Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform

ABSTRACT An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.

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System Architecture for IIoT-Based POC Molecular Diagnostic Device

Engineering Proceedings ◽

10.3390/i3s2021dresden-10147 ◽

2021 ◽

Vol 6 (1) ◽

pp. 60

Author(s):

Byeong-Heon Kil ◽

Ji-Seong Park ◽

Chan-Young Park ◽

Yu-Seop Kim ◽

Jong-Dae Kim

Keyword(s):

Point Of Care ◽

Low Cost ◽

Diagnostic System ◽

Pcr Amplification ◽

Dna Amplification ◽

System Structure ◽

World Health ◽

Molecular Diagnostic ◽

Target System ◽

Open Platform

In this paper, we investigate an efficient structure for a point-of-care (POC) molecular diagnostic system based on the industrial Internet of things (IIoT). The target system can perform automated molecular diagnosis including DNA extraction, PCR amplification, and fluorescence detection. Samples and reagents are placed in a multi-room cartridge and loaded into the system. A rotating motor and a syringe motor control the cartridge to extract DNA from the sample. The extracted DNA is transferred to a polymerase chain reaction (PCR) chamber for DNA amplification and detection. The proposed system provides multiplexing of up to four colors. For POC molecular diagnostics, the World Health Organization demands features such as low volume, low cost, fast results, and a user-friendly interface. In this paper, we propose a system structure that can satisfy these requirements by using a PCR chip and open platform. A distributed structure is adopted for the convenience of maintenance, and a web-based GUI is adopted for the user’s convenience. We also investigated communication problems that may occur between system components. Using the proposed structure, the user can conveniently control from standard computing devices including a smartphone.

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Rapid and Sensitive Detection of miRNA Based on AC Electrokinetic Capacitive Sensing for Point-of-Care Applications

Sensors ◽

10.3390/s21123985 ◽

2021 ◽

Vol 21 (12) ◽

pp. 3985

Author(s):

Nan Wan ◽

Yu Jiang ◽

Jiamei Huang ◽

Rania Oueslati ◽

Shigetoshi Eda ◽

...

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Clinical Diagnostics ◽

Detection Methods ◽

Capacitive Sensing ◽

Application Potential ◽

Mirna Detection ◽

Mirna 25 ◽

Low Sensitivity

A sensitive and efficient method for microRNAs (miRNAs) detection is strongly desired by clinicians and, in recent years, the search for such a method has drawn much attention. There has been significant interest in using miRNA as biomarkers for multiple diseases and conditions in clinical diagnostics. Presently, most miRNA detection methods suffer from drawbacks, e.g., low sensitivity, long assay time, expensive equipment, trained personnel, or unsuitability for point-of-care. New methodologies are needed to overcome these limitations to allow rapid, sensitive, low-cost, easy-to-use, and portable methods for miRNA detection at the point of care. In this work, to overcome these shortcomings, we integrated capacitive sensing and alternating current electrokinetic effects to detect specific miRNA-16b molecules, as a model, with the limit of detection reaching 1.0 femto molar (fM) levels. The specificity of the sensor was verified by testing miRNA-25, which has the same length as miRNA-16b. The sensor we developed demonstrated significant improvements in sensitivity, response time and cost over other miRNA detection methods, and has application potential at point-of-care.

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Demonstration of a Label-Free and Low-Cost Optical Cavity-Based Biosensor Using Streptavidin and C-Reactive Protein

Biosensors ◽

10.3390/bios11010004 ◽

2020 ◽

Vol 11 (1) ◽

pp. 4

Author(s):

Donggee Rho ◽

Seunghyun Kim

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Optical Cavity ◽

Sample Volume ◽

Small Sample ◽

Label Free ◽

C Reactive Protein ◽

Reactive Protein ◽

Cavity Structure

An optical cavity-based biosensor (OCB) has been developed for point-of-care (POC) applications. This label-free biosensor employs low-cost components and simple fabrication processes to lower the overall cost while achieving high sensitivity using a differential detection method. To experimentally demonstrate its limit of detection (LOD), we conducted biosensing experiments with streptavidin and C-reactive protein (CRP). The optical cavity structure was optimized further for better sensitivity and easier fluid control. We utilized the polymer swelling property to fine-tune the optical cavity width, which significantly improved the success rate to produce measurable samples. Four different concentrations of streptavidin were tested in triplicate, and the LOD of the OCB was determined to be 1.35 nM. The OCB also successfully detected three different concentrations of human CRP using biotinylated CRP antibody. The LOD for CRP detection was 377 pM. All measurements were done using a small sample volume of 15 µL within 30 min. By reducing the sensing area, improving the functionalization and passivation processes, and increasing the sample volume, the LOD of the OCB are estimated to be reduced further to the femto-molar range. Overall, the demonstrated capability of the OCB in the present work shows great potential to be used as a promising POC biosensor.

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Whole Blood Immunoassay Based on Centrifugal Bead Sedimentation

Clinical Chemistry ◽

10.1373/clinchem.2011.162206 ◽

2011 ◽

Vol 57 (5) ◽

pp. 753-761 ◽

Cited By ~ 44

Author(s):

Ulrich Y Schaff ◽

Greg J Sommer

Keyword(s):

Whole Blood ◽

Single Channel ◽

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Microfluidic System ◽

Blood Samples ◽

Reactive Protein ◽

Immunoassay Technique ◽

Microfluidic Immunoassay

BACKGROUND Centrifugal “lab on a disk” microfluidics is a promising avenue for developing portable, low-cost, automated immunoassays. However, the necessity of incorporating multiple wash steps results in complicated designs that increase the time and sample/reagent volumes needed to run assays and raises the probability of errors. We present proof of principle for a disk-based microfluidic immunoassay technique that processes blood samples without conventional wash steps. METHODS Microfluidic disks were fabricated from layers of patterned, double-sided tape and polymer sheets. Sample was mixed on-disk with assay capture beads and labeling antibodies. Following incubation, the assay beads were physically separated from the blood cells, plasma, and unbound label by centrifugation through a density medium. A signal-laden pellet formed at the periphery of the disk was analyzed to quantify concentration of the target analyte. RESULTS To demonstrate this technique, the inflammation biomarkers C-reactive protein and interleukin-6 were measured from spiked mouse plasma and human whole blood samples. On-disk processing (mixing, labeling, and separation) facilitated direct assays on 1-μL samples with a 15-min sample-to-answer time, <100 pmol/L limit of detection, and 10% CV. We also used a unique single-channel multiplexing technique based on the sedimentation rate of different size or density bead populations. CONCLUSIONS This portable microfluidic system is a promising method for rapid, inexpensive, and automated detection of multiple analytes directly from a drop of blood in a point-of-care setting.

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Application of Flexible Display Technology to Low Cost Multiplexed Point-of-Care Medical Diagnostic Testing

Journal of Global Oncology ◽

10.1200/jgo.2016.003855 ◽

2016 ◽

Vol 2 (3_suppl) ◽

pp. 14s-14s

Author(s):

Benjamin A. Katchman ◽

Joseph T. Smith ◽

Jennifer Blain Christen ◽

Karen S. Anderson

Keyword(s):

Intellectual Property ◽

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Manufacturing Technology ◽

Analytical Sensitivity ◽

State University ◽

Arizona State University ◽

New Approach ◽

Display Technology

Abstract 62 One of the key roadblocks limiting the transition of high-sensitivity and high-specificity point-of-care technologies from the research laboratory to wide spread use is the availability of a low-cost-high-volume manufacturing technology. This work presents a new interdisciplinary approach combining low cost commercial display manufacturing technology with programmable high density protein microarray printing technology to fabricate disposable point-of-care immunosensors with clinical level sensitivity. Our approach is designed to leverage advances in commercial display technology to reduce pre-functionalized biosensor substrate costs to pennies per cm2, as well as to leverage the display industry’s ability to manufacture an immense number of low cost consumer electronic products annually. For this work, we demonstrate that our new approach can offer diagnostic sensitivity at or below 10 pg/mL, which approaches the lower limit of detection of typical clinical laboratory instrumentation. Our new approach is also designed to overcome the limited analytical sensitivity of existing POC devices (>100x improved sensitivity). It also contains new capability for multiplexed biomarker detection (>10 antigens) in a single low cost POC device through an innovative disposable and scalable architecture, based on flat panel display technology. Here, we demonstrate multiplexed detection of antibodies to the HPV16 proteins E2, E6, and E7, which are circulating biomarkers for cervical as well as head and neck cancers. This detection technology has 100 percent correlation to our current laboratory-based measurement instrumentation. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST: Benjamin A. Katchman Patents, Royalties, Other Intellectual Property: Arizona State University Joseph T. Smith Patents, Royalties, Other Intellectual Property: Arizona State University Jennifer Blain Christen Patents, Royalties, Other Intellectual Property: Arizona State University Karen S. Anderson Stock or Other Ownership: Provista Diagnostics Consulting or Advisory Role: Provista Diagnostics Patents, Royalties, Other Intellectual Property: Arizona State University

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Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System

Journal of Clinical Microbiology ◽

10.1128/jcm.00466-17 ◽

2017 ◽

Vol 55 (10) ◽

pp. 2964-2971 ◽

Cited By ~ 4

Author(s):

Padmapriya P. Banada ◽

Srinidhi Deshpande ◽

Riccardo Russo ◽

Eric Singleton ◽

Darshini Shah ◽

...

Keyword(s):

Bacillus Anthracis ◽

Dynamic Range ◽

Limit Of Detection ◽

Point Of Care ◽

Severe Disease ◽

Automated System ◽

Bacterial Isolates ◽

Sample Processing ◽

Blood Samples ◽

Content Type

ABSTRACT Bacillus anthracis is a tier 1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect B. anthracis bacteremia using a system that is suitable for point-of-care testing. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. The assay limit of detection (LOD) and dynamic range were determined by spiking B. anthracis DNA into individual PCR mixtures and B. anthracis CFU into human blood. One-milliliter blood samples were added to the filter-based detection cartridge and tested for B. anthracis on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates or by testing blood samples drawn from patients with concurrent non- B. anthracis bacteremia or nonbacteremic controls. The assay LODs were 5 genome equivalents per reaction and 10 CFU/ml blood for both the B. anthracis Sterne and V1B strains. There was a 6-log 10 dynamic range. Assay specificity was 100% for tests of non- B. anthracis bacterial isolates and patient blood samples. Assay time was less than 90 min. This automated system suitable for point-of-care detection rapidly identifies B. anthracis directly from blood with high sensitivity. This assay might lead to early detection and more rapid therapy in the event of a bioterrorism attack.

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Alleviating the Neglected Tropical Diseases: Recent Developments in Diagnostics and Detection

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666181106124015 ◽

2018 ◽

Vol 18 (18) ◽

pp. 1559-1574 ◽

Cited By ~ 3

Author(s):

Satakshi Hazra ◽

Sanjukta Patra

Keyword(s):

Pathogen Detection ◽

Neglected Tropical Diseases ◽

Point Of Care ◽

Low Cost ◽

Holistic Approach ◽

Diagnostic Tools ◽

Tropical Diseases ◽

Antimicrobial Drug Resistance ◽

Diagnostic Measures ◽

Specificity And Sensitivity

Background: Neglected tropical diseases (NTDs) are communicable diseases caused by a group of bacteria, viruses, protozoa and helminths prevalent in more than 145 countries that affect the world’s poverty stricken populations. WHO enlists 18 NTDs amongst people living in endemic areas having inaccessibility to preventive measures. Steps to reduce the global disease burden of the NTDs need attention at multi-factorial levels. Control programmes, mass drug administrations, transmission checks, eradication surveillances and diagnoses are some of them. The foremost in this list is confirmatory diagnosis. A comprehensive summary of the innovative, high-impact, multiplexed, low-cost diagnostic tools developed in the last decade that helped to meet the needs of users can depict a holistic approach to further evaluate potential technologies and reagents currently in research. Major Advancements: A literature survey based on developing nano-biotechnological platforms to meet the diagnostic challenges in NTDs towards development of a useful point-of-care (POC) unit is reported. However, in order to pave the way for complete eradication more sensitive tools are required that are user-friendly and applicable for use in endemic and low-resource settings. There are various novel research progresses/advancements made for qualitative and quantitative measurement of infectious load in some diseases like dengue, Chagas disease and leishmaniasis; though further improvements on the specificity and sensitivity front are still awaited. Strategies to combat the problem of antimicrobial drug resistance in diagnosis of NTDs have also been put forward by various research groups and organizations. Moreover, the state-of-the-art “omics” approaches like metabolomics and metagenomics have also started to contribute constructively towards diagnosis and prevention of the NTDs. Conclusion: A concrete solution towards a single specimen based common biomarker detection platform for NTDs is lacking. Identifying robust biomarkers and implementing them on simple diagnostic tools to ease the process of pathogen detection can help us understand the obstacles in current diagnostic measures of the NTDs.

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Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.02483-16 ◽

2017 ◽

Vol 55 (5) ◽

pp. 1285-1298 ◽

Cited By ~ 169

Author(s):

Antonina A. Votintseva ◽

Phelim Bradley ◽

Louise Pankhurst ◽

Carlos del Ojo Elias ◽

Matthew Loose ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antibiotic Susceptibility ◽

Point Of Care ◽

Low Cost ◽

Illumina Miseq ◽

Reference Laboratory ◽

Whole Genome ◽

Content Type ◽

Phylogenetic Placement

ABSTRACT Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

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Inkjet printing of paraffin on paper allows low-cost point-of-care diagnostics for pathogenic fungi

Cellulose ◽

10.1007/s10570-020-03314-3 ◽

2020 ◽

Vol 27 (13) ◽

pp. 7691-7701 ◽

Cited By ~ 1

Author(s):

Anusha Prabhu ◽

M. S. Giri Nandagopal ◽

Prakash Peralam Yegneswaran ◽

Hardik Ramesh Singhal ◽

Naresh Kumar Mani

Keyword(s):

Filter Paper ◽

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

Pathogenic Fungi ◽

Sem Analysis ◽

Qualitative Assessment ◽

Resource Limited ◽

Hydrophobic Barrier ◽

Pore Blockage

Abstract We present a high resolution, ultra-frugal printing of paper microfluidic devices using in-house paraffin formulation on a simple filter paper. The patterns printed using an office inkjet printer formed a selective hydrophobic barrier of 4 ± 1 µm thickness with a hydrophilic channel width of 275 µm. These printed patterns effectively confine common aqueous solutions and solvents, which was verified by solvent compatibility studies. SEM analysis reveals that the solvent confinement is due to pore blockage in the filter paper. The fabricated paper-based device was validated for qualitative assessment of Candida albicans (pathogenic fungi) by using a combination of L-proline β-naphthylamide as the substrate and cinnamaldehyde as an indicator. Our studies reveal that the pathogenic fungi can be detected within 10 min with the limit of detection (LOD) of 0.86 × 106 cfu/mL. Owing to its simplicity, this facile method shows high potential and can be scaled up for developing robust paper-based devices for biomarker detection in resource-limited settings. Graphic abstract

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Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System

Journal of Clinical Microbiology ◽

10.1128/jcm.00036-19 ◽

2019 ◽

Vol 57 (5) ◽

Cited By ~ 2

Author(s):

Padmapriya P. Banada ◽

Srinidhi Deshpande ◽

Sukalyani Banik ◽

Darshini Shah ◽

Ranie Koshy ◽

...

Keyword(s):

Rapid Detection ◽

Dynamic Range ◽

Limit Of Detection ◽

Point Of Care ◽

Severe Disease ◽

High Sensitivity ◽

Negative Control ◽

Blood Samples ◽

Content Type ◽

Clinical Patient

ABSTRACT Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis. The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.

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