cell detection. This system demonstrates the ability to perform reliable flow cytometry toward PoC diagnostics to benefit NCD control plans.

Electrochemical POC device for fast malaria quantitative diagnosis in whole blood by using magnetic beads, Poly-HRP and microfluidic paper electrodes.

Malaria, a parasitic infection caused by Plasmodium parasites and transmitted through the bite of infected female Anopheles mosquitos, is one of the main causes of mortality in many developing countries. Over 200 million new infections and nearly half a million deaths are reported each year, and more than three billion people are at risk of acquiring malaria worldwide. Nevertheless, most malaria cases could be cured if detected early. Malaria eradication is a top priority of the World Health Organisation. However, achieving this goal will require mass population screening and treatment, which will be hard to accomplish with current diagnostic tools. We report an electrochemical point-of-care device for the fast, simple and quantitative detection of Plasmodiumfalciparum lactate dehydrogenase (PfLDH) in whole blood samples. Sample analysis includes 5-min lysis to release intracellular parasites, and stirring for 5 more min with immuno-modified magnetic beads (MB) along with an immuno-modified signal amplifier. The rest of the magneto-immunoassay, including sample filtration, MB washing and electrochemical detection, is performed at a disposable paper electrode microfluidic device. The sensor provides PfLDH quantitation down to 2.47 ng mL-1 in spiked samples and for 0.006-1.5% parasitemias in Plasmodium-infected cultured red blood cells, and discrimination between healthy individuals and malaria patients presenting parasitemias >0.3%. Quantitative malaria diagnosis is attained with little user intervention, which is not achieved by other diagnostic methods.

3D-printed rolling circle amplification chip for on-site colorimetric detection of inorganic mercury in drinking water.

A point-of-care testing chip was developed for the colorimetric detection of inorganic mercury ion (Hg2+). The disposable chip fabricated by three-dimensional printing technology contains DNAzymes produced by rolling circle amplification (RCA); a color change caused by the enzymatic reaction between DNAzymes and the peroxidase substrate 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) is measured using a portable spectrophotometer. In the "turn-off"-type RCA reaction, the annealing of the T(12) primer that initiates the RCA reaction is blocked by the interaction of thymine with Hg2+; thus, the amount of amplified DNAzymes causing a color change is decreased depending on Hg2+ concentration. The colorimetric signal is enhanced by amplifying double-repeat DNAzymes from a circular DNA template. The chip detected Hg2+ in tap drinking water samples with high sensitivity (lowest validated value: 3.6 µg/L) and showed better selectivity, precision, and reproducibility than conventional analysis instruments. This low-cost easy-to-use platform can reduce the risk of accidental Hg2+ poisoning.

Single-step, high-specificity detection of single nucleotide mutation by primer-activatable loop-mediated isothermal amplification (PA-LAMP).

Loop-mediated isothermal amplification (LAMP) is a useful platform for nucleic acids detection in point-of-care (POC) situations, and development of single-step, close-tube LAMP reactions for specific detection of single nucleotide mutations (SNMs) remains a challenge. We develop a novel primer-activatable LAMP (PA-LAMP) strategy that enables highly specific and sensitive SNM detection using single-step, close-tube reactions. This strategy designs a terminal-blocked inner primer with a ribonucleotide insertion, which is cleaved and activated specifically to perfectly matched targets by ribonuclease (RNase) H2, to realize efficient amplification of mutant genes. It has shown dynamic responses of mutant target in a linear range from 220 aM to 22 pM with a lowest detectable concentration of 22 aM. It also demonstrates very high specificity in identifying the mutant in a large excess of the wild-type with a discrimination ratio as high as ∼10,000. It has been successfully applied to mutation detection of genomic DNA in tumor cells. The PA-LAMP strategy provides a useful, portable and affordable POC platform for highly sensitive and specific detection of genetic mutations in clinical applications.

The universe of ANA testing: a case for point-of-care ANA testing.

Testing for total antinuclear antibodies (ANA) is a critical tool for diagnosis and management of autoimmune diseases at both the primary care and subspecialty settings. Repurposing of ANA from a test for lupus to a test for any autoimmune condition has driven the increase in ANA requests. Changes in ANA referral patterns include early or subclinical autoimmune disease detection in patients with low pre-test probability and use of negative ANA results to rule out underlying autoimmune disease. A positive result can lead to further diagnostic considerations. Currently, ANA tests are performed in centralized laboratories; an alternative would be ANA testing at the clinical point-of-care (POC). By virtue of its near real-time data collection capability, low cost, and ease of use, we believe the POC ANA has the potential to enable a new paradigm shift in autoimmune serology testing.

Detection of autoantibodies in a point-of-care rheumatology setting.

Autoimmune rheumatic diseases are common and confront society with serious medical, social, and financial burdens imposed by their debilitating nature. Many autoimmune diseases are associated with a particular set of autoantibodies, which have emerged as highly useful to define and classify disease, predict flares, or monitor efficacy of therapy. However, current practice for monitoring autoantibodies is protracted, labor-intensive, and expensive. This review provides an overview on the value of point-of-care (POC) biosensor technology in the diagnosis and management of patients with autoimmune rheumatic diseases. Real-time measurement of autoantibodies will clearly benefit the rheumatology practice in emergency and urgent care settings, where definitive diagnosis is essential for initiation of correct critical care therapy. Immediate serological information in clinic will provide considerable value for long-term patient care and an opportunity for an instant, result-deduced therapeutic action, avoiding delays and improving compliance, especially in field-based and remote areas. We describe the particular autoantibodies that are useful disease and activity markers and would, therefore, be attractive to POC applications. Already existing biosensors and platforms that show promise for autoantibody testing are summarized and comparatively evaluated. As POC assessment is gaining momentum in several areas of patient care, we propose that rheumatology is poised to benefit from this innovative and affordable technology.