Microfluidic droplet application for bacterial surveillance in fresh-cut produce wash waters.

Foodborne contamination and associated illness in the United States is responsible for an estimated 48 million cases per year. Increased food demand, global commerce of perishable foods, and the growing threat of antibiotic resistance are driving factors elevating concern for food safety. Foodborne illness is often associated with fresh-cut, ready-to-eat produce commodities due to the perishable nature of the product and relatively minimal processing from farm to the consumer. The research presented here optimizes and evaluates the utility of microfluidic droplets, also termed ultra-miniaturized bioreactors, for rapid detection of viable Salmonella enterica ser. Typhimurium in a shredded lettuce wash water acquired from a major Mid-Atlantic produce processing facility (denoted as Producer) in the U.S. Using a fluorescently-labeled anti-S. Typhimurium antibody and relative fluorescence intensities, paired with in-droplet incubation, S. Typhimurium was detected and identified with 100% specificity in less than 5 h. In initial optimization experiments using S. Typhimurium-spiked sterile water, the relative fluorescence intensity of S. Typhimurium was approximately two times that of the observed relative intensities of five non-S. Typhimurium negative controls at 4-h incubation in droplets containing Rappaport-Vasiliadis (RV) broth at 37°C: relative fluorescence intensity for S. Typhimurium = 2.36 (95% CI: 2.15-2.58), Enterobacter aerogens 1.12 (95% CI: 1.09-1.16), Escherichia coli 700609 = 1.13 (95% CI: 1.09-1.17), E. coli 13706 1.13 (95% CI: 1.07-1.19), E. coli 700891 1.05 (95% CI: 1.03-1.07) and Citrobacter freundii 1.04 (95% CI: 1.03-1.05). S. Typhimurium- and E. aerogens-spiked shredded lettuce wash waters acquired from the Producer were then incubated 4 h in-droplet at 37°C with RV broth. The observed relative fluorescence of S. Typhimurium was significantly higher than that of E. aerogens, 1.56 (95% CI: 1.42-1.71) and 1.10 (95% CI: 1.08-1.12), respectively. While further optimization focusing on compatible concentration methodologies for highly-dilute produce water samples is needed, this application of droplet microfluidics shows great promise in dramatically shortening the time necessary-from days to hours-to confirm viable bacterial contamination in ready-to-eat produce wash waters used throughout the domestic and international food industry.

Next-generation Diagnostics for Melioidosis: Evaluation of a Prototype i-STAT Cartridge to Detect Burkholderia pseudomallei Biomarkers.

BACKGROUND: Infection with the gram-negative bacterium Burkholderia pseudomallei can result in melioidosis, a life-threatening disease that can be difficult to diagnose. Culture remains the gold standard for diagnosis but requires laboratory resources not available in many endemic regions. A lateral flow immunoassay has shown promise for POC diagnostics but suffers from low sensitivity when used on blood samples. PCR also has low sensitivity on blood, attributed to the low bacterial numbers in blood observed in melioidosis patients, even when bacteraemic. METHODS: A prototype i-STAT cartridge was developed to utilize the monoclonal antibody specific for the capsule of pathogenic Burkholderia species employed on the LFI. The resulting POC assay was evaluated on 414 clinical specimens from Darwin, Australia and Cambodia. RESULTS: The i-STAT assay accurately distinguished Australian blood culture positive melioidosis patients from Australian patients hospitalized with other infections (AUC = 0.91, 95% CI 0.817 - 1.0). We derived an assay cutoff with 76% sensitivity and 94% specificity that correctly classified 88% (n = 74) of the Australian patients. Interestingly, only 46% (6/13) of the culture-positive melioidosis patients in Cambodia were classified correctly. Of great importance however, the assay detected capsule from blood samples for 32% of blood culture negative melioidosis patients in both cohorts and previously undiagnosed melioidosis patients in Cambodia. In addition the assay showed high sensitivity and specificity for urine, pus and sputum. CONCLUSIONS: Diagnostic tools that are not dependent upon the growth kinetics or the levels of bacteremia of B. pseudomallei represent the next-generation of diagnostics and must be pursued further.

Far-Forward Diagnostics in Toxic Industrial Chemical and Material Exposure Scenarios and Biomarker Identification.

: This study describes key technical solutions for detecting environmental toxicants and diagnosing adverse health effects in military operational settings as outlined at a symposium cosponsored by the Department of Defense and the Johns Hopkins University-Applied Physics Laboratory (October 27 to 28, 2015). Such technologies are urgently needed in order to provide critical decision-aid tools and prognostic assessment of potential clinical sequelae. This review summarizes the state-of-the-science on (1) prioritization of adverse health effects, (2) existing technologies and diagnostic tools available for use in theater, (3) challenges to advancing diagnostic tools far-forward, and (4) the potential utility of anchoring diagnostic tools to adverse outcome pathways. Emerging technologies are increasingly available for physiological, environmental, and individual exposure monitoring. Challenges to overcome in austere environments include cold chain requirements and determination of adequate sampling intervals.

An activity-dependent assay for ricin and related RNA N-glycosidases based on electrochemiluminescence.

Synthetic biotinylated RNA substrates were cleaved by the combined actions of ricin holotoxin and a chemical agent, N,N'-dimethylethylenediamine. The annealing of the product with a ruthenylated oligodeoxynucleotide resulted in the capture of ruthenium chelate onto magnetic beads, enabling the electrochemiluminescence (ECL)-based detection of RNA N-glycosidase activities of toxins. ECL immunoassays and the activity assay exhibited similar limits of detection just below signals with 0.1 ng/ml of ricin; the ECL response was linear as the ricin concentration increased by two orders of magnitude. Activities were detected with other adenine-specific RNA N-glycosidases, including Ricinus communis agglutinin (RCA), saporin, and abrin II. The substrate that provided the greatest sensitivity was composed of a four-residue loop, GdAGA, in a hairpin structure. When the 2'-deoxyadenosine (dA) was substituted with adenosine (A), 2'-deoxyinosine, or 2'-deoxyuridine, toxin-dependent signals were abolished. Placing the GdAGA motif in a six-residue loop or replacing it with GdAdGA or GdAAA resulted in measurable activities and signal patterns that were reproducible for a given toxin. Data indicated that saporin and abrin II shared one pattern, while ricin and RCA shared a distinct pattern. A monoclonal antibody that enhanced the activities of ricin, RCA, and abrin II to different extents, thus improving the diagnostic potential of the assay, was identified .