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Title: 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. This paper describes a portable system for rapidly identifyingmore » 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.« less

Authors:
 [1];  [2];  [2];  [3];  [4];  [2];  [5];  [6];  [7];  [7];  [8]
+ Show Author Affiliations
  1. Univ. of Wisconsin, Madison, WI (United States). Dept. of Biochemistry; Leibniz Inst. of Polymer Research (IPF), Dresden (Germany)
  2. Univ. of Wisconsin, Madison, WI (United States). Dept. of Biochemistry
  3. Univ. of Puerto Rico, Mayagüez, PR (United States). Dept. of Mechanical Engineering
  4. Univ. of Wisconsin, Madison, WI (United States). Dept. of Biochemistry; Univ. of Puerto Rico, Mayagüez, PR (United States). Dept. of Mechanical Engineering
  5. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Computations Directorate
  6. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Physical and Life Sciences Directorate
  7. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Global Security Principal Directorate
  8. Univ. of Wisconsin, Madison, WI (United States). Dept. of Biochemistry. Dept. of Biomedical Engineering. Dept. of Chemistry
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org.:
USDOE; USDOD; National Science Foundation (NSF); Bill and Melinda Gates Foundation (United States)
OSTI Identifier:
1513157
Report Number(s):
LLNL-JRNL-701140
Journal ID: ISSN 0099-2240; 834172
Grant/Contract Number:  
AC52-07NA27344; B603628; DMR-1121288; OPP1068092
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 83; Journal Issue: 4; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 47 OTHER INSTRUMENTATION; 60 APPLIED LIFE SCIENCES

Citation Formats

Renner, Lars D., Zan, Jindong, Hu, Linda I., Martinez, Manuel, Resto, Pedro J., Siegel, Adam C., Torres, Clint, Hall, Sara B., Slezak, Tom R., Nguyen, Tuan H., and Weibel, Douglas B. Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform. United States: N. p., 2016. Web. doi:10.1128/AEM.02449-16.
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Renner, Lars D., Zan, Jindong, Hu, Linda I., Martinez, Manuel, Resto, Pedro J., Siegel, Adam C., Torres, Clint, Hall, Sara B., Slezak, Tom R., Nguyen, Tuan H., & Weibel, Douglas B. Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform. United States. https://doi.org/10.1128/AEM.02449-16
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Renner, Lars D., Zan, Jindong, Hu, Linda I., Martinez, Manuel, Resto, Pedro J., Siegel, Adam C., Torres, Clint, Hall, Sara B., Slezak, Tom R., Nguyen, Tuan H., and Weibel, Douglas B. Fri . "Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform". United States. https://doi.org/10.1128/AEM.02449-16. https://www.osti.gov/servlets/purl/1513157.
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@article{osti_1513157,
title = {Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform},
author = {Renner, Lars D. and Zan, Jindong and Hu, Linda I. and Martinez, Manuel and Resto, Pedro J. and Siegel, Adam C. and Torres, Clint and Hall, Sara B. and Slezak, Tom R. and Nguyen, Tuan H. and Weibel, Douglas B.},
abstractNote = {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. 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.},
doi = {10.1128/AEM.02449-16},
journal = {Applied and Environmental Microbiology},
number = 4,
volume = 83,
place = {United States},
year = {Fri Dec 16 00:00:00 EST 2016},
month = {Fri Dec 16 00:00:00 EST 2016}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.1128/AEM.02449-16
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Citation Metrics:
Cited by: 38 works
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Figures / Tables:

FIG 1 FIG 1: Images depicting the B-chip and electronic reader. A) An image of a B-chip containing 16 microchambers (‘C’, each with a volume of 1 $μ$L). B) An image of a degassed B-chip that has been loaded with an aqueous solution of crystal violet (50 $μ$L) at the inlet ‘I’more » after 20 min. Air fills the main and side channels (‘MC’ and ‘SC’), physically separates reactions, and prevents cross-contamination between the chambers. Excess sample is collected in the reservoir ‘R’ (scale bar: 10 mm). C) Representative image of fluorescence from on-chip RPAs for S. aureus, imaged with an ImageQuant; green circles indicate the presence of S. aureus specific primers and red circles indicate the absence of primers (scale bar: 2 mm). D-E) images of the B-chip reader highlighting the housing, optical components, and a Peltier plate to heat the sample.« less
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    The Oral Bacterium Fusobacterium nucleatum Binds Staphylococcus aureus and Alters Expression of the Staphylococcal Accessory Regulator sarA
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