- Emergency Medicine
- Infectious Diseases
- Systems Biology
- Biomedical Engineering
- Predictive Analytics
Boards, Advisory Committees, Professional Organizations
Grant Reviewer, NIAID, DoD-CDMRP, MRC-UK (2011 - Present)
Research Committee Member, Society of Academic Emergency Medicine (2012 - Present)
Research Committee Member, American College of Emergency Physicians (2012 - Present)
Board Certification: Emergency Medicine, American Board of Emergency Medicine (2007)
Fellowship:Johns Hopkins University School of Medicine (2004) MD
Residency:Johns Hopkins University School of Medicine (2002) MD
Medical Education:University of California, Los Angeles (1999) CA
Undergraduate, Massachusetts Institute of Technology, B.S. (1994)
Current Research and Scholarly Interests
Dr. Yang's research is focused on bridging the translational gap at the interface of molecular biology, genome science, engineering, and acute care medicine. The investigative interest of the Yang lab falls within the general theme of developing integrative systems-level approaches for precision diagnostics, as well as data driven knowledge discoveries, to improve the health outcome and our understanding of complex critical illnesses. Using sepsis as the disease model with complex host-pathogen dynamics, the goals of the Yang lab are divided into 2 areas:
1) Developing high-content, near-patient, diagnostic system for rapid broad pathogen detection and characterization.
2) Integrating multi-omics molecular and phenotypic data layers with novel computational approaches into AI-assisted diagnostics and predictive analytics for sepsis.
SEP-SEQ Trial - Determining the Etiology of Sepsis Using an Infectious Disease Diagnostic Sequencing Assay
The purpose of this study is to evaluate the performance of the Karius Infectious Diseases Plasma Sequencing Assay in patients who present to the emergency room with sepsis.
Stanford is currently not accepting patients for this trial.
Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease.
Thousands of pathogens are known to infect humans, but only a fraction are readily identifiable using current diagnostic methods. Microbial cell-free DNA sequencing offers the potential to non-invasively identify a wide range of infections throughout the body, but the challenges of clinical-grade metagenomic testing must be addressed. Here we describe the analytical and clinical validation of a next-generation sequencing test that identifies and quantifies microbial cell-free DNA in plasma from 1,250 clinically relevant bacteria, DNA viruses, fungi and eukaryotic parasites. Test accuracy, precision, bias and robustness to a number of metagenomics-specific challenges were determined using a panel of 13 microorganisms that model key determinants of performance in 358 contrived plasma samples, as well as 2,625 infections simulated in silico and 580 clinical study samples. The test showed 93.7% agreement with blood culture in a cohort of 350 patients with a sepsis alert and identified an independently adjudicated cause of the sepsis alert more often than all of the microbiological testing combined (169 aetiological determinations versus 132). Among the 166 samples adjudicated to have no sepsis aetiology identified by any of the tested methods, sequencing identified microbial cell-free DNA in 62, likely derived from commensal organisms and incidental findings unrelated to the sepsis alert. Analysis of the first 2,000 patient samples tested in the CLIA laboratory showed that more than 85% of results were delivered the day after sample receipt, with 53.7% of reports identifying one or more microorganisms.
View details for DOI 10.1038/s41564-018-0349-6
View details for PubMedID 30742071
A "Culture" Shift: Broad Bacterial Detection, Identification, and Antimicrobial Susceptibility Testing Directly from Whole Blood.
BACKGROUND: The time required for bloodstream pathogen detection, identification (ID), and antimicrobial susceptibility testing (AST) does not satisfy the acute needs of disease management. Conventional methods take up to 3 days for ID and AST. Molecular diagnostics have reduced times for ID, but their promise to supplant culture is unmet because AST times remain slow. We developed a combined quantitative PCR (qPCR)-based ID+AST assay with sequential detection, ID, and AST of leading nosocomial bacterial pathogens.METHODS: ID+AST was performed on whole blood samples by (a) removing blood cells, (b) brief bacterial enrichment, (c) bacterial detection and ID, and (d) species-specific antimicrobial treatment. Broad-spectrum qPCR of the internal transcribed spacer between the 16S and 23S was amplified for detection. High-resolution melting identified the species with a curve classifier. AST was enabled by Ct differences between treated and untreated samples.RESULTS: A detection limit of 1 CFU/mL was achieved for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. All species were accurately identified by unique melting curves. Antimicrobial minimum inhibitory concentrations were identified with Ct differences of ≥1 cycle. Using an RNA target allowed reduction of AST incubation time from 60 min to 5 min. Rapid-cycle amplification reduced qPCR times by 83% to 30 min.CONCLUSIONS: Combined, sequential ID+AST protocols allow rapid and reliable detection, ID, and AST for the diagnosis of bloodstream infections, enabling conversion of empiric to targeted therapy by the second dose of antimicrobials.
View details for DOI 10.1373/clinchem.2018.290189
View details for PubMedID 30087140
Microbial Typing by Machine Learned DNA Melt Signatures
There is still an ongoing demand for a simple broad-spectrum molecular diagnostic assay for pathogenic bacteria. For this purpose, we developed a single-plex High Resolution Melt (HRM) assay that generates complex melt curves for bacterial identification. Using internal transcribed spacer (ITS) region as the phylogenetic marker for HRM, we observed complex melt curve signatures as compared to 16S rDNA amplicons with enhanced interspecies discrimination. We also developed a novel Naïve Bayes curve classification algorithm with statistical interpretation and achieved 95% accuracy in differentiating 89 bacterial species in our library using leave-one-out cross-validation. Pilot clinical validation of our method correctly identified the etiologic organisms at the species-level in 59 culture-positive mono-bacterial blood culture samples with 90% accuracy. Our findings suggest that broad bacterial sequences may be simply, reliably and automatically profiled by ITS HRM assay for clinical adoption.
View details for DOI 10.1038/srep42097
View details for Web of Science ID 000393494300001
View details for PubMedID 28165067
Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples
NUCLEIC ACIDS RESEARCH
2013; 41 (18)
Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.
View details for DOI 10.1093/nar/gkt684
View details for Web of Science ID 000325776600006
View details for PubMedID 23935121
Advances in microfluidic PCR for point-of-care infectious disease diagnostics
2011; 29 (6): 830-839
Global burdens from existing or emerging infectious diseases emphasize the need for point-of-care (POC) diagnostics to enhance timely recognition and intervention. Molecular approaches based on PCR methods have made significant inroads by improving detection time and accuracy but are still largely hampered by resource-intensive processing in centralized laboratories, thereby precluding their routine bedside- or field-use. Microfluidic technologies have enabled miniaturization of PCR processes onto a chip device with potential benefits including speed, cost, portability, throughput, and automation. In this review, we provide an overview of recent advances in microfluidic PCR technologies and discuss practical issues and perspectives related to implementing them into infectious disease diagnostics.
View details for DOI 10.1016/j.biotechadv.2011.06.017
View details for Web of Science ID 000296821900024
View details for PubMedID 21741465
View details for PubMedCentralID PMC3186827
Continuous dielectrophoretic bacterial separation and concentration from physiological media of high conductivity
LAB ON A CHIP
2011; 11 (17): 2893-2900
Biological sample processing involves purifying target analytes from various sample matrices and concentrating them to a small volume from a large volume of crude sample. This complex process is the major obstacle for developing a microfluidic diagnostic platform. In this study, we present a microfluidic device that can continuously separate and concentrate pathogenic bacterial cells from complex sample matrices such as cerebrospinal fluid and whole blood. Having overcome critical limitations of dielectrophoretic (DEP) operation in physiological media of high conductivity, we utilized target specific DEP techniques to incorporate cell separation, medium exchange, and target concentration into an integrated platform. The proposed microfluidic device can uptake mL volumes of crude biological sample and selectively concentrate target cells into a submicrolitre volume, providing ~10(4) fold of concentration. We designed the device based on the electrokinetic theory and electric field simulation, and tested the device performance with different sample types. The separation efficiency of the device was as high as 97.0% for a bead mixture in TAE buffer and 94.3% and 87.2% for E. coli in human cerebrospinal fluid and blood, respectively. A capture efficiency of 100% was achieved in the concentration chamber. With a relatively simple configuration, the proposed device provides a robust method of continuous sample processing, which can be readily integrated into a fully automated microfluidic diagnostic platform for pathogen detection and quantification.
View details for DOI 10.1039/c1lc20307j
View details for Web of Science ID 000293651100012
View details for PubMedID 21776517
- Molecular methods for pathogen detection in blood LANCET 2010; 375 (9710): 178-179
PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings
LANCET INFECTIOUS DISEASES
2004; 4 (6): 337-348
Molecular diagnostics are revolutionising the clinical practice of infectious disease. Their effects will be significant in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes. PCR is the most well-developed molecular technique up to now, and has a wide range of already fulfilled, and potential, clinical applications, including specific or broad-spectrum pathogen detection, evaluation of emerging novel infections, surveillance, early detection of biothreat agents, and antimicrobial resistance profiling. PCR-based methods may also be cost effective relative to traditional testing procedures. Further advancement of technology is needed to improve automation, optimise detection sensitivity and specificity, and expand the capacity to detect multiple targets simultaneously (multiplexing). This review provides an up-to-date look at the general principles, diagnostic value, and limitations of the most current PCR-based platforms as they evolve from bench to bedside.
View details for Web of Science ID 000221799100020
View details for PubMedID 15172342
Factors Associated With Influenza in an Emergency Department Setting.
The Journal of emergency medicine
BACKGROUND: Emergency departments (EDs) become more overcrowded during peak respiratory virus season. Distinguishing influenza from other viruses is crucial to implement social distancing practices, early treatment, and prompt disposition.OBJECTIVES: We sought to determine factors associated with influenza among a prospective cohort of consecutive ED patients with acute respiratory illness (ARI).METHODS: Between December 2016 and March 2017, trained research assistants screened consecutive ED patients with ARI symptoms. ARI criteria included measured fever at home or in the ED >38°C and a cough, sore throat, or rhinorrhea with a duration of symptoms >12hours and <1week. After consent, research assistants collected demographics and clinical history using a standardized data form, and patients had a polymerase chain reaction-based assay that is nearly 100% sensitive for influenza. Univariate analysis was conducted on all predictor variables. Significant variables were entered into a multivariate logistic regression model to find factors that were independently associated with influenza.RESULTS: One hundred nineteen patients consented to enrollment and 31% were found to be positive for influenza. Myalgia, the absence of gastrointestinal symptoms (no diarrhea or vomiting), sore throat, chills, headache, and oxygen saturation ≥97% were significant on univariate analysis and were entered into the multivariate model. Myalgia (adjusted odds ratio [AOR] 3.9), the absence of gastrointestinal symptoms (AOR 4.7), and oxygen saturation ≥97% (AOR 2.8) were significant independent factors of influenza.CONCLUSION: The presence of myalgia, the absence of gastrointestinal symptoms, and oxygen saturation ≥97% are factors that can help distinguish influenza from other acute respiratory illnesses in the ambulatory ED population.
View details for DOI 10.1016/j.jemermed.2018.12.012
View details for PubMedID 30803847
A 'culture' shift: Application of molecular techniques for diagnosing polymicrobial infections.
With the advancement of microbiological discovery, it is evident that many infections, particularly bloodstream infections, are polymicrobial in nature. Consequently, new challenges have emerged in identifying the numerous etiologic organisms in an accurate and timely manner using the current diagnostic standard. Various molecular diagnostic methods have been utilized as an effort to provide a fast and reliable identification in lieu or parallel to the conventional culture-based methods. These technologies are mostly based on nucleic acid, proteins, or physical properties of the pathogens with differing advantages and limitations. This review evaluates the different molecular methods and technologies currently available to diagnose polymicrobial infections, which will help determine the most appropriate option for future diagnosis.
View details for DOI 10.1016/j.biotechadv.2019.02.013
View details for PubMedID 30797092
Using a novel rapid viral test to improve triage of emergency department patients with acute respiratory illness during flu season.
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology
2018; 108: 72–76
BACKGROUND: Acute respiratory illnesses (ARI) are mostly viral in etiology and cause significant morbidity and mortality. Point of care PCR (POC-PCR) is a promising new technology for rapid virus identification but utility in the Emergency Department (ED) is not yet defined.OBJECTIVES: Primarily, to investigate the value of POC-PCR in rapidly identifying RSV and influenza in the setting of ED triage. Additionally, to assess whether rapid knowledge of accurate test results would improve patient management by preventing nosocomial transmission and optimizing the prescription of antimicrobials for ARIs.STUDY DESIGN: A prospective cohort study of consecutive ED patients with ARI symptoms during peak flu season was conducted. Patient nasopharyngeal swabs were collected and tested using a POC-PCR device; physicians and patients were blinded to results. Virus positive and negative groups were compared by ED patient room placement and antimicrobial therapy ordered. Specificity and sensitivity were calculated using laboratory-PCR as the gold standard.RESULTS: Of 119 participants, 52.9% were POC-PCR positive - Influenza A (42.9%), RSV (41.3%), influenza B (15.9%). Nearly 70% of virus positive patients were placed rooms shared with non-ARI patients. Antibiotics were prescribed for 27.3% of virus positive patients, and 77.8% of oseltamivir-eligible patients did not receive therapy. POC-PCR was 100% sensitive (95% CI, 80.5-100.0%) and 95.2% specific (95% CI, 76.2-99.9%).CONCLUSIONS: Rapid POC-PCR for influenza and RSV in ED triage has excellent sensitivity and specificity and the potential to improve social distancing practices through better triage and increase appropriate prescription of antimicrobials.
View details for DOI 10.1016/j.jcv.2018.09.008
View details for PubMedID 30261422
A review on cluster estimation methods and their application to neural spike data
JOURNAL OF NEURAL ENGINEERING
2018; 15 (3): 031003
The extracellular action potentials recorded on an electrode result from the collective simultaneous electrophysiological activity of an unknown number of neurons. Identifying and assigning these action potentials to their firing neurons-'spike sorting'-is an indispensable step in studying the function and the response of an individual or ensemble of neurons to certain stimuli. Given the task of neural spike sorting, the determination of the number of clusters (neurons) is arguably the most difficult and challenging issue, due to the existence of background noise and the overlap and interactions among neurons in neighbouring regions. It is not surprising that some researchers still rely on visual inspection by experts to estimate the number of clusters in neural spike sorting. Manual inspection, however, is not suitable to processing the vast, ever-growing amount of neural data. To address this pressing need, in this paper, thirty-three clustering validity indices have been comprehensively reviewed and implemented to determine the number of clusters in neural datasets. To gauge the suitability of the indices to neural spike data, and inform the selection process, we then calculated the indices by applying k-means clustering to twenty widely used synthetic neural datasets and one empirical dataset, and compared the performance of these indices against pre-existing ground truth labels. The results showed that the top five validity indices work consistently well across variations in noise level, both for the synthetic datasets and the real dataset. Using these top performing indices provides strong support for the determination of the number of neural clusters, which is essential in the spike sorting process.
View details for DOI 10.1088/1741-2552/aab385
View details for Web of Science ID 000430324400001
View details for PubMedID 29498353
Impact of Rapid Molecular Respiratory Virus Testing on Real-Time Decision Making in a Pediatric Emergency Department
JOURNAL OF MOLECULAR DIAGNOSTICS
2017; 19 (3): 460-467
Acute respiratory illnesses (ARIs) are usually viral [influenza, respiratory syncytial virus (RSV)] and account for 25% of emergency department (ED) peak-season visits. Laboratory PCR testing is accurate albeit slow, whereas rapid antigen testing is inaccurate. We determined the impact of bedside PCR (molecular point-of-care test; mPOCT) on pediatric ARI management. This was a prospective cohort study of consecutive pediatric patients with ED-ordered respiratory PCR test, enrolled over 9 weeks during peak flu season. On ordering, ED physicians were interviewed to ascertain real-time plans if given immediate influenza/RSV PCR results for the current patient. Two groups were compared: actual management and management adjusted for mPOCT results. We compared ED length of stay (LOS), tests ordered, and antibiotic/antiviral ordering. One-hundred thirty-six respiratory PCR panels were ordered, 71 by admitting team, 61 for ED management. Of 61 ED-initiated tests, physicians indicated in 39 cases (64%) they would change patient management were bedside viral results available. Physicians would have decreased ED LOS by 33 minutes, ordered fewer tests (18%; P < 0.001) with average patient charge savings of $669, fewer antibiotics among discharged patients (17%; P = 0.043), and increased appropriate antiviral use (13%; P = 0.023). Rapid bedside ARI mPOCT PCR has the potential to decrease ED LOS, reduce diagnostic tests and patient charges, and increase appropriate use of antibiotics and antiviral agents.
View details for DOI 10.1016/j.jmoldx.2017.01.009
View details for Web of Science ID 000400314100011
View details for PubMedID 28341587
View details for PubMedCentralID PMC5417039
Umbilical Cord Blood NOS1 as a Potential Biomarker of Neonatal Encephalopathy.
Frontiers in pediatrics
2017; 5: 112
There are no definitive markers to aid in diagnosis of neonatal encephalopathy (NE). The purpose of our study was (1) to identify and evaluate the utility of neuronal nitric oxide synthase (NOS1) in umbilical cord blood as a NE biomarker and (2) to identify the source of NOS1 in umbilical cord blood.This was a nested case-control study of neonates >35 weeks of gestation. ELISA for NOS1 in umbilical cord blood was performed. Sources of NOS1 in umbilical cord were investigated by immunohistochemistry, western blot, ELISA, and quantitative PCR. Furthermore, umbilical cords of full-term neonates were subjected to 1% hypoxia ex vivo.NOS1 was present in umbilical cord blood and increased in NE cases compared with controls. NOS1 was expressed in endothelial cells of the umbilical cord vein, but not in artery or blood cells. In ex vivo experiments, hypoxia was associated with increased levels of NOS1 in venous endothelial cells of the umbilical cord as well as in ex vivo culture medium.This is the first study to investigate an early marker of NE. NOS1 is elevated with hypoxia, and further studies are needed to investigate it as a valuable tool for early diagnosis of neonatal brain injury.
View details for DOI 10.3389/fped.2017.00112
View details for PubMedID 28649562
View details for PubMedCentralID PMC5466059
Integrated Bacterial Identification and Antimicrobial Susceptibility Testing Using PCR and High-Resolution Melt.
Accurate and timely diagnostics are critical for managing bacterial infections. The current gold standard, culture-based diagnostics, can provide clinicians with comprehensive diagnostic information including bacterial identity and antimicrobial susceptibility, but they often require several days of turnaround time, which leads to compromised clinical outcome and promotes the spread of antibiotic resistance. Nucleic acid amplification tests such as PCR have significantly accelerated the detection of specific bacteria but generally lack the capacities for broad-based bacterial identification or antimicrobial susceptibility testing. Here, we report an integrated assay based on PCR and high-resolution melt (HRM) for rapid diagnosis for bacterial infections. In our assay, we measure bacterial growth in the presence or absence of certain antibiotics with real-time quantitative PCR or digital PCR to determine antimicrobial susceptibility. In addition, we use HRM and a machine learning algorithm to identify bacterial species based on melt-curve profiles of the 16S rRNA gene in an automated fashion. As a demonstration, we correctly identified the bacterial species and their antimicrobial susceptibility profiles for multiple unknown samples in blinded tests within ∼6.5 h.
View details for DOI 10.1021/acs.analchem.7b02809
View details for PubMedID 29027789
RNA-Seq Count Data Modelling by Grey Relational Analysis and Nonparametric Gaussian Process
2016; 11 (10)
This paper introduces an approach to classification of RNA-seq read counts using grey relational analysis (GRA) and Bayesian Gaussian process (GP) models. Read counts are transformed to microarray-like data to facilitate normal-based statistical methods. GRA is designed to select differentially expressed genes by integrating outcomes of five individual feature selection methods including two-sample t-test, entropy test, Bhattacharyya distance, Wilcoxon test and receiver operating characteristic curve. GRA performs as an aggregate filter method through combining advantages of the individual methods to produce significant feature subsets that are then fed into a nonparametric GP model for classification. The proposed approach is verified by using two benchmark real datasets and the five-fold cross-validation method. Experimental results show the performance dominance of the GRA-based feature selection method as well as GP classifier against their competing methods. Moreover, the results demonstrate that GRA-GP considerably dominates the sparse Poisson linear discriminant analysis classifiers, which were introduced specifically for read counts, on different number of features. The proposed approach therefore can be implemented effectively in real practice for read count data analysis, which is useful in many applications including understanding disease pathogenesis, diagnosis and treatment monitoring at the molecular level.
View details for DOI 10.1371/journal.pone.0164766
View details for Web of Science ID 000389602800048
View details for PubMedID 27783633
View details for PubMedCentralID PMC5082617
- Nested Machine Learning Facilitates Increased Sequence Content for Large-Scale Automated High Resolution Melt Genotyping SCIENTIFIC REPORTS 2015
A rabbit model of non-typhoidal Salmonella bacteremia
COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES
2014; 37 (4): 211-220
Bacteremia is an important cause of morbidity and mortality in humans. In this study, we focused on the development of an animal model of bacteremia induced by non-typhoidal Salmonella. New Zealand White rabbits were inoculated with a human isolate of non-typhoidal Salmonella strain CVD J73 via the intra-peritoneal route. Blood samples were collected at specific time points and at euthanasia from infected rabbits. Additionally, tissue samples from the heart, lungs, spleen, gastrointestinal tract, liver and kidneys were obtained at euthanasia. All experimentally infected rabbits displayed clinical signs of disease (fever, dehydration, weight loss and lethargy). Tissues collected at necropsy from the animals exhibited histopathological changes indicative of bacteremia. Non-typhoidal Salmonella bacteria were detected in the blood and tissue samples of infected rabbits by microbiological culture and real-time PCR assays. The development of this animal model of bacteremia could prove to be a useful tool for studying how non-typhoidal Salmonella infections disseminate and spread in humans.
View details for DOI 10.1016/j.cimid.2014.05.004
View details for Web of Science ID 000343351700002
View details for PubMedID 25033732
Sensitive Detection and Serovar Differentiation of Typhoidal and Nontyphoidal Salmonella enterica Species Using 16S rRNA Gene PCR Coupled with High-Resolution Melt Analysis
JOURNAL OF MOLECULAR DIAGNOSTICS
2014; 16 (2): 261-266
Salmonella enterica species infections are a significant public health problem causing high morbidity rates worldwide and high mortality rates in the developing world. These infections are not always rapidly diagnosed as a cause of bloodstream infections because of the limitations of blood culture, which greatly affects clinical care as a result of treatment delays. A molecular diagnostic assay that could rapidly detect and identify S. enterica species infections as a cause of sepsis is needed. Nine typhoidal and nontyphoidal S. enterica serovars were used to establish the limit of detection (LOD) of a previously published 16S rRNA gene PCR (16S PCR) in mock whole blood specimens. In addition, 16 typhoidal and nontyphoidal S. enterica serovars were used to evaluate the serovar differentiation capability of 16S PCR coupled with high-resolution melt analysis. The overall LOD of 16S PCR for the nine typhoidal and nontyphoidal S. enterica serovars analyzed was <10 colony-forming units per milliliter (CFU/mL) in mock whole blood specimens, with the lowest and highest LOD at <1 CFU/mL and 9 CFU/mL, respectively. By high-resolution melt analysis, the typhoidal and nontyphoidal S. enterica serovar groups analyzed each generated a unique grouping code, allowing for serovar-level identification. 16S PCR coupled with high-resolution melt analysis could be a useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of S. enterica bloodstream infections.
View details for DOI 10.1016/j.jmoldx.2013.10.011
View details for Web of Science ID 000332193300013
View details for PubMedID 24365382
Trainable high resolution melt curve machine learning classifier for large-scale reliable genotyping of sequence variants.
2014; 9 (9): e109094
High resolution melt (HRM) is gaining considerable popularity as a simple and robust method for genotyping sequence variants. However, accurate genotyping of an unknown sample for which a large number of possible variants may exist will require an automated HRM curve identification method capable of comparing unknowns against a large cohort of known sequence variants. Herein, we describe a new method for automated HRM curve classification based on machine learning methods and learned tolerance for reaction condition deviations. We tested this method in silico through multiple cross-validations using curves generated from 9 different simulated experimental conditions to classify 92 known serotypes of Streptococcus pneumoniae and demonstrated over 99% accuracy with 8 training curves per serotype. In vitro verification of the algorithm was tested using sequence variants of a cancer-related gene and demonstrated 100% accuracy with 3 training curves per sequence variant. The machine learning algorithm enabled reliable, scalable, and automated HRM genotyping analysis with broad potential clinical and epidemiological applications.
View details for DOI 10.1371/journal.pone.0109094
View details for PubMedID 25275518
Reverse Transcription-PCR-Electrospray Ionization Mass Spectrometry for Rapid Detection of Biothreat and Common Respiratory Pathogens
JOURNAL OF CLINICAL MICROBIOLOGY
2013; 51 (10): 3300-3307
Electrospray ionization mass spectrometry (ESI-MS) analysis of reverse transcription (RT)-PCR amplicons from human respiratory samples allows for broad pathogen identification approximately 8 h after collection. We investigated the performance characteristics of a high-throughput RT-PCR-coupled ESI-MS assay for distinguishing biothreat (BT) agents from common bacterial, fungal, and viral respiratory pathogens in bronchoalveolar lavage (BAL) fluid specimens from subjects with suspected respiratory infections. In a retrospective case series, 202 BAL fluid specimens were collected at the Johns Hopkins Hospital between August 2010 and February 2011 from patients with suspected acute respiratory infections. Samples were processed using standard bacterial, viral, and fungal testing in the clinical microbiology laboratory as part of routine care and then were blindly spiked with either water or nucleic acids from BT organisms (Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella spp., Burkholderia spp., and Rickettsia prowazekii) and tested by RT-PCR-ESI-MS. The sensitivities and specificities of RT-PCR-ESI-MS versus standard clinical methods were as follows: for mock BT DNA, 98.5% sensitivity (95% confidence interval [CI], 94.2 to 99.7%) and 100% specificity (95% CI, 93.1 to 100.0%); for bacterial pathogens, 81.8% sensitivity (95% CI, 74.3 to 87.6%) and 73.6% specificity (95% CI, 64.2 to 81.4%); for viral pathogens, 93.3% sensitivity (95% CI, 66.0 to 99.7%) and 97.3% specificity (95% CI, 89.7 to 99.5%); for fungal pathogens, 42.6% sensitivity (95% CI, 29.5 to 56.7%) and 97.8% specificity (95% CI, 91.8 to 99.6%). Our data suggest that RT-PCR-ESI-MS is a useful adjunct to standard culture protocols for rapid detection of both BT and common respiratory pathogens; further study is required for assay validation, especially for fungal detection, and potential implementation.
View details for DOI 10.1128/JCM.01443-13
View details for Web of Science ID 000324624300021
View details for PubMedID 23903543
State of virtual reality based disaster preparedness and response training.
The advent of technologically-based approaches to disaster response training through Virtual Reality (VR) environments appears promising in its ability to bridge the gaps of other commonly established training formats. Specifically, the immersive and participatory nature of VR training offers a unique realistic quality that is not generally present in classroom-based or web-based training, yet retains considerable cost advantages over large-scale real-life exercises and other modalities and is gaining increasing acceptance. Currently, numerous government departments and agencies including the U.S. Department of Homeland Security (DHS), the Centers for Disease Control and Prevention (CDC) as well as academic institutions are exploring the unique advantages of VR-based training for disaster preparedness and response. Growing implementation of VR-based training for disaster preparedness and response, conducted either independently or combined with other training formats, is anticipated. This paper reviews several applications of VR-based training in the United States, and reveals advantages as well as potential drawbacks and challenges associated with the implementation of such training platform.
View details for DOI 10.1371/currents.dis.1ea2b2e71237d5337fa53982a38b2aff
View details for PubMedID 23653102
- Harnessing Genomic Approaches for Infectious Disease Diagnosis in Emergency Medicine: Getting Closer to Prime Time ANNALS OF EMERGENCY MEDICINE 2012; 60 (5): 621-623
Comparative Analysis of Two Broad-Range PCR Assays for Pathogen Detection in Positive-Blood-Culture Bottles: PCR-High-Resolution Melting Analysis versus PCR-Mass Spectrometry
JOURNAL OF CLINICAL MICROBIOLOGY
2012; 50 (10): 3287-3292
Detection of pathogens in bloodstream infections is important for directing antimicrobial treatment, but current culture-based approaches can be problematic. Broad-range PCR assays which target conserved genomic motifs for postamplification amplicon analysis permit detection of sepsis-causing pathogens. Comparison of different broad-range assays is important for informing future implementation strategies. In this study, we compared positive-blood-culture bottles processed by PCR coupled to high-resolution melting curve analysis (PCR/HRMA) and PCR coupled to electrospray ionization-mass spectrometry (PCR/ESI-MS) to microbiology culture results. Genus-level concordance was 90% (confidence interval [CI], 80 to 96%) for PCR/HRMA and 94% (CI, 85 to 98%) for PCR/ESI-MS. Species-level concordance was 90% (CI, 80 to 96%) for PCR/HRMA and 86% (CI, 75 to 93%) for PCR/ESI-MS. Unlike PCR/HRMA, PCR/ESI-MS was able to resolve polymicrobial samples. Our results demonstrated that the two assays have similar overall concordance rates but may have different roles as potential adjunctive tests with standard blood culture, since each method has different capabilities, advantages, and disadvantages.
View details for DOI 10.1128/JCM.00677-12
View details for Web of Science ID 000308870200022
View details for PubMedID 22855511
A broad range assay for rapid detection and etiologic characterization of bacterial meningitis: performance testing in samples from sub-Sahara
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
2012; 74 (1): 22-27
This study aimed to conduct a pilot evaluation of broad-based multiprobe polymerase chain reaction (PCR) in clinical cerebrospinal fluid (CSF) samples compared to local conventional PCR/culture methods used for bacterial meningitis surveillance. A previously described PCR consisting of initial broad-based detection of Eubacteriales by a universal probe, followed by Gram typing, and pathogen-specific probes was designed targeting variable regions of the 16S rRNA gene. The diagnostic performance of the 16S rRNA assay in "127 CSF samples was evaluated in samples from patients from Togo, Africa, by comparison to conventional PCR/culture methods. Our probes detected Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Uniprobe sensitivity and specificity versus conventional PCR were 100% and 54.6%, respectively. Sensitivity and specificity of uniprobe versus culture methods were 96.5% and 52.5%, respectively. Gram-typing probes correctly typed 98.8% (82/83) and pathogen-specific probes identified 96.4% (80/83) of the positives. This broad-based PCR algorithm successfully detected and provided species level information for multiple bacterial meningitis agents in clinical samples.
View details for DOI 10.1016/j.diagmicrobio.2012.05.015
View details for Web of Science ID 000308116900005
View details for PubMedID 22809694
Identification of Bacterial Pathogens in Ascitic Fluids from Patients with Suspected Spontaneous Bacterial Peritonitis by Use of Broad-Range PCR (16S PCR) Coupled with High-Resolution Melt Analysis
JOURNAL OF CLINICAL MICROBIOLOGY
2012; 50 (7): 2428-2432
Spontaneous bacterial peritonitis (SBP) can be a severe complication occurring in patients with cirrhosis and ascites, with associated mortality often as high as 40%. Traditional diagnostics for SBP rely on culture techniques for proper diagnosis, although recent reports suggest that the presence of bacterial DNA in peritoneal fluid in patients with cirrhosis and ascites is an indicator of SBP. A previously published broad-range PCR (16S PCR) coupled with high-resolution melt analysis (HRMA) was compared with standard culture techniques for diagnosis of SBP in 106 peritoneal fluid samples from patients with suspected SBP. The sensitivity and specificity for 16S PCR for detecting eubacterial DNA compared with those of standard culture techniques were 100% (17/17) and 91.5% (85/89), respectively. Overall, HRMA concordance with species identification was 70.6% (12/17), although the 5 samples that were discordant at the species level were SBP resulting from a polymicrobial infection, and species-level identification for polymicrobial infections is outside the capability of HRMA. Both the broad-range 16S PCR and HRMA analysis provide useful diagnostic adjunctive assays for clinicians in detecting and identifying pathogens responsible for SBP.
View details for DOI 10.1128/JCM.00345-12
View details for Web of Science ID 000307360800040
View details for PubMedID 22573594
Application of a 16S rRNA PCR-High-Resolution Melt Analysis Assay for Rapid Detection of Salmonella Bacteremia
JOURNAL OF CLINICAL MICROBIOLOGY
2012; 50 (3): 1122-1124
Current culture and phenotypic protocols for diagnosing Salmonella infections can be time-consuming. Here, we describe the application of a 16S rRNA PCR coupled to high-resolution melt analysis (HRMA) for species and serotype identification within 6 h of blood sample collection from a patient with Salmonella enterica serotype Enteritidis bacteremia.
View details for DOI 10.1128/JCM.05121-11
View details for Web of Science ID 000300997800098
View details for PubMedID 22205823
A surface topography assisted droplet manipulation platform for biomarker detection and pathogen identification
LAB ON A CHIP
2011; 11 (3): 398-406
This paper reports a droplet microfluidic, sample-to-answer platform for the detection of disease biomarkers and infectious pathogens using crude biosamples. The platform exploited the dual functionality of silica superparamagnetic particles (SSP) for solid phase extraction of DNA and magnetic actuation. This enabled the integration of sample preparation and genetic analysis within discrete droplets, including the steps of cell lysis, DNA binding, washing, elution, amplification and detection. The microfluidic device was self contained, with all reagents stored in droplets, thereby eliminating the need for fluidic coupling to external reagent reservoirs. The device incorporated unique surface topographic features to assist droplet manipulation. Pairs of micro-elevations were created to form slits that facilitated efficient splitting of SSP from droplets. In addition, a compact sample handling stage, which integrated the magnet manipulator, the droplet microfluidic device and a Peltier thermal cycler, was built for convenient droplet manipulation and real-time detection. The feasibility of the platform was demonstrated by analysing ovarian cancer biomarker Rsf-1 and detecting Escherichia coli with real time polymerase chain reaction and real time helicase dependent amplification.
View details for DOI 10.1039/c0lc00296h
View details for Web of Science ID 000286326700007
View details for PubMedID 21046055
An all-in-one microfluidic device for parallel DNA extraction and gene analysis
2010; 12 (6): 1043-1049
We have developed a microfluidic device capable of fully integrated sample preparation and gene analysis from crude biosamples such as whole blood. Our platform takes the advantage of the silica superparamagnetic particle based solid phase extraction to develop an all-in-one scheme that performs cell lysis, DNA binding, washing, elution and the PCR in the same reaction chamber. The device also employs a unique reagent loading scheme, allowing efficient preparation of multiple reactions via a single injection channel. In addition, PCR is performed in a droplet-in-oil manner, eliminating the need for chamber sealing. The combination of these design features greatly reduces the complexity in implementing fully integrated lab-on-a-chip systems for genetic detection, facilitating parallel analysis of multiple samples or genes on a single microchip. The capability of the device is demonstrated by performing DNA isolation from the human whole blood sample and analyzing the Rsf-1 gene using the TaqMan probe based gene specific PCR assays.
View details for DOI 10.1007/s10544-010-9458-6
View details for Web of Science ID 000283246500009
View details for PubMedID 20632111
Rapid Identification of Bacterial Pathogens in Positive Blood Culture Bottles by Use of a Broad-Based PCR Assay Coupled with High-Resolution Melt Analysis
JOURNAL OF CLINICAL MICROBIOLOGY
2010; 48 (9): 3410-3413
We evaluated a broad-based PCR assay coupled with high-resolution melt analysis for rapid bacterial identification in patients with bacterial sepsis. With a reference library of 60 clinically relevant bacterial species, 52 positive blood culture samples were tested. Our assay identified 46/52 samples at the species level, with 100% concordance to culture findings.
View details for DOI 10.1128/JCM.00718-10
View details for Web of Science ID 000281480400068
View details for PubMedID 20631110
Use of Quantitative Broad-based Polymerase Chain Reaction for Detection and Identification of Common Bacterial Pathogens in Cerebrospinal Fluid
ACADEMIC EMERGENCY MEDICINE
2010; 17 (7): 741-747
Conventional laboratory diagnosis of bacterial meningitis based on microscopy followed by culture is time-consuming and has only moderate sensitivity.The objective was to define the limit of detection (LOD), analytic specificity, and performance characteristics of a broad-based quantitative multiprobe polymerase chain reaction (PCR) assay for rapid bacterial detection and simultaneous pathogen-specific identification in patients with suspected meningitis.A PCR algorithm consisting of initial broad-based detection of Eubacteriales by a universal probe, followed by pathogen identification using either pathogen-specific probes or Gram-typing probes, was employed to detect pathogens. The 16S rRNA gene, which contains both conserved and variable regions, was chosen as the target. Pathogen-specific probes were designed for Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes. Gram-positive and -negative typing probes were designed based on conserved regions across all eubacteria. The LOD and time to detection were assessed by dilutional mocked-up samples. A total of 108 convenience cerebrospinal fluid (CSF) clinical samples obtained from the Johns Hopkins Hospital (JHH) microbiology laboratory were tested, and results were compared with hospital microbiologic culture reports.The LOD of the assay ranged from 10(1) to 10(2) colony-forming units (CFU)/mL. Pathogen-specific probes showed no cross-reactivity with other organisms. Time to detection was 3 hours. In clinical specimens, the universal probe correctly detected 16 of 22 culture-positive clinical specimens (sensitivity = 72.7%; 95% confidence interval [CI] = 49.8% to 89.3%), which were all correctly characterized by either pathogen-specific or Gram-typing probes. Adjusted sensitivity after removing probable microbiologic laboratory contaminants was 88.9% (95% CI = 65.3% to 98.6%). The universal probe was negative for 86 of 86 culture-negative specimens.A broad-based multiprobe PCR assay demonstrated strong analytic performance characteristics. Findings from a pilot clinical study showed promise in translation to human subjects, supporting potential utility of the assay as an adjunct to traditional diagnostics for early identification of bacterial meningitis.
View details for DOI 10.1111/j.1553-2712.2010.00790.x
View details for Web of Science ID 000279613400017
View details for PubMedID 20653589
Rapid Identification of Biothreat and Other Clinically Relevant Bacterial Species by Use of Universal PCR Coupled with High-Resolution Melting Analysis
JOURNAL OF CLINICAL MICROBIOLOGY
2009; 47 (7): 2252-2255
A rapid assay for eubacterial species identification is described using high-resolution melt analysis to characterize PCR products. Unique melt profiles generated from multiple hypervariable regions of the 16S rRNA gene for 100 clinically relevant bacterial pathogens, including category A and B biothreat agents and their surrogates, allowed highly specific species identification.
View details for DOI 10.1128/JCM.00033-09
View details for Web of Science ID 000267713000038
View details for PubMedID 19458181
HIV Seropositivity Predicts Longer Duration of Stay and Rehospitalization Among Nonbacteremic Febrile Injection Drug Users With Skin and Soft Tissue Infections
Annual Meeting of the Society-for-Academic-Emergency-Medicine
LIPPINCOTT WILLIAMS & WILKINS. 2008: 398–405
Skin/soft tissue infections (SSTIs) are the leading cause of hospital admissions among injection drug users (IDUs).We performed a retrospective investigation to determine the epidemiology of SSTIs (ie, cellulitis and/or abscesses) in febrile IDUs, with a focus on bacteriology and potential predictors of increased health care utilization measured by longer length of stay and rehospitalization. Subjects were drawn from a cohort of febrile IDUs presenting to an inner-city emergency department from 1998 to 2004.Of the 295 febrile IDUs with SSTIs, specific discharge diagnoses were cellulitis only (n = 143, 48.5%), abscesses only (n = 113, 38.3%), and both (n = 39, 13.2%). Documented HIV infection rate was 28%. Of note, 10 subjects were newly diagnosed with HIV infection during their visits. Staphylococcus aureus was the leading pathogen, and increasing rates of methicillin-resistant S. aureus emerged over time (before 2001: 4%, 2001-2004: 56%, P < 0.01). HIV seropositivity predicted rehospitalization within 90 days [adjusted hazard ratios and 95% confidence intervals: 2.90 (1.20 to 7.02)]. HIV seropositivity also predicted increased length of stay in those who were nonbacteremic [adjusted hazard ratios and 95% confidence intervals: 1.49 (1.11 to 2.01)].Among febrile IDUs with SSTIs, a strong association between HIV seropositivity and health care resource utilization was found. Accordingly, attention to HIV serostatus should be considered in clinical disposition decisions for this vulnerable high-risk population.
View details for Web of Science ID 000260844300008
View details for PubMedID 19186352
Rapid polymerase chain reaction-based screening assay for bacterial biothreat agents
Annual Meeting of the Society-for-Academic-Emergency-Medicine
BLACKWELL PUBLISHING. 2008: 388–92
To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens.The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria.The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus.A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.
View details for DOI 10.1111/j.1553-2712.2008.00061.x
View details for Web of Science ID 000254412600014
View details for PubMedID 18370996
Rapid PCR-based diagnosis of septic arthritis by early gram-type classification and pathogen identification
JOURNAL OF CLINICAL MICROBIOLOGY
2008; 46 (4): 1386-1390
Septic arthritis (SA) is a rheumatologic emergency associated with significant morbidity and mortality. Delayed or inadequate treatment of SA can lead to irreversible joint destruction and disability. Current methods of diagnosing SA rely on synovial fluid analysis and culture which are known to be imprecise and time-consuming. We report a novel adaptation of a probe-based real-time PCR assay targeting the 16S rRNA gene for early and accurate diagnosis of bacterial SA. The assay algorithm consists of initial broad-range eubacterial detection, followed by Gram typing and species characterization of the pathogen. The platform demonstrated a high analytical sensitivity with a limit of detection of 10(1) CFU/ml with a panel of SA-related organisms. Gram typing and pathogen-specific probes correctly identified their respective targets in a mock test panel of 36 common clinically relevant pathogens. One hundred twenty-one clinical synovial fluid samples from patients presenting with suspected acute SA were tested. The sensitivity and specificity of the assay were 95% and 97%, respectively, versus synovial fluid culture results. Gram-typing probes correctly identified 100% of eubacterial positive samples as to gram-positive or gram-negative status, and pathogen-specific probes correctly identified the etiologic agent in 16/20 eubacterial positive samples. The total assay time from sample collection to result is 3 h. We have demonstrated that a real-time broad-based PCR assay has high analytical and clinical performance with an improved time to detection versus culture for SA. This assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute care setting where rapid pathogen detection and identification would assist in disposition and treatment decisions.
View details for DOI 10.1128/JCM.02305-07
View details for Web of Science ID 000254866400035
View details for PubMedID 18305128
Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry
2007; 2 (5)
Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology.Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution.Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.
View details for DOI 10.1371/journal.pone.0000489
View details for Web of Science ID 000207448800019
View details for PubMedID 17534439
Communicable respiratory threats in the ED: Tuberculosis, influenza, SARS, and other aerosolized infections
EMERGENCY MEDICINE CLINICS OF NORTH AMERICA
2006; 24 (4): 989-?
Respiratory infections are the most common communicable infectious diseases. EDs are the front line for patients with respiratory infections because of their acute nature and because the ED is the principal site of health care for those at highest risk. These diseases include influenza, tuberculosis, and measles, together accounting for 25% of infectious causes of death worldwide. These are emerging and biothreat agents that follow the same route of transmission, such as pneumonic plague. We discuss epidemiology, pathogenesis, diagnosis, and treatment of each agent. Emphasis is on the ED's role as a public health prevention arena, with attention to education and disease prevention, early identification of disease in patients at risk, and reduction of illnesses.
View details for DOI 10.1016/j.emc.2006.06.006
View details for Web of Science ID 000241062500012
View details for PubMedID 16982349
Multiplexed hybridization detection with multicolor colocalization of quantum dot nanoprobes
2005; 5 (9): 1693-1697
We demonstrate a hybridization detection method using multicolor oligonucleotide-functionalized quantum dots as nanoprobes. In the presence of various target sequences, combinatorial self-assembly of the nanoprobes via independent hybridization reactions leads to the generation of discernible sequence-specific spectral codings. Detection of single-molecule hybridization is achieved by measuring colocalization of individual nanoprobes. Genetic analysis for anthrax pathogenicity through simultaneous detection of multiple relevant sequences is demonstrated using this novel biosensing method as proof-of-concept.
View details for DOI 10.1021/nl050888v
View details for Web of Science ID 000231945500011
View details for PubMedID 16159207
Quantitative PCR assay using sputum samples for rapid diagnosis of pneumococcal pneumonia in adult emergency department patients
JOURNAL OF CLINICAL MICROBIOLOGY
2005; 43 (7): 3221-3226
Accurate diagnosis of pneumococcal pneumonia in the acute-care setting remains a challenge due to the inadequate sensitivity of conventional diagnostic tests. Sputum cultures, which are likely to have the highest diagnostic yields of all specimen types, have been considered unreliable, due to their inability to differentiate colonization from infection. Our objective was to evaluate the potential clinical utility of a rapid quantitative real-time PCR assay using sputum samples for Streptococcus pneumoniae in adult patients with community-acquired pneumonia (CAP). A prospective clinical observational study of consecutively enrolled emergency department patients with CAP was performed; only those patients with excess good-quality sputum samples were included for evaluation. Sputum samples were tested for the presence of S. pneumoniae by using a quantitative PCR that targets the pneumolysin gene. PCR findings were compared with those of a composite reference standard comprising Gram staining of sputum samples and sputum/blood cultures. The area under the curve (AUC) and a log-transformed threshold, which provides the maximal sensitivity and specificity, were calculated. Of 487 subjects enrolled, 129 were evaluable. Receiver operating characteristic curve analysis demonstrated an AUC of 0.87. Sensitivity and specificity were 90.0 percent and 80.0 percent, respectively; positive and negative predictive values were 58.7 percent and 96.2 percent, respectively. We have demonstrated that a quantitative rapid pneumolysin PCR assay has favorable accuracy for diagnosis of pneumococcal pneumonia in adult patients with CAP; this assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute-care setting, where rapid pathogen identification may assist in selection of the most appropriate antibiotics.
View details for DOI 10.1128/JCM.43.7.3221-3226.2005
View details for Web of Science ID 000230614900030
View details for PubMedID 16000439
Real-time PCR for Chlamydia pneumoniae utilizing the Roche lightcycler and a 16S rRNA gene target
JOURNAL OF MOLECULAR DIAGNOSTICS
2004; 6 (2): 132-136
Chlamydia pneumoniae (CPN) causes pneumonia in humans, and has emerged as an important respiratory pathogen. There are also established links between CPN infection and coronary artery disease. Traditional culture methods for CPN detection can be time consuming and difficult. There are a variety of molecular-based amplification methods for CPN detection. These methods are more sensitive than culture, but have the disadvantage of being inconsistent and non-comparable across studies. In this paper, we describe the adaptation of the existing primer set CPN 90/CPN91 for use in a real-time PCR assay using the Roche Lightcycler and a Taqman probe. This assay had an analytical sensitivity of between 4 and 0.4 infection-forming units (IFUs)/PCR reaction. A total of 355 samples were tested for validation of the assay. Tested samples included two standardized panels of blinded samples from culture (N = 70), archived specimens consisting of a CPN dilution series, CPN-spiked porcine aortal tissue and endarterectomy specimens (N = 87). The third group consisted of prospectively collected PBMCs from clinical samples (N = 198). Results were compared to nested PCR, which targets the ompA gene of CPN; TETR PCR, which targets the 16S rRNA gene of CPN; or the known result for the sample. Overall, the assay had a sensitivity of 88.5% (69 of 78) and a specificity of 99.3% (275 of 277). This method should prove useful for accurate, high throughput detection of CPN.
View details for Web of Science ID 000221002500009
View details for PubMedID 15096569
Use of the Roche LightCycler instrument in a real-time PCR for Trichomonas vaginalis in urine samples from females and males
JOURNAL OF CLINICAL MICROBIOLOGY
2003; 41 (12): 5619-5622
Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted infection (STI) that can result in vaginitis, urethritis, and preterm birth. Traditional methods of diagnosis, including wet preparation, can be unreliable. In this study, we describe the adaptation of an existing PCR method for specific detection of T. vaginalis DNA into a rapid real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe chemistry. The FRET-based assay described demonstrated high sensitivity with a detection limit of 1.06 organisms, as well as a high specificity. A total of 253 urine samples collected prospectively from both men and women were tested for T. vaginalis DNA with both the FRET-based assay and a previously validated PCR assay. When the validated PCR assay was used as the "gold standard" and after discrepancies had been resolved, our FRET-based assay demonstrated an analytical sensitivity and specificity of 90.1 and 100%, respectively. Overall results suggest that FRET-based assays can provide rapid, accurate, and high-throughput detection of T. vaginalis and may prove useful in clinical settings and for large-scale screening programs.
View details for DOI 10.1128/JCM.41.12.5619-5622.2003
View details for Web of Science ID 000187228800041
View details for PubMedID 14662951
Quantitative multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens
JOURNAL OF CLINICAL MICROBIOLOGY
2002; 40 (9): 3449-3454
We describe a novel adaptation of the TaqMan PCR assay which potentially allows for highly sensitive detection of any eubacterial species with simultaneous species identification. Our system relies on a unique multiprobe design in which a single set of highly conserved sequences encoded by the 16S rRNA gene serves as the primer pair and is used in combination with both an internal highly conserved sequence, the universal probe, and an internal variable region, the species-specific probe. A pre-PCR ultrafiltration step effectively decontaminates or removes background DNA. The TaqMan system described reliabAly detected 14 common bacterial species with a detection limit of 50 fg. Further, highly sensitive and specific pathogen detection was demonstrated with a prototype species-specific probe designed to detect Staphylococcus aureus. This assay has broad potential in the clinical arena for rapid and specific diagnosis of infectious diseases.
View details for DOI 10.1128/JCM.40.9.3449-3454.2002
View details for Web of Science ID 000177829900053
View details for PubMedID 12202592
LACZ EXPRESSION IN GERMLINE TRANSGENIC ZEBRAFISH CAN BE DETECTED IN LIVING EMBRYOS
1994; 161 (1): 77-83
Use of transgenic technology in zebrafish has been limited by the inability to efficiently express transgenes in early embryos of F1 and subsequent generations and to rapidly detect transgenic fish. We generated transgenic fish by injecting fertilized eggs with the Escherichia coli lacZ gene under the control of the Xenopus elongation factor 1 alpha transcriptional regulatory element. Four of five lines of transgenic fish we obtained express the lacZ gene in early embryos. The pattern of expression was distinct for each line, with two lines showing extensive expression beginning at approximately the midblastula transition, one showing patchy expression and one showing expression almost exclusively in motor neurons. Expression patterns were stable through the F2 generation in the three lines studied to date. The availability of these lines facilitated the development of a reliable and rapid method for live-staining lacZ-expressing embryos using the substrate fluorescein-di-beta-D-galactopyranoside (FDG). Positive embryos of the two most highly lacZ-expressing lines could be identified after 2-3 min of staining in FDG and then picked out and raised. These observations should prove useful for a variety of studies in zebrafish.
View details for Web of Science ID A1994MQ97400009
View details for PubMedID 8293887