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 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.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Leibniz Institute of Polymer Research, Dresden, Germany.
authors.forename: Lars D
authors.initials: LD
authors.lastname: Renner
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Jindong
authors.initials: J
authors.lastname: Zan
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Linda I
authors.initials: LI
authors.lastname: Hu
authors.affiliation: Department of Mechanical Engineering University of Puerto Rico at Mayagüez, Mayagüez, Puerto Rico, USA.
authors.forename: Manuel
authors.initials: M
authors.lastname: Martinez
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Department of Mechanical Engineering University of Puerto Rico at Mayagüez, Mayagüez, Puerto Rico, USA.
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Resto
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Adam C
authors.initials: AC
authors.lastname: Siegel
authors.affiliation: Computations Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA.
authors.forename: Clint
authors.initials: C
authors.lastname: Torres
authors.affiliation: Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA.
authors.forename: Sara B
authors.initials: SB
authors.lastname: Hall
authors.affiliation: Global Security Principal Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA.
authors.forename: Tom R
authors.initials: TR
authors.lastname: Slezak
authors.affiliation: Global Security Principal Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA thn@llnl.gov weibel@biochem.wisc.edu.
authors.forename: Tuan H
authors.initials: TH
authors.lastname: Nguyen
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA thn@llnl.gov weibel@biochem.wisc.edu.|Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Weibel
mesh_terms: D040981:Acinetobacter baumannii / Q000145:classification / Q000235:genetics; D001424:Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology; D003428:Cross Infection / Q000175:diagnosis* / Q000382:microbiology; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial; D004754:Enterobacter / Q000145:classification / Q000235:genetics; D016984:Enterococcus faecium / Q000145:classification / Q000235:genetics; D006801:Humans; D007711:Klebsiella pneumoniae / Q000145:classification / Q000235:genetics; D056656:Lab-On-A-Chip Devices; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D019095:Point-of-Care Systems; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000235:genetics; D013211:Staphylococcus aureus / Q000145:classification / Q000235:genetics
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial
keywords: ESKAPE pathogens; bacteria; detection; infection; isothermal PCR; lab-on-a-chip; point of care