Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Hibernacula Microclimate and White-nose Syndrome Susceptibility in the Little Brown Myotis (Myotis lucifugus)

The objective of this project was to determine the relationship between hibernacula microclimate and White-nose Syndrome (WNS), an emerging infectious disease in bats. Microclimate was examined on a species scale and at the level of the individual bat to determine if there was a difference in microclimate preference between healthy and WNS-affected little brown myotis (Myotis lucifugus) and to determine the role of microclimate in disease progression. There is anecdotal evidence that colder, drier hibernacula are less affected by WNS. This was tested by placing rugged temperature and humidity dataloggers in field sites throughout the eastern USA, experimentally determining the response to microclimate differences in captive bats, and testing microclimate roosting preference. This study found that microclimate significantly differed from the entrance of a hibernaculum versus where bats traditionally roost. It also found hibernaculum temperature and sex had significant impacts on survival in WNS-affected bats. Male bats with WNS had increased survivability over WNS-affected female bats and WNS bats housed below the ideal growth range of the fungus that causes WNS, Geomyces destructans, had increased survival over those housed at warmer temperatures. The results from this study are immediately applicable to (1) predict which hibernacula are more likely to be infected next winter, (2) further our understanding of WNS, and (3) determine if direct mitigation strategies, such as altering the microclimate of mines, will be effective ways to combat the spread of the fungus.

FPGA BASED DESIGN FOR ACCELERATED FAULT-TESTING OF INTEGRATED CIRCUITS

In the past few decades, integrated circuits have become a major part of everyday life. Every circuit that is created needs to be tested for faults so faulty circuits are not sent to end-users. The creation of these tests is time consuming, costly and difficult to perform on larger circuits. This research presents a novel method for fault detection and test pattern reduction in integrated circuitry under test. By leveraging the FPGA's reconfigurability and parallel processing capabilities, a speed up in fault detection can be achieved over previous computer simulation techniques. This work presents the following contributions to the field of Stuck-At-Fault detection: We present a new method for inserting faults into a circuit net list. Given any circuit netlist, our tool can insert multiplexers into a circuit at correct internal nodes to aid in fault emulation on reconfigurable hardware. We present a parallel method of fault emulation. The benefit of the FPGA is not only its ability to implement any circuit, but its ability to process data in parallel. This research utilizes this to create a more efficient emulation method that implements numerous copies of the same circuit in the FPGA. A new method to organize the most efficient faults. Most methods for determinin the minimum number of inputs to cover the most faults require sophisticated softwareprograms that use heuristics. By utilizing hardware, this research is able to process data faster and use a simpler method for an efficient way of minimizing inputs.

Molecular investigation of lymph nodes in colon cancer patients using one-step nucleic acid amplification (OSNA): a new road to better staging?

A new diagnostic system, called one-step nucleic acid amplification (OSNA), has recently been designed to detect cytokeratin 19 mRNA as a surrogate for lymph node metastases. The objective of this prospective investigation was to compare the performance of OSNA with both standard hematoxylin and eosin (H&E) analysis and intensive histopathology in the detection of colon cancer lymph node metastases.

Technetium-99m-HDP uptake characteristics in equine fractures: a retrospective study

Bone scintigraphy is a very sensitive diagnostic tool to detect elevated bone metabolism. In cases of fractures and fissure fractures, the radiopharmaceutical uptake in the bone is said to be increased within a few hours after the injury. In this retrospective study, the scintigraphic uptake characteristics at the fracture site of 36 horses with radiographically confirmed fractures or fissure fractures were evaluated. Uptake ratios between the fracture region and adjacent normal bone or soft tissue activity respectively were calculated and compared to different anamnestic and radiographic data. The overall sensitivity of bone scintigraphy was 94.4% (34 positive cases out of 36). In the 36 horses, no correlation between the age of the fracture and the radiopharmaceutical uptake was found. However, there seems to be a lack of sensitivity in early detection of equine pelvic fractures when a standing bone scintigraphy examination protocol is used.

Distribution of RTX toxin genes in strains of [Actinobacillus] rossii and [Pasteurella] mairii

Strains of [Actinobacillus] rossii, [Pasteurella] mairii and [Pasteurella] aerogenes can be isolated from abortion in swine. The RTX toxin Pax has previously been found only in those [P.] aerogenes strains isolated from abortion. Nothing is known about RTX toxins in field isolates of the other two species. To gain insight into the distribution of selected RTX toxin genes and their association with abortion, PCR screening for the pax, apxII and apxIII operons on 21 [A.] rossii and seven [P.] mairii isolates was done. Since species can be phenotypically misidentified, the study was backed up by a phylogenetic analysis of all strains based on 16S rRNA, rpoB and infB genes. The pax gene was detected in all [P.] mairii but not in [A.] rossii strains. No apx genes were found in [P.] mairii but different gene combinations for apx were detected in [A.] rossii strains. Most of these strains were positive for apxIII, either alone or in combination with apxII. Whereas pax was found to be associated to strains from abortion no such indication could be found with apx in [A.] rossii strains. Phylogenetically [A.] rossii strains formed a heterogeneous cluster separated from Actinobacillus sensu stricto. [P.] mairii strains clustered with [P.] aerogenes but forming a separate branch. The fact that [P.] aerogenes, [P.] mairii and [A.] rossii can phylogenetically clearly be identified and might contain distinct RTX toxin genes allows their proper diagnosis and will further help to investigate their role as pathogens.

Antibiotic susceptibility patterns and resistance genes of starter cultures and probiotic bacteria used in food

A survey of starter and probiotic cultures was carried out to determine the current antibiotic resistance situation in microbial food additives in Switzerland. Two hundred isolates from 90 different sources were typed by molecular and other methods to belong to the genera Lactobacillus (74 samples), Staphylococcus (33 samples), Bifidobacterium (6 samples), Pediococcus (5 samples), or were categorized as lactococci or streptococci (82 samples). They were screened for phenotypic resistances to 20 antibiotics by the disk diffusion method. Twenty-seven isolates exhibiting resistances that are not an intrinsic feature of the respective genera were further analyzed by microarray hybridization as a tool to trace back phenotypic resistances to specific genetic determinants. Their presence was finally verified by PCR amplification or Southern hybridization. These studies resulted in the detection of the tetracycline resistance gene tet(K) in 5 Staphylococcus isolates used as meat starter cultures, the tetracycline resistance gene tet(W) in the probiotic cultures Bifidobacterium lactis DSM 10140 and Lactobacillus reuteri SD 2112 (residing on a plasmid), and the lincosamide resistance gene lnu(A) (formerly linA) in L. reuteri SD 2112.

Screening and testing for endocrine disruption in fish-biomarkers as "signposts," not "traffic lights," in risk assessment

Biomarkers are currently best used as mechanistic "signposts" rather than as "traffic lights" in the environmental risk assessment of endocrine-disrupting chemicals (EDCs). In field studies, biomarkers of exposure [e.g., vitellogenin (VTG) induction in male fish] are powerful tools for tracking single substances and mixtures of concern. Biomarkers also provide linkage between field and laboratory data, thereby playing an important role in directing the need for and design of fish chronic tests for EDCs. It is the adverse effect end points (e.g., altered development, growth, and/or reproduction) from such tests that are most valuable for calculating adverseNOEC (no observed effect concentration) or adverseEC10 (effective concentration for a 10% response) and subsequently deriving predicted no effect concentrations (PNECs). With current uncertainties, biomarkerNOEC or biomarkerEC10 data should not be used in isolation to derive PNECs. In the future, however, there may be scope to increasingly use biomarker data in environmental decision making, if plausible linkages can be made across levels of organization such that adverse outcomes might be envisaged relative to biomarker responses. For biomarkers to fulfil their potential, they should be mechanistically relevant and reproducible (as measured by interlaboratory comparisons of the same protocol). VTG is a good example of such a biomarker in that it provides an insight to the mode of action (estrogenicity) that is vital to fish reproductive health. Interlaboratory reproducibility data for VTG are also encouraging; recent comparisons (using the same immunoassay protocol) have provided coefficients of variation (CVs) of 38-55% (comparable to published CVs of 19-58% for fish survival and growth end points used in regulatory test guidelines). While concern over environmental xenoestrogens has led to the evaluation of reproductive biomarkers in fish, it must be remembered that many substances act via diverse mechanisms of action such that the environmental risk assessment for EDCs is a broad and complex issue. Also, biomarkers such as secondary sexual characteristics, gonadosomatic indices, plasma steroids, and gonadal histology have significant potential for guiding interspecies assessments of EDCs and designing fish chronic tests. To strengthen the utility of EDC biomarkers in fish, we need to establish a historical control database (also considering natural variability) to help differentiate between statistically detectable versus biologically significant responses. In conclusion, as research continues to develop a range of useful EDC biomarkers, environmental decision-making needs to move forward, and it is proposed that the "biomarkers as signposts" approach is a pragmatic way forward in the current risk assessment of EDCs.

Induction of haem oxygenase-1 causes cortical non-haem iron increase in experimental pneumococcal meningitis: evidence that concomitant ferritin up-regulation prevents iron-induced oxidative damage

Desferrioxamine inhibits cortical necrosis in neonatal rats with experimental pneumococcal meningitis, suggesting that iron-induced oxidative damage might be responsible for neuronal damage. We therefore examined the spatial and temporal profile of changes in cortical iron and iron homeostatic proteins during pneumococcal meningitis. Infection was associated with a steady and global increase of non-haem iron in the cortex, particularly in neuronal cell bodies of layer II and V, and in capillary endothelial cells. The non-haem iron increase was associated with induction of haem oxygenase (HO)-1 in neurones, microglia and capillary endothelial cells, whereas HO-2 levels remained unchanged, suggesting that the non-haem iron increase might be the result of HO-1-mediated haem degradation. Indeed, treatment with the haem oxygenase inhibitor tin protoporphyrin (which completely blocked the accumulation of bilirubin detected in HO-1-positive cells) completely prevented the infection-associated non-haem iron increase. The same cells also displayed markedly increased ferritin staining, the increase of which occurred independently of HO activity. At the same time, no increase in DNA/RNA oxidation was observed in infected animals (as assessed by in situ detection of 8-hydroxy[deoxy]guanosine), strongly suggesting that ferritin up-regulation protected the brain from iron-induced oxidative damage. Thus, although pneumococcal meningitis leads to an increase of cortical non-haem iron, protective mechanisms up-regulated in parallel prevent iron-induced oxidative damage. Cortical damage does not appear to be a direct consequence of increased iron, therefore.

Virtopsy hemorrhage of the posterior cricoarytenoid muscle by blunt force to the neck in postmortem multislice computed tomography and magnetic resonance imaging

In forensic autopsies, one of the most important and common signs of violence to the neck is hemorrhages of the soft tissues. The Institute of Forensic Medicine in Bern evaluates the usefulness of postmortem multislice computed tomography (MSCT) and magnetic resonance imaging (MRI) of forensic cases prior to autopsy. The aim of this study was to prove the sensitivity of postmortem MSCT and MRI in the detection of hemorrhages of the neck muscles. A full body scan prior to and a detailed scan of the explanted larynx after autopsy were performed. MSCT detected multiple fractures of the larynx. Detailed MRI was able to demonstrate the hemorrhage of the left posterior cricoarytenoid muscle. The minor hemorrhage of the right posterior cricoarytenoid muscle could not be detected with certainty. Although more experience is required, we conclude that combined MRI and MSCT examination is a useful tool for documentation and examination of neck muscle hemorrhages in forensic cases.

Factors Influencing Spatial Variability in Nitrogen Processing in Nitrogen-Saturated Soils

Nitrogen (N) saturation is an environmental concern for forests in the eastern U.S. Although several watersheds of the Fernow Experimental Forest (FEF), West Virginia exhibit symptoms of Nsaturation, many watersheds display a high degree of spatial variability in soil N processing. This study examined the effects of temperature on net N mineralization and nitrification in N-saturatedsoils from FEF, and how these effects varied between high N-processing vs. low N-processingsoils collected from two watersheds, WS3 (fertilized with [NH4]2SO4) and WS4 (untreated control). Samples of forest floor material (O2 horizon) and mineral soil (to a 5-cm depth) were taken from three subplots within each of four plots that represented the extremes of highest and lowest ratesof net N mineralization and nitrification (hereafter, high N and low N, respectively) of untreated WS4 and N-treated WS3: control/low N, control/high N, N-treated/low N, N-treated/high N. Forest floor material was analyzed for carbon (C), lignin,and N. Subsamples of mineral soil were extractedimmediately with 1 N KCl and analyzed for NH4+and NO3– to determine preincubation levels. Extracts were also analyzed for Mg, Ca, Al, and pH. To test the hypothesis that the lack of net nitrification observed in field incubations on the untreated/low N plot was the result of absence ofnitrifier populations, we characterized the bacterial community involved in N cycling by amplification of amoA genes. Remaining soil was incubated for 28 d at three temperatures (10, 20, and30°C), followed by 1 N KCl extraction and analysis for NH4+ and NO3–. Net nitrification was essentially 100% of net N mineralization for all samples combined. Nitrification rates from lab incubation sat all temperatures supported earlier observations based on field incubations. At 30°C, rates from N- t reated/high N were three times those of N-treated/low N. Highest rates were found for untreated/high N (two times greater than those of N-treated/high N), whereas untreated/low N exhibited no net nitrification. However, soils exhibitingno net nitrification tested positive for presence of nitrifying bacteria, causing us to reject our initial hypothesis. We hypothesize that nitrifier populations in such soil are being inhibited by a combination of low Ca:Al ratios in mineral soil and allelopathic interactions with mycorrhizae of ericaceous species in the herbaceous layer.

Detection of hepatic portal venous gas: its clinical impact and outcome

The clinical impact and outcome of a rare radiographic finding of hepatic portal venous gas (HPVG) as well as the effectiveness of computed tomography (CT), CT scanogram, and conventional radiography in the detection of HPVG were retrospectively analyzed. CT scans, CT scanogram, and plain film radiographs of 11 patients with HPVG were reviewed and compared with their medical records and surgical and pathology reports. Eight of the 11 patients underwent plain film radiographs 1 day before or after the CT scan. HPVG was detected at CT in all 11 patients, on CT scanogram in three (3 of 11, 27.3%), and on plain films in one (one of eight, 12.5%). In nine of 11 patients (81.8%), CT revealed an associated pneumatosis intestinalis. In six of the 11 patients (54.6%), acute mesenteric ischemia was the underlying disease for HPVG. Seven patients (63.6%) underwent emergency exploratory laparotomy. The mortality rate for HPVG alone was 27.3% (3 of 11) and for HPVG related to mesenteric bowel disease 50% (three of six). Acute mesenteric ischemia is the most common cause of HPVG, which continues to have a predictably higher mortality. CT is superior to CT scanograms and radiographs in the detection of HPVG and its underlying diseases and, therefore, should be used as the primary diagnostic tool.

Accuracy of in vivo coronary plaque morphology assessment: a validation study of in vivo virtual histology compared with in vitro histopathology

OBJECTIVES: The goal of the present study was to compare the accuracy of in vivo tissue characterization obtained by intravascular ultrasound (IVUS) radiofrequency (RF) data analysis, known as Virtual Histology (VH), to the in vitro histopathology of coronary atherosclerotic plaques obtained by directional coronary atherectomy. BACKGROUND: Vulnerable plaque leading to acute coronary syndrome (ACS) has been associated with specific plaque composition, and its characterization is an important clinical focus. METHODS: Virtual histology IVUS images were performed before and after a single debulking cut using directional coronary atherectomy. Debulking region of in vivo histology image was predicted by comparing pre- and post-debulking VH images. Analysis of VH images with the corresponding tissue cross section was performed. RESULTS: Fifteen stable angina pectoris (AP) and 15 ACS patients were enrolled. The results of IVUS RF data analysis correlated well with histopathologic examination (predictive accuracy from all patients data: 87.1% for fibrous, 87.1% for fibro-fatty, 88.3% for necrotic core, and 96.5% for dense calcium regions, respectively). In addition, the frequency of necrotic core was significantly higher in the ACS group than in the stable AP group (in vitro histopathology: 22.6% vs. 12.6%, p = 0.02; in vivo virtual histology: 24.5% vs. 10.4%, p = 0.002). CONCLUSIONS: Correlation of in vivo IVUS RF data analysis with histopathology shows a high accuracy. In vivo IVUS RF data analysis is a useful modality for the classification of different types of coronary components, and may play an important role in the detection of vulnerable plaque.

Active caspase 3 and DNA fragmentation as markers for apoptotic cell death in primary and metastatic liver tumours

AIMS: The induction of tumour cell death by apoptosis is a major goal of cancer therapy and the in situ detection of apoptosis in tumour tissue has become an important diagnostic parameter. Different apoptosis detection methods assess distinct biochemical processes in the dying cell. Thus, their direct comparison is mandatory to evaluate their diagnostic value. The aim of this study was to compare the immunohistochemical detection of active caspase 3 and single-stranded DNA in primary and metastatic liver tumours as markers of apoptotic cell death. METHODS: We studied detection of active caspase 3 and single-stranded DNA in 20 primary hepatocellular carcinomas (HCC) and 20 liver metastases from colorectal carcinomas (CRC) using immunohistochemistry on paraffin sections. RESULTS: Our results reveal that both methods are suitable and sensitive techniques for the in situ detection of apoptosis, however, they also demonstrate that immunohistochemistry for active caspase 3 and single-stranded DNA have differential sensitivities in HCC and CRC. CONCLUSION: The sensitivity of apoptosis detection using immunohistochemistry for active caspase 3 and single-stranded DNA may be tumour cell type dependent.

Cadaver dogs--a study on detection of contaminated carpet squares

INTRODUCTION: Cadaver dogs are known as valuable forensic tools in crime scene investigations. Scientific research attempting to verify their value is largely lacking, specifically for scents associated with the early postmortem interval. The aim of our investigation was the comparative evaluation of the reliability, accuracy, and specificity of three cadaver dogs belonging to the Hamburg State Police in the detection of scents during the early postmortem interval. MATERIAL AND METHODS: Carpet squares were used as an odor transporting media after they had been contaminated with the scent of two recently deceased bodies (PMI<3h). The contamination occurred for 2 min as well as 10 min without any direct contact between the carpet and the corpse. Comparative searches by the dogs were performed over a time period of 65 days (10 min contamination) and 35 days (2 min contamination). RESULTS: The results of this study indicate that the well-trained cadaver dog is an outstanding tool for crime scene investigation displaying excellent sensitivity (75-100), specificity (91-100), and having a positive predictive value (90-100), negative predictive value (90-100) as well as accuracy (92-100).