The Experts below are selected from a list of 18138 Experts worldwide ranked by ideXlab platform
Dayong Jin - One of the best experts on this subject based on the ideXlab platform.
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Orthogonal Scanning Automated Microscopy Speeds Up Time-Gated Luminescence Detection
Optics in the Life Sciences, 2013Co-Authors: James A. Piper, Dayong JinAbstract:We report a versatile platform of orthogonal scanning Automated Microscopy (OSAM) cooperated with time-gated detection technique. Featuring rapid processing and background-free detection, it offers ultra-high sensitivity to spot rare-event cells and quantitate low-expression surface molecules.
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time gated orthogonal scanning Automated Microscopy osam for high speed cell detection and analysis
Scientific Reports, 2012Co-Authors: James A. Piper, Yujing Huo, Dayong JinAbstract:We report a new development of orthogonal scanning Automated Microscopy (OSAM) incorporating time-gated detection to locate rare-event organisms regardless of autofluorescent background. The necessity of using long-lifetime (hundreds of microseconds) luminescent biolabels for time-gated detection implies long integration (dwell) time, resulting in slow scan speed. However, here we achieve high scan speed using a new 2-step orthogonal scanning strategy to realise on-the-fly time-gated detection and precise location of 1-μm lanthanide-doped microspheres with signal-to-background ratio of 8.9. This enables analysis of a 15 mm × 15 mm slide area in only 3.3 minutes. We demonstrate that detection of only a few hundred photoelectrons within 100 μs is sufficient to distinguish a target event in a prototype system using ultraviolet LED excitation. Cytometric analysis of lanthanide labelled Giardia cysts achieved a signal-to-background ratio of two orders of magnitude. Results suggest that time-gated OSAM represents a new opportunity for high-throughput background-free biosensing applications.
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Resolving low-expression cell surface antigens by time-gated orthogonal scanning Automated Microscopy.
Analytical chemistry, 2012Co-Authors: Jody Martin, James A. Piper, Jiangbo Zhao, Jingli Yuan, Martin Ostrowski, Ian T. Paulsen, Dayong JinAbstract:We report a highly sensitive method for rapid identification and quantification of rare-event cells carrying low-abundance surface biomarkers. The method applies lanthanide bioprobes and time-gated detection to effectively eliminate both nontarget organisms and background noise and utilizes the europium containing nanoparticles to further amplify the signal strength by a factor of ∼20. Of interest is that these nanoparticles did not correspondingly enhance the intensity of nonspecific binding. Thus, the dramatically improved signal-to-background ratio enables the low-expression surface antigens on single cells to be quantified. Furthermore, we applied an orthogonal scanning Automated Microscopy (OSAM) technique to rapidly process a large population of target-only cells on Microscopy slides, leading to quantitative statistical data with high certainty. Thus, the techniques together resolved nearly all false-negative events from the interfering crowd including many false-positive events.
Steven W. Metzger - One of the best experts on this subject based on the ideXlab platform.
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Rapid Automated Microscopy for Microbiological Surveillance of Ventilator-associated Pneumonia
American journal of respiratory and critical care medicine, 2015Co-Authors: Ivor S. Douglas, Connie S. Price, Katherine H. Overdier, Robert Wolken, Steven W. Metzger, Kenneth Robert Hance, David C. HowsonAbstract:Rationale: Diagnosis of ventilator-associated pneumonia (VAP) is imprecise.Objectives: To (1) determine whether alternate-day surveillance mini–bronchoalveolar lavage (mini-BAL) in ventilated adults could reduce time to initiation of targeted treatment and (2) evaluate the potential for Automated Microscopy to reduce analysis time.Methods: Adult intensive care unit patients who were anticipated to require ventilation for at least a further 48 hours were included. Mini-BALs were processed for identification, quantitation, and antibiotic susceptibility, using (1) clinical culture (50 ± 7 h) and (2) Automated Microscopy (∼5 h plus offline analysis).Measurements and Main Results: Seventy-seven mini-BALs were performed in 33 patients. One patient (3%) was clinically diagnosed with VAP. Of 73 paired samples, culture identified 7 containing pneumonia panel bacteria (>104 colony-forming units/ml) from five patients (15%) (4 Staphylococcus aureus [3 methicillin-resistant S. aureus], 2 Stenotrophomonas maltophilia,...
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Rapid antibiotic susceptibility phenotypic characterization of Staphylococcus aureus using Automated Microscopy of small numbers of cells.
Journal of microbiological methods, 2014Co-Authors: Connie S. Price, Shelley E. Kon, Steven W. MetzgerAbstract:Staphylococcus aureus remains a leading, virulent pathogen capable of expressing complex drug resistance that requires up to 2-4 days for laboratory analysis. In this study, we evaluate the ability of Automated Microscopy of immobilized live bacterial cells to differentiate susceptible from non-susceptible responses of S. aureus isolates (MRSA/MSSA, clindamycin resistance/susceptibility and VSSA/hVISA/VISA) to an antibiotic based on the characterization of as few as 10 growing clones after 4 h of growth, compared to overnight growth required for traditional culture based methods. Isolates included 131 characterized CDC isolates, 3 clinical isolates and reference strains. MRSA phenotype testing used 1 h of 1 μg/mL cefoxitin induction followed by 3 h of 6 μg/mL cefoxitin. Clindamycin susceptibility testing used 1h of induction by 0.1 μg/mL erythromycin followed by 3h of 0.5 μg/mL clindamycin. An Automated Microscopy system acquired time-lapse dark-field images, and then computed growth data for individual immobilized progenitor cells and their progeny clones while exposed to different test conditions. Results were compared to concurrent cefoxitin disk diffusion and D-test references. For CDC organisms, Microscopy detected 77/77 MRSA phenotypes and 54/54 MSSA phenotypes, plus 53/56 clindamycin-resistant and 75/75 clindamycin susceptible strains. Automated Microscopy was used to characterize heterogeneous and inducible resistance, and perform population analysis profiles. Microscopy-based hVISA population analysis profiles (PAPs) were included as an extended proof of concept, and successfully differentiated VSSA from hVISA and VISA phenotypes compared to plate-based PAP.
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5-Hour Antibiotic Susceptibility Testing of Enterococcus faecium and E. faecalis, and Acinetobacter baumannii Directly from Positive Blood Cultures Using Automated Microscopy
2013Co-Authors: Alena Shamsheyeva, Steven W. Metzger, David C. Howson, Dulini Gamage, Ben Turng, Connie S. PriceAbstract:Objectives: Healthcare-associated Acinetobacter baumannii and enterococcal bloodstream infections are of increasing concern. Septic patients infected with these organisms have been shown to face elevated risk of receiving inappropriate initial therapy with broad-spectrum antibiotics. Even a few hours of delay in initiating appropriate antibiotic treatment increases the risk of severe morbidity and mortality. An innovative technology using Automated Microscopy was evaluated for its performance for rapid antibiotic susceptibility testing (AST) of A. baumannii, E. faecium, and E. faecalis directly from positive blood culture. Methods: A total of 228 clinical isolates selected for having MICs near the CLSI breakpoints were tested; 88 A. baumannii isolates (45 with imipenem (IPM) and 43 with minocycline (MIN)), and 140 E. faecium or E. faecalis isolates (45 with ampicillin (AMP), 48 with vancomycin (VAN) and 47 with linezolid (LZD)). Ten to one hundred colony forming units of bacterial suspension were spiked into simulated blood culture bottles (1 part of healthy donor blood + 4 parts of BD BACTEC Standard Aerobic media), and incubated for approximate 20-24 h. One milliliter of the positive blood culture was removed and lysed. Automated gel electrofi ltration (20 min) was performed to reduce sample debris. Samples were pipetted into independent fl owcells of a disposable multichannel cassette, and electrokinetic concentration immobilized cells on the transparent lower surface of each fl owcell channel (5 min). Immobilized cells were challenged with single concentration solutions of antibiotic prepared in cation-adjusted Mueller Hinton broth with 0.85% agar. Automated Microscopy with image analysis software scanned and analyzed growth rates from changes in the biomass of each immobilized progenitor cell as it grew into a clone of daughter cells. A computer algorithm converted bacterial growth or inhibition in the presence of antibiotic into a minimum inhibitory concentration (MIC). A growth control was also performed. Standard CLSI frozen broth microdilution (BMD) was performed in parallel as a reference using overnight plated pure culture colonies. Microscopy AST results were compared to BMD results. Results: The time from positive blood culture sample preparation to obtain MIC results for the Microscopy system ranged from 3.5 to 5 h. Essential agreement (± 1 dilution) for A. baumannii with IPM and MIN was 98% and 95%, respectively. Essential agreement for Enterococcus spp. for AMP, VAN and LZD was 96%, 92%, and 94%, respectively. Conclusion: Data in this study suggests that the new Automated Microscopy technology is feasible for providing rapid AST results directly from positive blood culture within 5 h. AST results were concordant with overnight pure culture using BMD. Automated Microscopy offers a promising alternative for performing AST directly from positive blood culture bottles to provide rapid MIC results for tailoring treatment of A. baumannii and enterococcal blood stream infections.
James A. Piper - One of the best experts on this subject based on the ideXlab platform.
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Orthogonal Scanning Automated Microscopy Speeds Up Time-Gated Luminescence Detection
Optics in the Life Sciences, 2013Co-Authors: James A. Piper, Dayong JinAbstract:We report a versatile platform of orthogonal scanning Automated Microscopy (OSAM) cooperated with time-gated detection technique. Featuring rapid processing and background-free detection, it offers ultra-high sensitivity to spot rare-event cells and quantitate low-expression surface molecules.
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time gated orthogonal scanning Automated Microscopy osam for high speed cell detection and analysis
Scientific Reports, 2012Co-Authors: James A. Piper, Yujing Huo, Dayong JinAbstract:We report a new development of orthogonal scanning Automated Microscopy (OSAM) incorporating time-gated detection to locate rare-event organisms regardless of autofluorescent background. The necessity of using long-lifetime (hundreds of microseconds) luminescent biolabels for time-gated detection implies long integration (dwell) time, resulting in slow scan speed. However, here we achieve high scan speed using a new 2-step orthogonal scanning strategy to realise on-the-fly time-gated detection and precise location of 1-μm lanthanide-doped microspheres with signal-to-background ratio of 8.9. This enables analysis of a 15 mm × 15 mm slide area in only 3.3 minutes. We demonstrate that detection of only a few hundred photoelectrons within 100 μs is sufficient to distinguish a target event in a prototype system using ultraviolet LED excitation. Cytometric analysis of lanthanide labelled Giardia cysts achieved a signal-to-background ratio of two orders of magnitude. Results suggest that time-gated OSAM represents a new opportunity for high-throughput background-free biosensing applications.
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Resolving low-expression cell surface antigens by time-gated orthogonal scanning Automated Microscopy.
Analytical chemistry, 2012Co-Authors: Jody Martin, James A. Piper, Jiangbo Zhao, Jingli Yuan, Martin Ostrowski, Ian T. Paulsen, Dayong JinAbstract:We report a highly sensitive method for rapid identification and quantification of rare-event cells carrying low-abundance surface biomarkers. The method applies lanthanide bioprobes and time-gated detection to effectively eliminate both nontarget organisms and background noise and utilizes the europium containing nanoparticles to further amplify the signal strength by a factor of ∼20. Of interest is that these nanoparticles did not correspondingly enhance the intensity of nonspecific binding. Thus, the dramatically improved signal-to-background ratio enables the low-expression surface antigens on single cells to be quantified. Furthermore, we applied an orthogonal scanning Automated Microscopy (OSAM) technique to rapidly process a large population of target-only cells on Microscopy slides, leading to quantitative statistical data with high certainty. Thus, the techniques together resolved nearly all false-negative events from the interfering crowd including many false-positive events.
Alan R. Watson - One of the best experts on this subject based on the ideXlab platform.
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Automated Microscopy, dipsticks and the diagnosis of urinary tract infection
Archives of disease in childhood, 2009Co-Authors: Andrew Lunn, Stephen Holden, Tim Boswell, Alan R. WatsonAbstract:Objectives: Automated Microscopy (AM) is increasingly used to screen samples for suspected urinary tract infection (UTI). A 98.8% negative predictive value has been reported in adult studies. The aim of our study was to validate this method in a paediatric population. Methods: Urine samples were collected from children with known or suspected nephrourological disease attending nephrology and urology clinics over a 6 week time period. Samples were tested with dipstick, the UF100 flow cytometer (AM) and culture. A gold standard of a positive culture of 105 colony forming units per ml (cfu/ml) with a pathogenic organism was used and the sensitivity, specificity and likelihood ratios were calculated. Results: 280 urine samples were collected from 263 patients (143 male, median age 10.2 years, range 0.1-19.75 years). 221(79%) were midstream or clean-catch samples. AM identified 42 of 186 samples as requiring culture and 17 of 19 samples which had a pure growth > 105 cfu/ml. Two patients were not identified by AM, one was treated for vulvovaginitis, one commenced prophylactic antibiotics prior to the culture result being obtained. The sensitivity, specificity, positive and negative likelihood ratios were 0.89, 0.85, 5.98 and 0.17 respectively. This compared to 0.95, 0.72, 3.34 and 0.29 respectively with urine dipstick. Conclusion: AM performed comparably to urine dipstick in the diagnosis of UTI with improved specificity and likelihood ratios with a slight reduction in sensitivity. The data support the use of AM for screening urines for culture in children but different AM methods and algorithms require local evaluation.
Connie S. Price - One of the best experts on this subject based on the ideXlab platform.
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Rapid Automated Microscopy for Microbiological Surveillance of Ventilator-associated Pneumonia
American journal of respiratory and critical care medicine, 2015Co-Authors: Ivor S. Douglas, Connie S. Price, Katherine H. Overdier, Robert Wolken, Steven W. Metzger, Kenneth Robert Hance, David C. HowsonAbstract:Rationale: Diagnosis of ventilator-associated pneumonia (VAP) is imprecise.Objectives: To (1) determine whether alternate-day surveillance mini–bronchoalveolar lavage (mini-BAL) in ventilated adults could reduce time to initiation of targeted treatment and (2) evaluate the potential for Automated Microscopy to reduce analysis time.Methods: Adult intensive care unit patients who were anticipated to require ventilation for at least a further 48 hours were included. Mini-BALs were processed for identification, quantitation, and antibiotic susceptibility, using (1) clinical culture (50 ± 7 h) and (2) Automated Microscopy (∼5 h plus offline analysis).Measurements and Main Results: Seventy-seven mini-BALs were performed in 33 patients. One patient (3%) was clinically diagnosed with VAP. Of 73 paired samples, culture identified 7 containing pneumonia panel bacteria (>104 colony-forming units/ml) from five patients (15%) (4 Staphylococcus aureus [3 methicillin-resistant S. aureus], 2 Stenotrophomonas maltophilia,...
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Rapid antibiotic susceptibility phenotypic characterization of Staphylococcus aureus using Automated Microscopy of small numbers of cells.
Journal of microbiological methods, 2014Co-Authors: Connie S. Price, Shelley E. Kon, Steven W. MetzgerAbstract:Staphylococcus aureus remains a leading, virulent pathogen capable of expressing complex drug resistance that requires up to 2-4 days for laboratory analysis. In this study, we evaluate the ability of Automated Microscopy of immobilized live bacterial cells to differentiate susceptible from non-susceptible responses of S. aureus isolates (MRSA/MSSA, clindamycin resistance/susceptibility and VSSA/hVISA/VISA) to an antibiotic based on the characterization of as few as 10 growing clones after 4 h of growth, compared to overnight growth required for traditional culture based methods. Isolates included 131 characterized CDC isolates, 3 clinical isolates and reference strains. MRSA phenotype testing used 1 h of 1 μg/mL cefoxitin induction followed by 3 h of 6 μg/mL cefoxitin. Clindamycin susceptibility testing used 1h of induction by 0.1 μg/mL erythromycin followed by 3h of 0.5 μg/mL clindamycin. An Automated Microscopy system acquired time-lapse dark-field images, and then computed growth data for individual immobilized progenitor cells and their progeny clones while exposed to different test conditions. Results were compared to concurrent cefoxitin disk diffusion and D-test references. For CDC organisms, Microscopy detected 77/77 MRSA phenotypes and 54/54 MSSA phenotypes, plus 53/56 clindamycin-resistant and 75/75 clindamycin susceptible strains. Automated Microscopy was used to characterize heterogeneous and inducible resistance, and perform population analysis profiles. Microscopy-based hVISA population analysis profiles (PAPs) were included as an extended proof of concept, and successfully differentiated VSSA from hVISA and VISA phenotypes compared to plate-based PAP.
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5-Hour Antibiotic Susceptibility Testing of Enterococcus faecium and E. faecalis, and Acinetobacter baumannii Directly from Positive Blood Cultures Using Automated Microscopy
2013Co-Authors: Alena Shamsheyeva, Steven W. Metzger, David C. Howson, Dulini Gamage, Ben Turng, Connie S. PriceAbstract:Objectives: Healthcare-associated Acinetobacter baumannii and enterococcal bloodstream infections are of increasing concern. Septic patients infected with these organisms have been shown to face elevated risk of receiving inappropriate initial therapy with broad-spectrum antibiotics. Even a few hours of delay in initiating appropriate antibiotic treatment increases the risk of severe morbidity and mortality. An innovative technology using Automated Microscopy was evaluated for its performance for rapid antibiotic susceptibility testing (AST) of A. baumannii, E. faecium, and E. faecalis directly from positive blood culture. Methods: A total of 228 clinical isolates selected for having MICs near the CLSI breakpoints were tested; 88 A. baumannii isolates (45 with imipenem (IPM) and 43 with minocycline (MIN)), and 140 E. faecium or E. faecalis isolates (45 with ampicillin (AMP), 48 with vancomycin (VAN) and 47 with linezolid (LZD)). Ten to one hundred colony forming units of bacterial suspension were spiked into simulated blood culture bottles (1 part of healthy donor blood + 4 parts of BD BACTEC Standard Aerobic media), and incubated for approximate 20-24 h. One milliliter of the positive blood culture was removed and lysed. Automated gel electrofi ltration (20 min) was performed to reduce sample debris. Samples were pipetted into independent fl owcells of a disposable multichannel cassette, and electrokinetic concentration immobilized cells on the transparent lower surface of each fl owcell channel (5 min). Immobilized cells were challenged with single concentration solutions of antibiotic prepared in cation-adjusted Mueller Hinton broth with 0.85% agar. Automated Microscopy with image analysis software scanned and analyzed growth rates from changes in the biomass of each immobilized progenitor cell as it grew into a clone of daughter cells. A computer algorithm converted bacterial growth or inhibition in the presence of antibiotic into a minimum inhibitory concentration (MIC). A growth control was also performed. Standard CLSI frozen broth microdilution (BMD) was performed in parallel as a reference using overnight plated pure culture colonies. Microscopy AST results were compared to BMD results. Results: The time from positive blood culture sample preparation to obtain MIC results for the Microscopy system ranged from 3.5 to 5 h. Essential agreement (± 1 dilution) for A. baumannii with IPM and MIN was 98% and 95%, respectively. Essential agreement for Enterococcus spp. for AMP, VAN and LZD was 96%, 92%, and 94%, respectively. Conclusion: Data in this study suggests that the new Automated Microscopy technology is feasible for providing rapid AST results directly from positive blood culture within 5 h. AST results were concordant with overnight pure culture using BMD. Automated Microscopy offers a promising alternative for performing AST directly from positive blood culture bottles to provide rapid MIC results for tailoring treatment of A. baumannii and enterococcal blood stream infections.
