Proof of concept study of a novel pacemapping algorithm as a basis to guide ablation of ventricular arrhythmias.

Aims: To determine if a software algorithm can use an individualized distance-morphology difference model, built from three initial pacemaps, to prospectively locate the exit site (ES) of ventricular arrhythmias (VA). Methods and results: Consecutive patients undergoing ablation of VA from a single centre were recruited. During mapping, three initial pacing points were collected in the chamber of interest and the navigation algorithm applied to predict the ES, which was corroborated by conventional mapping techniques. Thirty-two patients underwent ES prediction over 35 procedures. Structural heart disease was present in 16 (7 ischaemic cardiomyopathy, 9 non-ischaemic cardiomyopathy), median ejection fraction 45% [Interquartile range (IQR) 26]. The remainder had normal hearts. The navigation algorithm was applied to 46 VA (24 left ventricle, 11 right ventricular outflow tract, 5 left ventricular outflow tract, 4 right ventricle, 2 epicardial) and successfully located the site of best pacemap match in 45 within a median area of 196.5 mm2 (IQR 161.3, range 46.6-1288.2 mm2). Conclusions: In a diverse population of patients with and without structural heart disease, the ES of VA can be accurately and reliably identified to within a clinically useful target area using a simple software navigation algorithm based on pacemapping.

Relationship Between Distance and Change in Surface ECG Morphology During Pacemapping as a Guide to Ablation of Ventricular Arrhythmias: Implications for the Spatial Resolution of Pacemapping.

BACKGROUND: Pacemapping is used to localize the exit site of ventricular arrhythmia. Although the relationship between distance and change in QRS morphology is its basis, this relationship has not been systematically quantified. METHODS AND RESULTS: Patients (n=68) undergoing ventricular arrhythmia ablation between March 2012 and July 2013 were recruited. Pacemapping was targeted to areas of voltage >0.5 mV. Linear mixed-effects models were constructed of distance against morphology difference measured by the root mean square error sum across all 12 ECG leads (E12). Forty of 68 (58%) patients had structural heart disease, and 21/40 (53%) patients were ischemic. Nine hundred thirty-five pacing points were collected, generating 6219 pacing site pair combinations (3087 [50%] ventricular bodies, 756 [12%] outflow tract, and 162 [3%] epicardial). In multivariable analysis, increase in E12 was predicted by increasing distance (0.07 per mm; 95% confidence interval 0.07-0.08; P<0.001). Compared with the left ventricle, E12 values were lower in the right ventricle (P=0.037) and left ventricular outflow tract (P<0.001) and higher in left ventricle-right ventricle pairs (P=0.021) and left ventricular epicardium (P=0.08). There was no difference in E12 in the right ventricular outflow tract compared with the right-left ventricular outflow tract (P=0.75) pairs. Structural heart disease or inadvertent pacing in scar was not associated with changes in E12; however, the presence of latency and split potentials were associated with higher and lower E12 values, respectively (P<0.001). CONCLUSIONS: A robust positive relationship exists between distance and QRS morphological change when restricting pacing points to areas of voltage >0.5 mV. Significant differences in the spatial resolution of pacemapping exist within the heart.

Nonpharmacologic approach to minimizing shivering during surface cooling: a proof of principle study.

PURPOSE: This study had 2 objectives: (1) to quantify the metabolic response to physical cooling in febrile patients with systemic inflammatory response syndrome (SIRS) and (2) to provide proof for the hypothesis that the efficiency of external cooling and the subsequent shivering response are influenced by site and temperature of surface cooling pads. METHODS: To quantify shivering thermogenesis during surface cooling for fever, we monitored oxygen consumption (VO(2)) in 6 febrile patients with SIRS during conventional cooling with cooling blankets and ice packs. To begin to determine how location and temperature of surface cooling influence shivering, we compared 5 cooling protocols for inducing mild hypothermia in 6 healthy volunteers. RESULTS: In the patients with SIRS, core temperature decreased 0.67 °C per hour, all patients shivered, VO(2) increased 57.6%, and blood pressure increased 15% during cooling. In healthy subjects, cooling with the 10 °C vest was most comfortable and removed heat most efficiently without shivering or VO(2) increase. Cooling with combined vest and thigh pads stimulated the most shivering and highest VO(2) and increased core temperature. Reducing vest temperature from 10 °C to 5 °C failed to increase heat removal secondary to cutaneous vasoconstriction. Capsaicin, an agonist for the transient receptor potential cation channel subfamily V member 1 (TRPV1) warm-sensing channels, partially reversed this effect in 5 subjects. CONCLUSIONS: Our results identify the hazards of surface cooling in febrile critically ill patients and support the concept that optimization of cooling pad temperature and position may improve cooling efficiency and reduce shivering.

Oligonucleotide microarrays for identification of microbial pathogens and detection of their virulence-associated or drug-resistance determinants.

Microarrays are spatially ordered arrays with ligands chemically immobilized in discrete spots on a solid matrix, usually a microscope slide. Microarrays are a high-throughput large-scale screening system enabling simultaneous identification of a large number of labeled target molecules (up to several hundred thousand) that bind specifically to the immobilized ligands of the array. DNA microarrays represent a promising tool for clinical, environmental, and industrial microbiology since the technology allows relatively rapid identification of large number of genetic determinants simultaneously, providing detailed genomic level information regarding the pathogen species, including identification of their virulence-associated factors and the presence of antibiotic resistance genes. In this chapter, we describe key aspects and methodologies important for the development and use of DNA microarrays for microbial diagnostics.

A simple portable electroluminescence illumination-based CCD detector.

In this chapter we describe a simple and relatively inexpensive Electroluminescence (EL) illumination and charged-coupled device (CCD) camera (EL-CCD) based detector for monitoring fluorescence and colorimetric assays. The portable battery-operated fluorescence detector includes an EL panel for fluoro-genic excitation at 490 nm, a cooled CCD digital camera to monitor emission at 523 nm, filters and a close up lens. The detector system is controlled by a laptop computer for camera operation, image acquisition and analysis. The system was tested using a fluorogenic peptide substrate (SNAP-25) for botulinum neurotoxin serotype A (BoNT-A) labeled with FITC. The level of detection of the system was found to be 1.25 nM of the peptide, similar to the detection level of a commercial photomultipler-based plate fluorometer. The multichannel EL-CCD was used with an assay plate capable of testing nine samples simultaneously in 1 min at this detection level. The portable system is small and is operated by a 12 V source. The modular detector was designed with easily interchangeable ELs, filters and lenses and can be used and adapted for a wide variety of fluorescence and colorimetric assays, fluorescence labels and assay formats.

Rapid DNA amplification using a battery-powered thin-film resistive thermocycler.

A prototype handheld, compact, rapid thermocycler was developed for multiplex analysis of nucleic acids in an inexpensive, portable configuration. Instead of the commonly used Peltier heating/cooling element, electric thin-film resistive heater and a miniature fan enable rapid heating and cooling of glass capillaries leading to a simple, low-cost Thin-Film Resistive Thermocycler (TFRT). Computer-based pulse width modulation control yields heating rates of 6-7 K/s and cooling rates of 5 K/s. The four capillaries are closely coupled to the heater, resulting in low power consumption. The energy required by a nominal PCR cycle (20 s at each temperature) was found to be 57+/-2 J yielding an average power of approximately 1.0 W (not including the computer and the control system). Thus the device can be powered by a standard 9 V alkaline battery (or other 9 V power supply). The prototype TFRT was demonstrated (in a benchtop configuration) for detection of three important food pathogens (E. coli ETEC, Shigella dysenteriae, and Salmonella enterica). PCR amplicons were analyzed by gel electrophoresis. The 35 cycle PCR protocol using a single channel was completed in less then 18 min. Simple and efficient heating/cooling, low cost, rapid amplification, and low power consumption make the device suitable for portable DNA amplification applications including clinical point of care diagnostics and field use.

Food microbial pathogen detection and analysis using DNA microarray technologies.

Culture-based methods used for microbial detection and identification are simple to use, relatively inexpensive, and sensitive. However, culture-based methods are too time-consuming for high-throughput testing and too tedious for analysis of samples with multiple organisms and provide little clinical information regarding the pathogen (e.g., antibiotic resistance genes, virulence factors, or strain subtype). DNA-based methods, such as polymerase chain reaction (PCR), overcome some these limitations since they are generally faster and can provide more information than culture-based methods. One limitation of traditional PCR-based methods is that they are normally limited to the analysis of a single pathogen, a small group of related pathogens, or a small number of relevant genes. Microarray technology enables a significant expansion of the capability of DNA-based methods in terms of the number of DNA sequences that can be analyzed simultaneously, enabling molecular identification and characterization of multiple pathogens and many genes in a single array assay. Microarray analysis of microbial pathogens has potential uses in research, food safety, medical, agricultural, regulatory, public health, and industrial settings. In this article, we describe the main technical elements of microarray technology and the application and potential use of DNA microarrays for food microbial analysis.

Biosensors for the analysis of food- and waterborne pathogens and their toxins.

Biosensors are devices which combine a biochemical recognition element with a physical transducer. There are various types of biosensors, including electrochemical, acoustical, and optical sensors. Biosensors are used for medical applications and for environmental testing. Although biosensors are not commonly used for food microbial analysis, they have great potential for the detection of microbial pathogens and their toxins in food. They enable fast or real-time detection, portability, and multipathogen detection for both field and laboratory analysis. Several applications have been developed for microbial analysis of food pathogens, including E. coli O157:H7, Staphylococcus aureus, Salmonella, and Listeria monocytogenes, as well as various microbial toxins such as staphylococcal enterotoxins and mycotoxins. Biosensors have several potential advantages over other methods of analysis, including sensitivity in the range of ng/mL for microbial toxins and <100 colony-forming units/mL for bacteria. Fast or real-time detection can provide almost immediate interactive information about the sample tested, enabling users to take corrective measures before consumption or further contamination can occur. Miniaturization of biosensors enables biosensor integration into various food production equipment and machinery. Potential uses of biosensors for food microbiology include online process microbial monitoring to provide real-time information in food production and analysis of microbial pathogens and their toxins in finished food. Biosensors can also be integrated into Hazard Analysis and Critical Control Point programs, enabling critical microbial analysis of the entire food manufacturing process. In this review, the main biosensor approaches, technologies, instrumentation, and applications for food microbial analysis are described.

Microarray analysis of Bacillus cereus group virulence factors.

Bacillus cereus, B. thuringiensis and B. anthracis are closely related medically and economically important bacterial species that belong to the B. cereus group. Members of the B. cereus group carry genes encoding several important virulence factors, including enterotoxins, phospholipases and exotoxins. Since it is difficult to differentiate among B. cereus group members, and because Bacillus virulence factors are very important for pathogenesis, we explored the use of microarray-based detection of virulence factor genes as a tool for strain identification and for determining virulence. Our method requires an initial multiplex PCR amplification step, followed by identification of the PCR amplicons by hybridization to an oligonucleotide microarray containing genes for all three types of Bacillus virulence factors including B. anthracis virulence factors. The DNA chip described here contains 21 identical arrays used for analysis of seven samples in triplicates. Using the arrays, we found that virulence factors are present in several combinations in the strains analyzed. This work also demonstrates the potential of oligonucleotide microarrays for medical, food safety and biodefense analysis of microbial pathogens.

Oligo Design: a computer program for development of probes for oligonucleotide microarrays.

Oligonucleotide microarrays have demonstrated potential for the analysis of gene expression, genotyping, and mutational analysis. Our work focuses primarily on the detection and identification of bacteria based on known short sequences of DNA. Oligo Design, the software described here, automates several design aspects that enable the improved selection of oligonucleotides for use with microarrays for these applications. Two major features of the program are: (i) a tiling algorithm for the design of short overlapping temperature-matched oligonucleotides of variable length, which are useful for the analysis of single nucleotide polymorphisms and (ii) a set of tools for the analysis of multiple alignments of gene families and related short DNA sequences, which allow for the identification of conserved DNA sequences for PCR primer selection and variable DNA sequences for the selection of unique probes for identification. Note that the program does not address the full genome perspective but, instead, is focused on the genetic analysis of short segments of DNA. The program is Internet-enabled and includes a built-in browser and the automated ability to download sequences from GenBank by specifying the GI number. The program also includes several utilities, including audio recital of a DNA sequence (useful for verifying sequences against a written document), a random sequence generator that provides insight into the relationship between melting temperature and GC content, and a PCR calculator.