Documents de treball / Informes (Genètica, Microbiologia i Estadística)

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Development of a standarised protocol for analyses somatic coliphages in sludge, soil and treated biowastes [Final Report]
(2006) Lucena Gutiérrez, Francisco; Blanch i Gisbert, Anicet; Jofre i Torroella, Joan
An initial desk study indicated that many biosolids contain human virus, but also that even in the more contaminated ones, they are not very abundant and that they are difficult to recover and quantify. Moreover, feasible methods for detecting infectious viruses are only available for enteroviruses, and it is well known that infectious viruses are needed for risk assessment. The need of indicators seemed obvious, as it was clear that the traditional bacterial indicators are not a good option for predicting presence and behaviour of human viruses in biosolids, biowastes and soils. Bacteriophages appeared as more suitable indicators, and among the proposed groups of phages, somatic coliphages aroused as those potentially more useful attending to their numbers in biosolids, feasibility of the detection methods and for sharing behaviour with viruses in biosolid and biowastes processing. Consequently, at present, somatic coliphages appear as a very useful indicator. Moreover, feasible standardised methods (ISO (adopted by CEN) and USEPA) for detection and quantification of somatic coliphages suspended in water, and consequently in aqueous solutions, are available. As well, literature available indicated that as human viruses, bacteriophages present in biosolids tend to be either included in or adsorbed to particles. Consequently, an extraction steep is necessary. There is a relatively abundant literature regarding extraction of human viruses and bacteriophages from solids. The methods for extracting bacteriophages (and also human viruses) from solids require the following steeps: homogenization, elution, clarification and decontamination. Described methods mostly vary in the elution steep. In a pair of papers comparing several elution methods showed elution with beef extract as the more efficient one. However the described methods required optimization in the various steeps. The optimization of the various steeps was the main aim of this work. Unfortunately, inoculation of biosolids with known concentrations of viruses (as it is feasible with water samples) are not mimicking what happens in the real world because of inclusion and adsorption of phages in/to solid particles. Therefore, it was decided to perform the experiments of optimization of the extraction method with matrixes containing concentrations as high as possible of naturally occurring somatic coliphages. The studied matrixes were raw sludge, digested and dewatered sludge, selected (for its content of somatic coliphages) compost and soil contaminated with raw sewage. Some of these matrixes contained very homogeneous and steady numbers of phages and consequently it was possible to compare modifications of each one of the steeps of the extraction method and how they affected the efficiency of recovery. The method was optimised, published in scientific literature (Guzman et al. 2007. J. Virol. Methods 144: 41-48) and a draft of a “standard method” for the extraction of somatic coliphages from biosolids, biowastes and soils has been presented for approval to the CEN/TC308/WG1/TG5. The non-convenience of inoculating these sort of matrices, since as said previously, the inoculated material does not mimic what happens in nature, complicated the performance of validation studies. Reference materials are needed for validation studies. To get round this problem, a few natural biosolids were tested as potential reference materials. For it, the matrices were distributed in a great number of containers, and placed at 4ºC. Then, somatic coliphages were enumerated after different days of storage from two containers and also from two subsamples of each one of the containers. This allowed testing the intra and inter-container homogeneity and the time elapsed without significant descent in the number of phages detected. Digested-dewatered sludge probed to be an excellent reference material lasting in perfect condition for at least 2 months. This will allow to make validation multilaboratory studies and have a reference material for “in lab” quality control. In fact, a small validation study of the extraction method with three laboratories was performed with satisfactory results. A few experiments done with other phages, as for example F-specific RNA bacteriophages, indicate that the method will also be applicable to other phages. As well, it may be useful for extracting human and animal viruses, though this should be further verified. In conclusion, a feasible, fast and low cost method for determining somatic coliphages from biosolids, biowastes and soils is available. Besides the feasibility of the methods for extraction, detection and enumeration, somatic coliphages have, in our opinion, several advantages to follow the higienization processes of sludges as well as to have an indication of the viral contamination of these solid matrixes.
Bacteriophages in bathing wàters: A feasibility study on the development of a method based on bacteriophages for the determination of microbiological quality of bathing waters
(2000) Jofre i Torroella, Joan; Lucena Gutiérrez, Francisco; Mooijman, K.; Pierzo, V.; Araujo Boira, Rosa Ma.; Bahar, M.; Demarquilly, C.; Havelar, A.
Methods for the detection and enumeration of somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis had been standardised and validated. Conditions for the preparation, transport and distribution of bacteriophage reference materials and preservation of samples had been defined. A method based on flocculation with Mg(OH2) with concentration efficiencies from about 40% was settled to concentrate phages from bathing waters. All methods were successfully implemented in routine laboratories all around the EU. Data on the occurrence of bacteriophages as compared to E. coli and Enterococci are available from diverse situations encountered in the EU. Results allow to conclude that the potential of phages for the determination of the microbiological quality of bathing waters merits to be considered since their determination is feasible and their behaviour in natural water differs from the behaviour of bacterial indicators and consequently they add valuable information.
Soils, sludges and treated bio-wastes — Extraction of bacteriophages from sludge, soils and treated biowastes
(2007-08) Working Group CEN TC308/WG1/TG5
This document is developed in the framework of the project 'Horizontal'. It is the result of a critical review "Methods for bacteriophages (and viruses) to be monitored in EU in sludges, soils and treated biowastes" and aims at evaluation of the latest developments in assessing bacteriophages in sludge, soil and biowastes. After discussion with all parties concerned in CEN (European Committee for Standardization) and selection of a number of test methods described in this study the standard has been developed further as a modular horizontal method and was validated within the project ”Horizontal”. Sludges, soils and biowastes can contain pathogens such as Enteroviruses, Salmonella spp. Most occur in the intestinal tract of humans and animals and can be transmitted through faecal contamination. The use of such contaminated materials in agriculture may cause outbreaks of infection due to the production of contaminated food and animal foodstocks. They may also be transmitted to wild animals. There is a need to monitor the efficacy of storage and treatment processes to control pathogens, and application rates to land. Viruses had been described to have a tendency to adsorb to solids. There are some differences between different viruses regarding adsorption efficiency, solids to which they can be adsorbed better and conditions that favour adsorptions. Once they are adsorbed to solids, they persist longer than when they are free. Consequently, viruses are expected to concentrate in sludges, biowastes, sediments and soils, where they will persist longer that in the contaminated waters. This standardised protocol specifies a procedure for the elution of bacteriophages (as viral indicators) from sewage sludges, compost and biowaste samples. The user should, prior to analysis, validate the method for the particular type of sample they wish to analyse: sludges, soils and biowastes.
R commands secondary production
(2011-11-02) Gaudes Saez, Ainhoa; Ocaña i Rebull, Jordi; Muñoz Gràcia, Isabel
R commands to calculate the secondary production estimates using the size-frequency method after Hynes and Coleman (1968), Benke (1979) and Huryn (1996).
Uso de la estadística en la oncología y la hematología
(2010) Monleón Getino, Toni
En este artículo abordamos el uso y la importancia de las herramientas estadísticas que se utilizan principalmente en los estudios médicos del ámbito de la oncología y la hematología, pero aplicables a muchos otros campos tanto médicos como experimentales o industriales. El objetivo del presente trabajo es presentar de una manera clara y precisa la metodología estadística necesaria para analizar los datos obtenidos en los estudios rigurosa y concisamente en cuanto a las hipótesis de trabajo planteadas por los investigadores. La medida de la respuesta al tratamiento elegidas en al tipo de estudio elegido determinarán los métodos estadísticos que se utilizarán durante el análisis de los datos del estudio y también el tamaño de muestra. Mediante la correcta aplicación del análisis estadístico y de una adecuada planificación se puede determinar si la relación encontrada entre la exposición a un tratamiento y un resultado es casual o por el contrario, está sujeto a una relación no aleatoria que podría establecer una relación de causalidad. Hemos estudiado los principales tipos de diseño de los estudios médicos más utilizados, tales como ensayos clínicos y estudios observacionales (cohortes, casos y controles, estudios de prevalencia y estudios ecológicos). También se presenta una sección sobre el cálculo del tamaño muestral de los estudios y cómo calcularlo, ¿Qué prueba estadística debe utilizarse?, los aspectos sobre fuerza del efecto ¿odds ratio¿ (OR) y riesgo relativo (RR), el análisis de supervivencia. Se presentan ejemplos en la mayoría de secciones del artículo y bibliografía más relevante.
A measure of stability as a criterion for the verification and analysis of simulation models
(2010) Monleón Getino, Toni; Ruiz de Villa, Carmen; Ocaña i Rebull, Jordi
The aim of this study is to define a new statistic, PVL, based on the relative distance between the likelihood associated with the simulation replications and the likelihood of the conceptual model. Our results coming from several simulation experiments of a clinical trial show that the PVL statistic range can be a good measure of stability to establish when a computational model verifies the underlying conceptual model. PVL improves also the analysis of simulation replications because only one statistic is associated with all the simulation replications. As well it presents several verification scenarios, obtained by altering the simulation model, that show the usefulness of PVL. Further simulation experiments suggest that a 0 to 20 % range may define adequate limits for the verification problem, if considered from the viewpoint of an equivalence test.
On the consequences of misspecifing assumptions concerning residuals distribution in a repeated measures and nonlinear mixed modelling context
(2004) El Halimi, Rachid; Ocaña i Rebull, Jordi
In this paper we describe the results of a simulation study performed to elucidate the robustness of the Lindstrom and Bates (1990) approximation method under non-normality of the residuals, under different situations. Concerning the fixed effects, the observed coverage probabilities and the true bias and mean square error values, show that some aspects of this inferential approach are not completely reliable. When the true distribution of the residuals is asymmetrical, the true coverage is markedly lower than the nominal one. The best results are obtained for the skew normal distribution, and not for the normal distribution. On the other hand, the results are partially reversed concerning the random effects. Soybean genotypes data are used to illustrate the methods and to motivate the simulation scenarios
Impact of incorrect assumptions on the covariance structure of random effects and/or residuals in nonlinear mixed models for repeated measures data
(2004) El Halimi, Rachid; Ocaña i Rebull, Jordi
In this paper we analyse, using Monte Carlo simulation, the possible consequences of incorrect assumptions on the true structure of the random effects covariance matrix and the true correlation pattern of residuals, over the performance of an estimation method for nonlinear mixed models. The procedure under study is the well known linearization method due to Lindstrom and Bates (1990), implemented in the nlme library of S-Plus and R. Its performance is studied in terms of bias, mean square error (MSE), and true coverage of the associated asymptotic confidence intervals. Ignoring other criteria like the convenience of avoiding over parameterised models, it seems worst to erroneously assume some structure than do not assume any structure when this would be adequate.
Supplementary material to Ocaña, J., Sánchez, M.P., Sánchez, A. and Carrasco, J.L. ¿On equivalence and bioequivalence testing¿, SORT, 32(2), 2008
(2008-06-05) Ocaña i Rebull, Jordi; Sánchez Olavarría, María Pilar; Sànchez, Àlex (Sànchez Pla); Carrasco Jordan, Josep Lluís
Equivalence testing is the natural approach to many statistical problems. First, its main application, bioequivalence testing, is reviewed. The basic concepts of bioequivalence testing (2×2 crossover designs, TOST, interval inclusion principle, etc.) and its problems (TOST biased character, the carry- over problem, etc.) are considered. Next, equivalence testing is discussed more generally. Some applications and methods are reviewed and the relation of equivalence testing and distance-based inference is highlighted. A new distance-based method to determine whether two gene lists are equivalent in terms of their annotations in the Gene Ontology illustrates these ideas. We end with a general discussion and some suggestions for future research.

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