Application of real-time PCR for total airborne bacterial assessment : comparison with epifluorescence microscopy and culture-dependent methods

Traditional culture-dependent methods to quantify and identify airborne microorganisms are limited by factors such as short-duration sampling times and inability to count nonculturableor non-viable bacteria. Consequently, the quantitative assessment of bioaerosols is often underestimated. Use of the real-time quantitative polymerase chain reaction (Q-PCR) to quantify bacteria in environmental samples presents an alternative method, which should overcome this problem. The aim of this study was to evaluate the performance of a real-time Q-PCR assay as a simple and reliable way to quantify the airborne bacterial load within poultry houses and sewage treatment plants, in comparison with epifluorescencemicroscopy and culture-dependent methods. The estimates of bacterial load that we obtained from real-time PCR and epifluorescence methods, are comparable, however, our analysis of sewage treatment plants indicate these methods give values 270-290 fold greater than those obtained by the ''impaction on nutrient agar'' method. The culture-dependent method of air impaction on nutrient agar was also inadequate in poultry houses, as was the impinger-culture method, which gave a bacterial load estimate 32-fold lower than obtained by Q-PCR. Real-time quantitative PCR thus proves to be a reliable, discerning, and simple method that could be used to estimate airborne bacterial load in a broad variety of other environments expected to carry high numbers of airborne bacteria. [Authors]

Comparison of the interobserver variability of 2 different methods of dietary assessment in a geriatric ward: a pilot study.

BACKGROUND: Protein-energy malnutrition is highly prevalent in aged populations. Associated clinical, economic, and social burden is important. A valid screening method that would be robust and precise, but also easy, simple, and rapid to apply, is essential for adequate therapeutic management. OBJECTIVES: To compare the interobserver variability of 2 methods measuring food intake: semiquantitative visual estimations made by nurses versus calorie measurements performed by dieticians on the basis of standardized color digital photographs of servings before and after consumption. DESIGN: Observational monocentric pilot study. SETTING/PARTICIPANTS: A geriatric ward. The meals were randomly chosen from the meal tray. The choice was anonymous with respect to the patients who consumed them. MEASUREMENTS: The test method consisted of the estimation of calorie consumption by dieticians on the basis of standardized color digital photographs of servings before and after consumption. The reference method was based on direct visual estimations of the meals by nurses. Food intake was expressed in the form of a percentage of the serving consumed and calorie intake was then calculated by a dietician based on these percentages. The methods were applied with no previous training of the observers. Analysis of variance was performed to compare their interobserver variability. RESULTS: Of 15 meals consumed and initially examined, 6 were assessed with each method. Servings not consumed at all (0% consumption) or entirely consumed by the patient (100% consumption) were not included in the analysis so as to avoid systematic error. The digital photography method showed higher interobserver variability in calorie intake estimations. The difference between the compared methods was statistically significant (P < .03). CONCLUSIONS: Calorie intake measures for geriatric patients are more concordant when estimated in a semiquantitative way. Digital photography for food intake estimation without previous specific training of dieticians should not be considered as a reference method in geriatric settings, as it shows no advantages in terms of interobserver variability.

Inferring transport characteristics in a fractured rock aquifer by combining single-hole ground-penetrating radar reflection monitoring and tracer test data

Investigations of solute transport in fractured rock aquifers often rely on tracer test data acquired at a limited number of observation points. Such data do not, by themselves, allow detailed assessments of the spreading of the injected tracer plume. To better understand the transport behavior in a granitic aquifer, we combine tracer test data with single-hole ground-penetrating radar (GPR) reflection monitoring data. Five successful tracer tests were performed under various experimental conditions between two boreholes 6 m apart. For each experiment, saline tracer was injected into a previously identified packed-off transmissive fracture while repeatedly acquiring single-hole GPR reflection profiles together with electrical conductivity logs in the pumping borehole. By analyzing depth-migrated GPR difference images together with tracer breakthrough curves and associated simplified flow and transport modeling, we estimate (1) the number, the connectivity, and the geometry of fractures that contribute to tracer transport, (2) the velocity and the mass of tracer that was carried along each flow path, and (3) the effective transport parameters of the identified flow paths. We find a qualitative agreement when comparing the time evolution of GPR reflectivity strengths at strategic locations in the formation with those arising from simulated transport. The discrepancies are on the same order as those between observed and simulated breakthrough curves at the outflow locations. The rather subtle and repeatable GPR signals provide useful and complementary information to tracer test data acquired at the outflow locations and may help us to characterize transport phenomena in fractured rock aquifers.

A comparison of methods for differential expression analysis of RNA-seq data.

BACKGROUND: Finding genes that are differentially expressed between conditions is an integral part of understanding the molecular basis of phenotypic variation. In the past decades, DNA microarrays have been used extensively to quantify the abundance of mRNA corresponding to different genes, and more recently high-throughput sequencing of cDNA (RNA-seq) has emerged as a powerful competitor. As the cost of sequencing decreases, it is conceivable that the use of RNA-seq for differential expression analysis will increase rapidly. To exploit the possibilities and address the challenges posed by this relatively new type of data, a number of software packages have been developed especially for differential expression analysis of RNA-seq data. RESULTS: We conducted an extensive comparison of eleven methods for differential expression analysis of RNA-seq data. All methods are freely available within the R framework and take as input a matrix of counts, i.e. the number of reads mapping to each genomic feature of interest in each of a number of samples. We evaluate the methods based on both simulated data and real RNA-seq data. CONCLUSIONS: Very small sample sizes, which are still common in RNA-seq experiments, impose problems for all evaluated methods and any results obtained under such conditions should be interpreted with caution. For larger sample sizes, the methods combining a variance-stabilizing transformation with the 'limma' method for differential expression analysis perform well under many different conditions, as does the nonparametric SAMseq method.

Workgroup report: incorporating in vitro alternative methods for developmental neurotoxicity into international hazard and risk assessment strategies.

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19-21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work.

Comparison of thinning methods in a Scots pine stand on drained peatland : a simulation study

Tiivistelmä: Harvennusmenetelmien vertailu ojitetun turvemaan männikössä. Simulointitutkimus

Calibration of high-resolution geophysical data with tracer test measurements to improve hydrological predictions

Relationships between porosity and hydraulic conductivity tend to be strongly scale- and site-dependent and are thus very difficult to establish. As a result, hydraulic conductivity distributions inferred from geophysically derived porosity models must be calibrated using some measurement of aquifer response. This type of calibration is potentially very valuable as it may allow for transport predictions within the considered hydrological unit at locations where only geophysical measurements are available, thus reducing the number of well tests required and thereby the costs of management and remediation. Here, we explore this concept through a series of numerical experiments. Considering the case of porosity characterization in saturated heterogeneous aquifers using crosshole ground-penetrating radar and borehole porosity log data, we use tracer test measurements to calibrate a relationship between porosity and hydraulic conductivity that allows the best prediction of the observed hydrological behavior. To examine the validity and effectiveness of the obtained relationship, we examine its performance at alternate locations not used in the calibration procedure. Our results indicate that this methodology allows us to obtain remarkably reliable hydrological predictions throughout the considered hydrological unit based on the geophysical data only. This was also found to be the case when significant uncertainty was considered in the underlying relationship between porosity and hydraulic conductivity.

A limited area model intercomparison on the "Montserrat-2000" flash-flood event using statistical and deterministic methods

In the scope of the European project Hydroptimet, INTERREG IIIB-MEDOCC programme, limited area model (LAM) intercomparison of intense events that produced many damages to people and territory is performed. As the comparison is limited to single case studies, the work is not meant to provide a measure of the different models' skill, but to identify the key model factors useful to give a good forecast on such a kind of meteorological phenomena. This work focuses on the Spanish flash-flood event, also known as "Montserrat-2000" event. The study is performed using forecast data from seven operational LAMs, placed at partners' disposal via the Hydroptimet ftp site, and observed data from Catalonia rain gauge network. To improve the event analysis, satellite rainfall estimates have been also considered. For statistical evaluation of quantitative precipitation forecasts (QPFs), several non-parametric skill scores based on contingency tables have been used. Furthermore, for each model run it has been possible to identify Catalonia regions affected by misses and false alarms using contingency table elements. Moreover, the standard "eyeball" analysis of forecast and observed precipitation fields has been supported by the use of a state-of-the-art diagnostic method, the contiguous rain area (CRA) analysis. This method allows to quantify the spatial shift forecast error and to identify the error sources that affected each model forecasts. High-resolution modelling and domain size seem to have a key role for providing a skillful forecast. Further work is needed to support this statement, including verification using a wider observational data set.

Introduction of sensor spectral response into image fusion methods. Application to wavelet-based methods

Usual image fusion methods inject features from a high spatial resolution panchromatic sensor into every low spatial resolution multispectral band trying to preserve spectral signatures and improve spatial resolution to that of the panchromatic sensor. The objective is to obtain the image that would be observed by a sensor with the same spectral response (i.e., spectral sensitivity and quantum efficiency) as the multispectral sensors and the spatial resolution of the panchromatic sensor. But in these methods, features from electromagnetic spectrum regions not covered by multispectral sensors are injected into them, and physical spectral responses of the sensors are not considered during this process. This produces some undesirable effects, such as resolution overinjection images and slightly modified spectral signatures in some features. The authors present a technique which takes into account the physical electromagnetic spectrum responses of sensors during the fusion process, which produces images closer to the image obtained by the ideal sensor than those obtained by usual wavelet-based image fusion methods. This technique is used to define a new wavelet-based image fusion method.

Measures of health and disease in Africa: are current methods giving us useful information about trends in cardiovascular diseases?

An enormous burst of interest in the public health burden from chronic disease in Africa has emerged as a consequence of efforts to estimate global population health. Detailed estimates are now published for Africa as a whole and each country on the continent. These data have formed the basis for warnings about sharp increases in cardiovascular disease (CVD) in the coming decades. In this essay we briefly examine the trajectory of social development on the continent and its consequences for the epidemiology of CVD and potential control strategies. Since full vital registration has only been implemented in segments of South Africa and the island nations of Seychelles and Mauritius - formally part of WHO-AFRO - mortality data are extremely limited. Numerous sample surveys have been conducted but they often lack standardization or objective measures of health status. Trend data are even less informative. However, using the best quality data available, age-standardized trends in CVD are downward, and in the case of stroke, sharply so. While acknowledging that the extremely limited available data cannot be used as the basis for inference to the continent, we raise the concern that general estimates based on imputation to fill in the missing mortality tables may be even more misleading. No immediate remedies to this problem can be identified, however bilateral collaborative efforts to strength local educational institutions and governmental agencies rank as the highest priority for near term development.

Novel methods improve prediction of species' distributions from occurrence data

Prediction of species' distributions is central to diverse applications in ecology, evolution and conservation science. There is increasing electronic access to vast sets of occurrence records in museums and herbaria, yet little effective guidance on how best to use this information in the context of numerous approaches for modelling distributions. To meet this need, we compared 16 modelling methods over 226 species from 6 regions of the world, creating the most comprehensive set of model comparisons to date. We used presence-only data to fit models, and independent presence-absence data to evaluate the predictions. Along with well-established modelling methods such as generalised additive models and GARP and BIOCLIM, we explored methods that either have been developed recently or have rarely been applied to modelling species' distributions. These include machine-learning methods and community models, both of which have features that may make them particularly well suited to noisy or sparse information, as is typical of species' occurrence data. Presence-only data were effective for modelling species' distributions for many species and regions. The novel methods consistently outperformed more established methods. The results of our analysis are promising for the use of data from museums and herbaria, especially as methods suited to the noise inherent in such data improve.

Crescent and star shapes of members of the Chlamydiales order: impact of fixative methods.

Members of the Chlamydiales order all share a biphasic lifecycle alternating between small infectious particles, the elementary bodies (EBs) and larger intracellular forms able to replicate, the reticulate bodies. Whereas the classical Chlamydia usually harbours round-shaped EBs, some members of the Chlamydia-related families display crescent and star-shaped morphologies by electron microscopy. To determine the impact of fixative methods on the shape of the bacterial cells, different buffer and fixative combinations were tested on purified EBs of Criblamydia sequanensis, Estrella lausannensis, Parachlamydia acanthamoebae, and Waddlia chondrophila. A linear discriminant analysis was performed on particle metrics extracted from electron microscopy images to recognize crescent, round, star and intermediary forms. Depending on the buffer and fixatives used, a mixture of alternative shapes were observed in varying proportions with stars and crescents being more frequent in C. sequanensis and P. acanthamoebae, respectively. No tested buffer and chemical fixative preserved ideally the round shape of a majority of bacteria and other methods such as deep-freezing and cryofixation should be applied. Although crescent and star shapes could represent a fixation artifact, they certainly point towards a diverse composition and organization of membrane proteins or intracellular structures rather than being a distinct developmental stage.

Bacterial contamination of platelet concentrates: pathogen detection and inactivation methods

Whereas the reduction of transfusion related viral transmission has been a priority during the last decade, bacterial infection transmitted by transfusion still remains associated to a high morbidity and mortality, and constitutes the most frequent infectious risk of transfusion. This problem especially concerns platelet concentrates because of their favorable bacterial growth conditions. This review gives an overview of platelet transfusion-related bacterial contamination as well as on the different strategies to reduce this problem by using either bacterial detection or inactivation methods.