4,521 research outputs found
Recommended from our members
A Rapid and Low-Cost Pathogen Detection Platform by Using a Molecular Agglutination Assay.
Rapid and low-cost pathogen diagnostic approaches are critical for clinical decision-making procedures. Cultivating bacteria often takes days to identify pathogens and provide antimicrobial susceptibilities. The delay in diagnosis may result in compromised treatment and inappropriate antibiotic use. Over the past decades, molecular-based techniques have significantly shortened pathogen identification turnaround time with high accuracy. However, these assays often use complex fluorescent labeling and nucleic acid amplification processes, which limit their use in resource-limited settings. In this work, we demonstrate a wash-free molecular agglutination assay with a straightforward mixing and incubation step that significantly simplifies procedures of molecular testing. By targeting the 16S rRNA gene of pathogens, we perform a rapid pathogen identification within 30 min on a dark-field imaging microfluidic cytometry platform. The dark-field images with low background noise can be obtained using a narrow beam scanning technique with off-the-shelf complementary metal oxide semiconductor (CMOS) imagers such as smartphone cameras. We utilize a machine learning algorithm to deconvolute topological features of agglutinated clusters and thus quantify the abundance of bacteria. Consequently, we unambiguously distinguish Escherichia coli positive from other E. coli negative among 50 clinical urinary tract infection samples with 96% sensitivity and 100% specificity. Furthermore, we also apply this quantitative detection approach to achieve rapid antimicrobial susceptibility testing within 3 h. This work exhibits easy-to-use protocols, high sensitivity, and short turnaround time for point-of-care testing uses
Taking connected mobile-health diagnostics of infectious diseases to the field.
Mobile Health or mHealth - The application of mobile devices, their components and related technologies to healthcare is improving patients’ access to treatment and advice. Now, in combination with connected diagnostic devices it offers new possibilities to diagnose, track and control infectious diseases and improve health system efficiencies. In this context we look at these technologies and highlight their promise but also the challenges in realising their potential to increase patient access to testing, aid in their treatment and improve the capability of public health authorities to monitor outbreaks, implement responses, and assess the impact of interventions across the world
Magnetic Bead Handling Using a Paper-Based Device for Quantitative Point-of-Care Testing
Low-cost assay automation; Malaria quantitative diagnosis; Smartphone colorimetric detectionAutomatització d'assaigs de baix cost; Diagnòstic quantitatiu de la malària; Detecció colorimètrica del telèfon intel·ligentAutomatización de ensayos de bajo costo; Diagnóstico cuantitativo de la malaria; Detección colorimétrica de teléfono inteligenteMicrofluidic paper-based analytical devices (μPADs) have been extensively proposed as ideal tools for point-of-care (POC) testing with minimal user training and technical requirements. However, most μPADs use dried bioreagents, which complicate production, reduce device reproducibility and stability, and require transport and storage under temperature and humidity-controlled conditions. In this work, we propose a μPAD produced using an affordable craft-cutter and stored at room temperature, which is used to partially automate a single-step colorimetric magneto-immunoassay. As a proof-of-concept, the μPAD has been applied to the quantitative detection of Plasmodium falciparum lactate dehydrogenase (Pf-LDH), a biomarker of malaria infection. In this system, detection is based on a single-step magneto-immunoassay that consists of a single 5-min incubation of the lysed blood sample with immuno-modified magnetic beads (MB), detection antibody, and an enzymatic signal amplifier (Poly-HRP). This mixture is then transferred to a single-piece paper device where, after on-chip MB magnetic concentration and washing, signal generation is achieved by adding a chromogenic enzyme substrate. The colorimetric readout is achieved by the naked eye or using a smartphone camera and free software for image analysis. This μPAD afforded quantitative Pf-LDH detection in <15 min, with a detection limit of 6.25 ng mL−1 when the result was interpreted by the naked eye and 1.4 ng mL−1 when analysed using the smartphone imaging system. Moreover, the study of a battery of clinical samples revealed concentrations of Pf-LDH that correlated with those provided by the reference ELISA and with better sensitivity than a commercial rapid diagnostic test (RDT). These results demonstrate that magneto-immunoassays can be partly automated by employing a μPAD, achieving a level of handling that approaches the requirements of POC testing.This research was funded by Fondo de Investigaciones Sanitarias of Instituto de Salud Carlos III (co-funded by the European Regional Development Fund; grants CPII18/00025, IFI18/00020, JR18/00022 and QUPID EuroNanoMed AC21_2/00021) and La Caixa Foundation (ID 100010434, fellowship LCF/BQ/DI18/11660061). Diagnostic Nanotools is a consolidated group supported by Secretaria d’Universitats i Recerca, Generalitat de Catalunya (2017 SGR 240)
Digital Platform for Automatic Qualitative and Quantitative Reading of a Cryptococcal Antigen Point-of-Care Assay Leveraging Smartphones and Artificial Intelligence
This work was presented in part at 31st European Congress of Clinical Microbiology & Infectious Diseases (ECCMID), which will take place online from 9 – 12 July 2021. Abstract number 03467.Cryptococcosis is a fungal infection that causes serious illness, particularly in immunocompromised individuals such as people living with HIV. Point of care tests (POCT) can help identify and diagnose patients with several advantages including rapid results and ease of use. The cryptococcal antigen (CrAg) lateral flow assay (LFA) has demonstrated excellent performance in diagnosing cryptococcosis, and it is particularly useful in resource-limited settings where laboratory-based tests may not be readily available. The use of artificial intelligence (AI) for the interpretation of rapid diagnostic tests can improve the accuracy and speed of test results, as well as reduce the cost and workload of healthcare professionals, reducing subjectivity associated with its interpretation. In this work, we analyze a smartphone-based digital system assisted by AI to automatically interpret CrAg LFA as well as to estimate the antigen concentration in the strip. The system showed excellent performance for predicting LFA qualitative interpretation with an area under the receiver operating characteristic curve of 0.997. On the other hand, its potential to predict antigen concentration based solely on a photograph of the LFA has also been demonstrated, finding a strong correlation between band intensity and antigen concentration, with a Pearson correlation coefficient of 0.953. The system, which is connected to a cloud web platform, allows for case identification, quality control, and real-time monitoring.CrAg LFA tests were provided by IMMY at no cost. This research was funded by Global Action For Fungal Infections (www.GAFFI.org), JYLAG, a charity Foundation based in Geneva, Switzerland, and Fondo de Investigación Sanitaria from Instituto de Salud Carlos III (PI20CIII/00043). D.B.-P. was supported by grant PTQ2020-011340/AEI/10.13039/501100011033 funded by the Spanish State Investigation Agency. J.C.S.-D. was supported by a fellowship from the Fondo de Investigación Sanitaria (grant FI17CIII/00027).S
Harnessing recombinase polymerase amplification for rapid multi-gene detection of SARS-CoV-2 in resource-limited settings
The COVID-19 pandemic is challenging diagnostic testing capacity worldwide. The mass testing needed to limit the spread of the virus requires new molecular diagnostic tests to dramatically widen access at the point-of-care in resource-limited settings. Isothermal molecular assays have emerged as a promising technology, given the faster turn-around time and minimal equipment compared to gold standard laboratory PCR methods. However, unlike PCR, they do not typically target multiple SARS-CoV-2 genes, risking sensitivity and specificity. Moreover, they often require multiple steps thus adding complexity and delays. Here we develop a multiplexed, 1-2 step, fast (20-30 minutes) SARS-CoV-2 molecular test using reverse transcription recombinase polymerase amplification to simultaneously detect two conserved targets - the E and RdRP genes. The agile multi-gene platform offers two complementary detection methods: real-time fluorescence or dipstick. The analytical sensitivity of the fluorescence test was 9.5 (95% CI: 7.0-18) RNA copies per reaction for the E gene and 17 (95% CI: 11-93) RNA copies per reaction for the RdRP gene. The analytical sensitivity for the dipstick was 130 (95% CI: 82-500) RNA copies per reaction. High specificity was found against common seasonal coronaviruses, SARS-CoV and MERS-CoV model samples. The dipstick readout demonstrated potential for point-of-care testing in decentralised settings, with minimal or equipment-free incubation methods and a user-friendly prototype smartphone application. This rapid, simple, ultrasensitive and multiplexed molecular test offers valuable advantages over gold standard tests and in future could be configurated to detect emerging variants of concern
Cellphone-Based Hand-Held Microplate Reader for Point-of-Care Testing of Enzyme-Linked Immunosorbent Assays
Standard microplate based enzyme-linked immunosorbent
assays (ELISA) are widely utilized for various nanomedicine,
molecular sensing, and disease screening applications, and this
multiwell plate batched analysis dramatically reduces diagnosis
costs per patient compared to nonbatched or nonstandard tests.
However, their use in resource-limited and field-settings is inhibited
by the necessity for relatively large and expensive readout instruments.
To mitigate this problem, we created a hand-held and cost-effective cellphone-based colorimetric microplate reader, which uses a 3D-printed optomechanical
attachment to hold and illuminate a 96-well plate using a light-emitting-diode (LED) array. This LED light is transmitted through each well, and
is then collected via 96 individual optical fibers. Captured images of this fiber-bundle are transmitted to our servers through a custom-designed app for
processing using a machine learning algorithm, yielding diagnostic results, which are delivered to the user within ∼1 min per 96-well plate, and are
visualized using the same app. We successfully tested this mobile platform in a clinical microbiology laboratory using FDA-approved mumps IgG, measles
IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests using a total of 567 and 571 patient samples for training and blind testing, respectively, and
achieved an accuracy of 99.6%, 98.6%, 99.4%, and 99.4% for mumps, measles, HSV-1, and HSV-2 tests, respectively. This cost-effective and hand-held
platform could assist health-care professionals to perform high-throughput disease screening or tracking of vaccination campaigns at the point-of-care,
even in resource-poor and field-settings. Also, its intrinsic wireless connectivity can serve epidemiological studies, generating spatiotemporal maps of
disease prevalence and immunity
Deep learning of HIV field-based rapid tests
Although deep learning algorithms show increasing promise for disease diagnosis, their use with rapid diagnostic tests performed in the field has not been extensively tested. Here we use deep learning to classify images of rapid human immunodeficiency virus (HIV) tests acquired in rural South Africa. Using newly developed image capture protocols with the Samsung SM-P585 tablet, 60 fieldworkers routinely collected images of HIV lateral flow tests. From a library of 11,374 images, deep learning algorithms were trained to classify tests as positive or negative. A pilot field study of the algorithms deployed as a mobile application demonstrated high levels of sensitivity (97.8%) and specificity (100%) compared with traditional visual interpretation by humans-experienced nurses and newly trained community health worker staff-and reduced the number of false positives and false negatives. Our findings lay the foundations for a new paradigm of deep learning-enabled diagnostics in low- and middle-income countries, termed REASSURED diagnostics1, an acronym for real-time connectivity, ease of specimen collection, affordable, sensitive, specific, user-friendly, rapid, equipment-free and deliverable. Such diagnostics have the potential to provide a platform for workforce training, quality assurance, decision support and mobile connectivity to inform disease control strategies, strengthen healthcare system efficiency and improve patient outcomes and outbreak management in emerging infections
Recommended from our members
Development and evaluation of point-of-care diagnostic technologies for providers and consumers
Point-of-care (POC) diagnostic technologies aim to expand access to traditional laboratory-based testing to near-patient settings. These settings can range from emergency or intensive care-units (ICUs) in the United States, to remote health posts in sub Saharan Africa. Differences in budget and infrastructure play a role in characterizing the wide array of possible “near patient” settings and must be taken into consideration in the engineering design process. In this dissertation we use translational engineering to develop practical and accessible microfluidic POC immunoassays for diverse settings, that include both provider and consumer facing applications.
First, we examined Lyme Disease in the U.S., where existing diagnostic technologies face the challenge of rapid and accurate serodiagnosis in the face of largely non-specific clinical symptoms. We developed a multiplexed rapid test that could replicate enzyme-linked immunosorbent assay (ELISA) performance for Lyme Disease diagnosis. After screening candidate biomarkers, we evaluated performance of the multiplexed microfluidic test against ELISA using clinical serum samples and illustrated the potential to streamline current clinical algorithms requiring two immunoassays (ELISA and Western Blot) into one standalone test suitable for physician’s offices or urgent care clinics in the U.S. We also showed exploratory work towards a similar multiplexed test design for another bacterial spirochete infection, Leptospirosis.
Next, we built on previous work towards a POC HIV-syphilis antenatal screening tool, to develop a smartphone-integrated, microfluidic assay for healthcare workers to use in low resource settings. The low-cost (34 to produce and provides results in 15 minutes. In this work, we focus on assay development efforts undertaken towards development of a fully integrated POC product suitable for deployment in the field, with practical considerations for the use of fingerstick blood, stability, scale-up and transport. We also streamlined the number of manual steps for end-user operation, through the use of lyophilized secondary antibodies, preloaded reagents on cassette, and an automatic result readout. While laboratory demonstration with clinical samples is important for initial characterization of POC devices, field evaluation reveals diagnostic performance under real-world conditions. We tested the device in the hands of minimally trained healthcare workers in Rwanda and saw comparable performance to other immunoassays run under field conditions. We also performed a follow-up pilot field study in Rwanda to evaluate the feasibility of the smartphone dongle platform for self-testing by patients/consumers in a low-resource setting, one of the most challenging use-cases for POC devices.
Finally, we sought to integrate intellectual frameworks from behavioral research and user-experience (UX) design in creating a new framework for evaluation of consumer-facing microfluidic devices, specifically towards HIV home-testing in the U.S. While overall rates of HIV are decreasing in the U.S., the population of gay, bisexual and other men who have sex with men (MSM) are disproportionately affected. Self-testing products for sexually transmitted infection (STI) testing could address unmet needs for these target populations in both increasing access and frequency of testing, as well as integrating use with sexual partners for early diagnosis or even prevention. We worked with a cohort of MSMs at high risk for HIV/STI transmission in New York City, and performed for the first time, a structured assessment of completely naïve users interacting with a smartphone interfaced microfluidic diagnostic device (“SMARTtest”). We integrated UX design value model of device usability, credibility, accessibility and acceptability into our evaluation framework, which influence user’s information, knowledge, motivation and behavioral skills towards engaging with a prevention method (“IMB” model). Thus far, such frameworks have rarely been applied to other consumer health monitoring devices, including microfluidic POC devices. As the microfluidic field moves towards more field demonstrations of devices, more untrained and minimally trained users will have access to such tools. It is important to understand how they use devices, what the device failure points are and what the most relevant design features are to spur user adoption and meaningful usage.
Underlying our work in creating accessible and practical POC immunoassay tools for infectious disease detection, is the illustration of the translational development roadmap from proof-of-concept assay development to field studies and user-based evaluations for intended end-use settings that range from U.S. based primary care clinics, rural health centers in low-resource settings as well as self-testing environments in both. Incorporating an understanding of the target use-case setting is critical in translating technologies for clinical use, whether in the infrastructure and services that are available, or end-user needs and constraints such as clinical workflow patterns, level of technical expertise and perceptions of usefulness and value. We show how user/use-case focused application of downstream translational engineering and testing informs upstream design choices and accelerates development of POC devices for real-world use. The sum of this work aims to illustrate tenets of translational engineering design and testing to advance insight into building POC products that are poised for greater adoption by target end users, whether they are health providers or consumers
The Development of a Primer Payload with Microparticles for UTI Pathogen Identification Using Polythymidine- Modified LAMP Primers in Droplet LAMP
Nucleic acid amplification tests (NAATs) are among the diagnostic tests with the highest sensitivity and specificity. However, they are more complex to develop than other diagnostic tests such as biochemical tests and lateral flow immunoassay tests. Polymerase chain reaction (PCR) is the gold standard for NAATs. PCR requires thermal cycling to achieve clonal amplification of the target pathogen DNA for diagnosis. Thermal cycling poses a challenge in the development of PCR diagnostics for point-of-care (POC) settings. Loop-mediated isothermal amplification (LAMP) offers an isothermal method for NAATs diagnostics. The advancement of the microfluidics field significantly enhances the development of LAMP diagnostics devices for POC testing. Another challenge with NAATs, is the limitation in the development of multiplex NAATs. Multiplexing however, occupies an important role in the efforts to address the antimicrobial resistance global crisis. Multiplexing will help to provide more thorough and complete diagnostics of infections, and enable doctors to prescribe the most effective antibiotics to the patients. This will help slow the emergence of antibiotic resistant pathogens. We are currently in a period of discovery void, with regards to antibiotics discovery. At this rate, more pathogens are becoming resistant to the antibiotics that we have, faster than we are developing new classes of antibiotics. According to the World Health Organization (WHO) interagency coordination group on AMR report to the secretary general of the United Nations, by 2050, there will be 10 million annual deaths globally, as a result of AMR-related events. There will also be 1 trillion in healthcare costs, and 28 million people will be living in poverty, as a result of the economic impact of uncontrolled AMR. Another area where multiplex diagnostics play a crucial role is infection control in the era of epidemics and pandemics. The increasing prevailing frequency of global pandemics stresses the need for the development of highly accurate and decentralized POC diagnostics. Over the last ten years, there have been more than 30 epidemics and pandemics around the world, including SARS-CoV-2, Monkey pox, India black fungus, Dengue fever, Measles, Zika, Avian influenza, Influenza A and Ebola. With advancing technology and international commerce and relations, we are now more connected than ever. This means that if there are no developments to make molecular tests more accessible at the POC, the future waves of epidemics and pandemics will have faster spread, further reach and more devastating impacts on the lives of the 8 billion people on our planet. We have developed a diagnostic method for executing droplet microfluidics LAMP via a microparticle primer payload mechanism and have demonstrated it with urinary tract infection (UTI) pathogens. With inspiration from overhang PCR and RNA-Seq, we engineered LAMP primers with 5’ polythymidine (PolyT) oligonucleotide (PolyT is placed in the middle of the Forward inner primers and Backward inner primers). The PolyT sequence is recognized by a biotinylated capture oligonucleotide engineered with a polyadenylated (PolyA) polynucleotide on the 3’ end. The streptavidin-coated microparticles functionalized with the PolyA oligonucleotide and PolyT primers, capture their specific target DNA and deliver the cargo into emulsion droplets of LAMP reagents for amplification. This platform provides the ability to multiplex by coding specific pathogen target DNA with different fluorescent signatures of the microparticles
Low-Cost, User-Friendly, All-Integrated Smartphone-Based Microplate Reader for Optical-Based Biological and Chemical Analyses
The quantitative detection of different molecular targets is of utmost importance for a variety of human activities, ranging from healthcare to environmental studies. Bioanalytical methods have been developed to solve this and to achieve the quantification of multiple targets from small volume samples. Generally, they can be divided into two different classes: point of care (PoC) and laboratory-based approaches. The former is rapid, low-cost, and user-friendly; however, the majority of the tests are semiquantitative, lacking in specificity and sensitivity. On the contrary, laboratory-based approaches provide high sensitivity and specificity, but the bulkiness of experimental instruments and complicated protocols hamper their use in resource-limited settings. In response, here we propose a smartphone-based device able to support laboratory-based optical techniques directly at the point of care. Specifically, we designed and fabricated a portable microplate reader that supports colorimetric, fluorescence, luminescence, and turbidity analyses. To demonstrate the potential of the device, we characterized its analytical performance by detecting a variety of relevant molecular targets (ranging from antibodies, toxins, drugs, and classic fluorophore dyes) and we showed how the estimated results are comparable to those obtained from a commercial microplate reader. Thanks to its low cost (< $3 00), portability (27 cm [length] × 18 cm [width] × 7 cm [height]), commercially available components, and open-source-based system, we believe it represents a valid approach to bring high-precision laboratory-based analysis at the point of care
- …