The identification of two Trypanosoma cruzi I genotypes from domestic and sylvatic transmission cycles in Colombia based on a single polymerase chain reaction amplification of the spliced-leader intergenic region

A single polymerase chain reaction (PCR) reaction targeting the spliced-leader intergenic region of Trypanosoma cruzi I was standardised by amplifying a 231 bp fragment in domestic (TcIDOM) strains or clones and 450 and 550 bp fragments in sylvatic strains or clones. This reaction was validated using 44 blind coded samples and 184 non-coded T. cruzi I clones isolated from sylvatic triatomines and the correspondence between the amplified fragments and their domestic or sylvatic origin was determined. Six of the nine strains isolated from acute cases suspected of oral infection had the sylvatic T. cruzi I profile. These results confirmed that the sylvatic T. cruzi I genotype is linked to cases of oral Chagas disease in Colombia. We therefore propose the use of this novel PCR reaction in strains or clones previously characterised as T. cruziI to distinguish TcIDOMfrom sylvatic genotypes in studies of transmission dynamics, including the verification of population selection within hosts or detection of the frequency of mixed infections by both T. cruzi I genotypes in Colombia.

The implementation of liquid chromatography tandem mass spectrometry for the official control of lipophilic toxins in seafood: Single-laboratory validation under four chromatographic conditions

We performed a comprehensive study to assess the fit for purpose of four chromatographic conditions for the determination of six groups of marine lipophilic toxins (okadaic acid and dinophysistoxins, pectenotoxins, azaspiracids, yessotoxins, gymnodimine and spirolides) by LC-MS/MS to select the most suitable conditions as stated by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). For every case, the elution gradient has been optimized to achieve a total run-time cycle of 12 min. We performed a single-laboratory validation for the analysis of three relevant matrices for the seafood aquaculture industry (mussels, pacific oysters and clams), and for sea urchins for which no data about lipophilic toxins have been reported before. Moreover, we have compared the method performance under alkaline conditions using two quantification strategies: the external standard calibration (EXS) and the matrix-matched standard calibration (MMS). Alkaline conditions were the only scenario that allowed detection windows with polarity switching in a 3200 QTrap mass spectrometer, thus the analysis of all toxins can be accomplished in a single run, increasing sample throughput. The limits of quantification under alkaline conditions met the validation requirements established by the EURLMB for all toxins and matrices, while the remaining conditions failed in some cases. The accuracy of the method and the matrix effects where generally dependent on the mobile phases and the seafood species. The MMS had a moderate positive impact on method accuracy for crude extracts, but it showed poor trueness for seafood species other than mussels when analyzing hydrolyzed extracts. Alkaline conditions with EXS and recovery correction for OA were selected as the most proper conditions in the context of our laboratory. This comparative study can help other laboratories to choose the best conditions for the implementation of LC-MS/MS according to their own necessities.

Effects of long-term averaging of quantitative blood pressure traits on the detection of genetic associations.

Blood pressure (BP) is a heritable, quantitative trait with intraindividual variability and susceptibility to measurement error. Genetic studies of BP generally use single-visit measurements and thus cannot remove variability occurring over months or years. We leveraged the idea that averaging BP measured across time would improve phenotypic accuracy and thereby increase statistical power to detect genetic associations. We studied systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP), and pulse pressure (PP) averaged over multiple years in 46,629 individuals of European ancestry. We identified 39 trait-variant associations across 19 independent loci (p < 5 × 10(-8)); five associations (in four loci) uniquely identified by our LTA analyses included those of SBP and MAP at 2p23 (rs1275988, near KCNK3), DBP at 2q11.2 (rs7599598, in FER1L5), and PP at 6p21 (rs10948071, near CRIP3) and 7p13 (rs2949837, near IGFBP3). Replication analyses conducted in cohorts with single-visit BP data showed positive replication of associations and a nominal association (p < 0.05). We estimated a 20% gain in statistical power with long-term average (LTA) as compared to single-visit BP association studies. Using LTA analysis, we identified genetic loci influencing BP. LTA might be one way of increasing the power of genetic associations for continuous traits in extant samples for other phenotypes that are measured serially over time.

Bioreporters: gfp versus lux revisited and single-cell response.

Genetically engineered organisms expressing spectroscopically active reporter molecules in response to chemical effectors display great potential as living transducers in sensing applications. Green fluorescent protein (gfp gene) bioreporters have distinct advantages over luminescent couterparts (lux gene), including applicability at the single-cell level, but are typically less sensitive. Here we describe a gfp-bearing bioreporter that is sensitive to naphthalene (a poorly water soluble pollutant behaving like a large class of hydrophobic compounds), is suitable for use in chemical assays and bioavailability studies, and has detection limits comparable to lux-bearing bioreporters for higher efficiency detection strategies. Simultaneously, we find that the exploitation of population response data from single-cell analysis is not an algorithmic conduit to enhanced signal detection and hence lower effector detection limits, as normally assumed. The assay reported functions to equal effect with or without biocide.

Development of In-Situ Detection Methods for Materials-Related Distress (MRD) in Concrete Pavements, July 2003

The purpose of this research was to summarize existing nondestructive test methods that have the potential to be used to detect materials-related distress (MRD) in concrete pavements. The various nondestructive test methods were then subjected to selection criteria that helped to reduce the size of the list so that specific techniques could be investigated in more detail. The main test methods that were determined to be applicable to this study included two stress-wave propagation techniques (impact-echo and spectral analysis of surface waves techniques), infrared thermography, ground penetrating radar (GPR), and visual inspection. The GPR technique was selected for a preliminary round of “proof of concept” trials. GPR surveys were carried out over a variety of portland cement concrete pavements for this study using two different systems. One of the systems was a state-of-the-art GPR system that allowed data to be collected at highway speeds. The other system was a less sophisticated system that was commercially available. Surveys conducted with both sets of equipment have produced test results capable of identifying subsurface distress in two of the three sites that exhibited internal cracking due to MRD. Both systems failed to detect distress in a single pavement that exhibited extensive cracking. Both systems correctly indicated that the control pavement exhibited negligible evidence of distress. The initial positive results presented here indicate that a more thorough study (incorporating refinements to the system, data collection, and analysis) is needed. Improvements in the results will be dependent upon defining the optimum number and arrangement of GPR antennas to detect the most common problems in Iowa pavements. In addition, refining highfrequency antenna response characteristics will be a crucial step toward providing an optimum GPR system for detecting materialsrelated distress.

Varicella Zoster Virus CNS disease in hematopoietic cell transplantation: A single center experience

Background: Varciella Zoster Virus (VZV) can lead to serious complications in Hematopoietic Cell Transplant (HCT) recipients. Central nervous system (CNS) VZV can be one of the most devastating infections in transplant recipients, yet little is known about this rare disease. Objectives: To describe CNS VZV in the post-transplant period and to define potential risk factors in the HCT population. Methods: We reviewed the course of all patients who received a first HCT at the Fred Hutchinson Cancer Center (FHCRC) in Seattle, WA from 1/1996 through 12/2007. Data were collected retrospectively using the Long-Term Follow-Up database, which includes on-site examinations, outside records, laboratory tests, and yearly questionnaires. Patients were classified as CNS VZV if they had laboratory confirmation of VZV in the cerebrospinal fluid (CSF), or had zoster with associated clinical and laboratory findings consistent with CNS disease. Results: A total of six patients developed VZV CNS disease during the evaluation period (table 1). Diagnosis was confirmed in 3/6 by detection of VZV in CSF by PCR. All other patients had a clinical diagnosis based on the presence of CNS symptoms, zoster, lymphocytic pleiocytosis, and response to IV acyclovir. Patients who developed CNS disease had a mean age of 42 years (range 34-51) at time of transplant. CNS disease developed at a mean of 9 months posttransplantation (range 0.5-24 months), and severity varied, ranging from meningitis (3/6) to encephalitis/myelitis (3/6). All had active graft-versus host disease (GHVD) and all were being treated with immunosuppressive therapy at time of diagnosis. Fever and headache were the most common symptoms, but patients who developed focal CNS findings or seizures (3/6) had a more complicated clinical course. While most patients presented with classic VZV/zoster skin lesions, 2/6 patients had no dermatologic findings associated with their presentation. Four (66%) of patients who developed VZV CNS disease died, two related to VZV complications despite aggressive antiviral therapy. Conclusions: In this cohort of HCT patients, VZV CNS disease was a rare complication. Mortality due to CNS VZV is high, particularly in patients who develop focal neurologic findings or seizures. Even in the absence of skin lesions, VZV CNS disease should be considered in patients who develop fevers and neurologic symptoms.

Landmark detection for fusion of fundus and MRI toward a patient-specific multimodal eye model.

Ophthalmologists typically acquire different image modalities to diagnose eye pathologies. They comprise, e.g., Fundus photography, optical coherence tomography, computed tomography, and magnetic resonance imaging (MRI). Yet, these images are often complementary and do express the same pathologies in a different way. Some pathologies are only visible in a particular modality. Thus, it is beneficial for the ophthalmologist to have these modalities fused into a single patient-specific model. The goal of this paper is a fusion of Fundus photography with segmented MRI volumes. This adds information to MRI that was not visible before like vessels and the macula. This paper contributions include automatic detection of the optic disc, the fovea, the optic axis, and an automatic segmentation of the vitreous humor of the eye.

Single-metal deposition: optimization of this fingermark enhancement technique

Following the introduction of single-metal deposition (SMD), a simplified fingermark detection technique based on multimetal deposition, optimization studies were conducted. The different parameters of the original formula were tested and the results were evaluated based on the contrast and overall aspect of the enhanced fingermarks. The new formula for SMD was found based on the most optimized parameters. Interestingly, it was found that important variations from the base parameters did not significantly affect the outcome of the enhancement, thus demonstrating that SMD is a very robust technique. Finally, a comparison of the optimized SMD with multi-metal deposition (MMD) was carried out on different surfaces. It was demonstrated that SMD produces comparable results to MMD, thus validating the technique.

Reference Cluster Normalization Improves Detection of Frontotemporal Lobar Degeneration by Means of FDG-PET.

Positron emission tomography with [18F] fluorodeoxyglucose (FDG-PET) plays a well-established role in assisting early detection of frontotemporal lobar degeneration (FTLD). Here, we examined the impact of intensity normalization to different reference areas on accuracy of FDG-PET to discriminate between patients with mild FTLD and healthy elderly subjects. FDG-PET was conducted at two centers using different acquisition protocols: 41 FTLD patients and 42 controls were studied at center 1, 11 FTLD patients and 13 controls were studied at center 2. All PET images were intensity normalized to the cerebellum, primary sensorimotor cortex (SMC), cerebral global mean (CGM), and a reference cluster with most preserved FDG uptake in the aforementioned patients group of center 1. Metabolic deficits in the patient group at center 1 appeared 1.5, 3.6, and 4.6 times greater in spatial extent, when tracer uptake was normalized to the reference cluster rather than to the cerebellum, SMC, and CGM, respectively. Logistic regression analyses based on normalized values from FTLD-typical regions showed that at center 1, cerebellar, SMC, CGM, and cluster normalizations differentiated patients from controls with accuracies of 86%, 76%, 75% and 90%, respectively. A similar order of effects was found at center 2. Cluster normalization leads to a significant increase of statistical power in detecting early FTLD-associated metabolic deficits. The established FTLD-specific cluster can be used to improve detection of FTLD on a single case basis at independent centers - a decisive step towards early diagnosis and prediction of FTLD syndromes enabling specific therapies in the future.

Integrating detection probabilities in species distribution models of amphibians breeding in Mediterranean temporary ponds

Aim  The imperfect detection of species may lead to erroneous conclusions about species-environment relationships. Accuracy in species detection usually requires temporal replication at sampling sites, a time-consuming and costly monitoring scheme. Here, we applied a lower-cost alternative based on a double-sampling approach to incorporate the reliability of species detection into regression-based species distribution modelling.Location  Doñana National Park (south-western Spain).Methods  Using species-specific monthly detection probabilities, we estimated the detection reliability as the probability of having detected the species given the species-specific survey time. Such reliability estimates were used to account explicitly for data uncertainty by weighting each absence. We illustrated how this novel framework can be used to evaluate four competing hypotheses as to what constitutes primary environmental control of amphibian distribution: breeding habitat, aestivating habitat, spatial distribution of surrounding habitats and/or major ecosystems zonation. The study was conducted on six pond-breeding amphibian species during a 4-year period.Results  Non-detections should not be considered equivalent to real absences, as their reliability varied considerably. The occurrence of Hyla meridionalis and Triturus pygmaeus was related to a particular major ecosystem of the study area, where suitable habitat for these species seemed to be widely available. Characteristics of the breeding habitat (area and hydroperiod) were of high importance for the occurrence of Pelobates cultripes and Pleurodeles waltl. Terrestrial characteristics were the most important predictors of the occurrence of Discoglossus galganoi and Lissotriton boscai, along with spatial distribution of breeding habitats for the last species.Main conclusions  We did not find a single best supported hypothesis valid for all species, which stresses the importance of multiscale and multifactor approaches. More importantly, this study shows that estimating the reliability of non-detection records, an exercise that had been previously seen as a naïve goal in species distribution modelling, is feasible and could be promoted in future studies, at least in comparable systems.

Detection of plant-modulated alterations in antifungal gene expression in Pseudomonas fluorescens CHA0 on roots by flow cytometry.

The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.

Photodynamic Diagnosis of Non-muscle-invasive Bladder Cancer with Hexaminolevulinate Cystoscopy: A Meta-analysis of Detection and Recurrence Based on Raw Data.

BACKGROUND: Studies on hexaminolevulinate (HAL) cystoscopy report improved detection of bladder tumours. However, recent meta-analyses report conflicting effects on recurrence. OBJECTIVE: To assess available clinical data for blue light (BL) HAL cystoscopy on the detection of Ta/T1 and carcinoma in situ (CIS) tumours, and on tumour recurrence. DESIGN, SETTING, AND PARTICIPANTS: This meta-analysis reviewed raw data from prospective studies on 1345 patients with known or suspected non-muscle-invasive bladder cancer (NMIBC). INTERVENTION: A single application of HAL cystoscopy was used as an adjunct to white light (WL) cystoscopy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We studied the detection of NMIBC (intention to treat [ITT]: n=831; six studies) and recurrence (per protocol: n=634; three studies) up to 1 yr. DerSimonian and Laird's random-effects model was used to obtain pooled relative risks (RRs) and associated 95% confidence intervals (CIs) for outcomes for detection. RESULTS AND LIMITATIONS: BL cystoscopy detected significantly more Ta tumours (14.7%; p<0.001; odds ratio [OR]: 4.898; 95% CI, 1.937-12.390) and CIS lesions (40.8%; p<0.001; OR: 12.372; 95% CI, 6.343-24.133) than WL. There were 24.9% patients with at least one additional Ta/T1 tumour seen with BL (p<0.001), significant also in patients with primary (20.7%; p<0.001) and recurrent cancer (27.7%; p<0.001), and in patients at high risk (27.0%; p<0.001) and intermediate risk (35.7%; p=0.004). In 26.7% of patients, CIS was detected only by BL (p<0.001) and was also significant in patients with primary (28.0%; p<0.001) and recurrent cancer (25.0%; p<0.001). Recurrence rates up to 12 mo were significantly lower overall with BL, 34.5% versus 45.4% (p=0.006; RR: 0.761 [0.627-0.924]), and lower in patients with T1 or CIS (p=0.052; RR: 0.696 [0.482-1.003]), Ta (p=0.040; RR: 0.804 [0.653-0.991]), and in high-risk (p=0.050) and low-risk (p=0.029) subgroups. Some subgroups had too few patients to allow statistically meaningful analysis. Heterogeneity was minimised by the statistical analysis method used. CONCLUSIONS: This meta-analysis confirms that HAL BL cystoscopy significantly improves the detection of bladder tumours leading to a reduction of recurrence at 9-12 mo. The benefit is independent of the level of risk and is evident in patients with Ta, T1, CIS, primary, and recurrent cancer.

Methylation of the hTERT Promoter: A Novel Cancer Biomarker for Leptomeningeal Metastasis Detection in Cerebrospinal Fluids.

PURPOSE: The diagnosis of leptomeningeal metastases is usually confirmed by the finding of malignant cells by cytologic examination in the cerebrospinal fluid (CSF). More sensitive and specific cancer biomarkers may improve the detection of tumor cells in the CSF. Promoter methylation of the human telomerase reverse transcriptase (hTERT) gene characterizes most cancer cells. The aim of this study was to develop a sensitive method to detect hTERT methylation and to explore its use as a cancer biomarker in CSF. EXPERIMENTAL DESIGN: In 77 CSF specimens from 67 patients, hTERT promoter methylation was evaluated using real-time methylation-sensitive high-resolution melting (MS-HRM) and real-time TaqMan PCR and MS-HRM in a single-tube assay. RESULTS: Real-time MS-HRM assay was able to detect down to 1% hTERT-methylated DNA in a background of unmethylated DNA. PCR products were obtained from 90% (69/77) of CSF samples. No false positive hTERT was detected in the 21 non-neoplastic control cases, given to the method a specificity of 100%. The sensitivity of the real-time MS-HRM compared with the cytologic gold standard analysis was of 92% (11/12). Twenty-six CSFs from 22 patients with an hTERT-methylated primary tumor showed cytologic results suspicious for malignancy; in 17 (65%) of them, a diagnosis of leptomeningeal metastases could be confirmed by the hTERT methylation test. CONCLUSION: The hTERT real-time MS-HRM approach is fast, specific, sensitive, and could therefore become a valuable tool for diagnosis of leptomeningeal metastases as an adjunct to the traditional examination of CSF. Clin Cancer Res; 19(8); 2216-23. ©2013 AACR.

Random assignment of intervention points in two phase single-case designs: data-division-specific distributions

The present study explores the statistical properties of a randomization test based on the random assignment of the intervention point in a two-phase (AB) single-case design. The focus is on randomization distributions constructed with the values of the test statistic for all possible random assignments and used to obtain p-values. The shape of those distributions is investigated for each specific data division defined by the moment in which the intervention is introduced. Another aim of the study consisted in testing the detection of inexistent effects (i.e., production of false alarms) in autocorrelated data series, in which the assumption of exchangeability between observations may be untenable. In this way, it was possible to compare nominal and empirical Type I error rates in order to obtain evidence on the statistical validity of the randomization test for each individual data division. The results suggest that when either of the two phases has considerably less measurement times, Type I errors may be too probable and, hence, the decision making process to be carried out by applied researchers may be jeopardized.