Development of molecular-based techniques for the detection, identification and quantification of food-borne pathogens

La presencia de microorganismos patógenos en alimentos es uno de los problemas esenciales en salud pública, y las enfermedades producidas por los mismos es una de las causas más importantes de enfermedad. Por tanto, la aplicación de controles microbiológicos dentro de los programas de aseguramiento de la calidad es una premisa para minimizar el riesgo de infección de los consumidores. Los métodos microbiológicos clásicos requieren, en general, el uso de pre-enriquecimientos no-selectivos, enriquecimientos selectivos, aislamiento en medios selectivos y la confirmación posterior usando pruebas basadas en la morfología, bioquímica y serología propias de cada uno de los microorganismos objeto de estudio. Por lo tanto, estos métodos son laboriosos, requieren un largo proceso para obtener resultados definitivos y, además, no siempre pueden realizarse. Para solucionar estos inconvenientes se han desarrollado diversas metodologías alternativas para la detección identificación y cuantificación de microorganismos patógenos de origen alimentario, entre las que destacan los métodos inmunológicos y moleculares. En esta última categoría, la técnica basada en la reacción en cadena de la polimerasa (PCR) se ha convertido en la técnica diagnóstica más popular en microbiología, y recientemente, la introducción de una mejora de ésta, la PCR a tiempo real, ha producido una segunda revolución en la metodología diagnóstica molecular, como pude observarse por el número creciente de publicaciones científicas y la aparición continua de nuevos kits comerciales. La PCR a tiempo real es una técnica altamente sensible -detección de hasta una molécula- que permite la cuantificación exacta de secuencias de ADN específicas de microorganismos patógenos de origen alimentario. Además, otras ventajas que favorecen su implantación potencial en laboratorios de análisis de alimentos son su rapidez, sencillez y el formato en tubo cerrado que puede evitar contaminaciones post-PCR y favorece la automatización y un alto rendimiento. En este trabajo se han desarrollado técnicas moleculares (PCR y NASBA) sensibles y fiables para la detección, identificación y cuantificación de bacterias patogénicas de origen alimentario (Listeria spp., Mycobacterium avium subsp. paratuberculosis y Salmonella spp.). En concreto, se han diseñado y optimizado métodos basados en la técnica de PCR a tiempo real para cada uno de estos agentes: L. monocytogenes, L. innocua, Listeria spp. M. avium subsp. paratuberculosis, y también se ha optimizado y evaluado en diferentes centros un método previamente desarrollado para Salmonella spp. Además, se ha diseñado y optimizado un método basado en la técnica NASBA para la detección específica de M. avium subsp. paratuberculosis. También se evaluó la aplicación potencial de la técnica NASBA para la detección específica de formas viables de este microorganismo. Todos los métodos presentaron una especificidad del 100 % con una sensibilidad adecuada para su aplicación potencial a muestras reales de alimentos. Además, se han desarrollado y evaluado procedimientos de preparación de las muestras en productos cárnicos, productos pesqueros, leche y agua. De esta manera se han desarrollado métodos basados en la PCR a tiempo real totalmente específicos y altamente sensibles para la determinación cuantitativa de L. monocytogenes en productos cárnicos y en salmón y productos derivados como el salmón ahumado y de M. avium subsp. paratuberculosis en muestras de agua y leche. Además este último método ha sido también aplicado para evaluar la presencia de este microorganismo en el intestino de pacientes con la enfermedad de Crohn's, a partir de biopsias obtenidas de colonoscopia de voluntarios afectados. En conclusión, este estudio presenta ensayos moleculares selectivos y sensibles para la detección de patógenos en alimentos (Listeria spp., Mycobacterium avium subsp. paratuberculosis) y para una rápida e inambigua identificación de Salmonella spp. La exactitud relativa de los ensayos ha sido excelente, si se comparan con los métodos microbiológicos de referencia y pueden serusados para la cuantificación de tanto ADN genómico como de suspensiones celulares. Por otro lado, la combinación con tratamientos de preamplificación ha resultado ser de gran eficiencia para el análisis de las bacterias objeto de estudio. Por tanto, pueden constituir una estrategia útil para la detección rápida y sensible de patógenos en alimentos y deberían ser una herramienta adicional al rango de herramientas diagnósticas disponibles para el estudio de patógenos de origen alimentario.

An Integrated Assessment of changes in the thermohaline circulation

This paper discusses the risks of a shutdown of the thermohaline circulation (THC) for the climate system, for ecosystems in and around the North Atlantic as well as for fisheries and agriculture by way of an Integrated Assessment. The climate model simulations are based on greenhouse gas scenarios for the 21st century and beyond. A shutdown of the THC, complete by 2150, is triggered if increased freshwater input from inland ice melt or enhanced runoff is assumed. The shutdown retards the greenhouse gas-induced atmospheric warming trend in the Northern Hemisphere, but does not lead to a persistent net cooling. Due to the simulated THC shutdown the sea level at the North Atlantic shores rises by up to 80 cm by 2150, in addition to the global sea level rise. This could potentially be a serious impact that requires expensive coastal protection measures. A reduction of marine net primary productivity is associated with the impacts of warming rather than a THC shutdown. Regional shifts in the currents in the Nordic Seas could strongly deteriorate survival chances for cod larvae and juveniles. This could lead to cod fisheries becoming unprofitable by the end of the 21st century. While regional socioeconomic impacts might be large, damages would be probably small in relation to the respective gross national products. Terrestrial ecosystem productivity is affected much more by the fertilization from the increasing CO2 concentration than by a THC shutdown. In addition, the level of warming in the 22nd to 24th century favours crop production in northern Europe a lot, no matter whether the THC shuts down or not. CO2 emissions corridors aimed at limiting the risk of a THC breakdown to 10% or less are narrow, requiring departure from business-as-usual in the next few decades. The uncertainty about THC risks is still high. This is seen in model analyses as well as in the experts’ views that were elicited. The overview of results presented here is the outcome of the Integrated Assessment project INTEGRATION.

Radon in homes and risk of lung cancer: collaborative analysis of individual data from 13 European case-control studies

Objective: To determine the risk of lung cancer associated with exposure at home to the radioactive disintegration products of naturally Occurring radon gas. Design: Collaborative analysis of individual data from 13 case-control studies of residential radon and lung cancer. Setting Nine European countries. Subjects 7148 cases Of lung cancer and 14 208 controls. Main outcome measures: Relative risks of lung cancer and radon gas concentrations in homes inhabited during the previous 5-34 years measured in becquerels (radon disintegrations per second) per cubic incite (Bq/m(3)) Of household air. Results: The mean measured radon concentration in homes of people in tire control group was 97 Bq/m(3), with 11% measuring > 200 and 4% measuring > 400 Bq/m(3). For cases of lung cancer the mean concentration was 104 Bq/m(3). The risk of lung cancer increased by 8.4% (95% confidence interval 3.0% to 15.8%) per 100 Bq/m(3) increase in measured radon (P = 0.0007). This corresponds to an increase of 16% (5% to 31%) per 100 Bq/m(3) increase in usual radon-that is, after correction for the dilution caused by random uncertainties in measuring radon concentrations. The dose-response relation seemed to be linear with no threshold and remained significant (P=0.04) in analyses limited to individuals from homes with measured radon < 200 Bq/m(3). The proportionate excess risk did not differ significantly with study, age, sex, or smoking. In the absence of other causes of death, the absolute risks of lung cancer by age 75 years at usual radon concentrations of 0, 100, and 400 Bq/m(3) would be about 0.4%, 0.5%, and 0.7%, respectively, for lifelong non-smokers, and about 25 times greater (10%, 12%, and 16%) for cigarette smokers. Conclusions: Collectively, though not separately, these studies show appreciable hazards from residential radon, particularly for smokers and recent ex-smokers, and indicate that it is responsible for about 2% of all deaths from cancer in Europe.

The effects of pre- and postnatal depression in fathers: a natural experiment comparing the effects of exposure to depression on offspring

Background: Depression in fathers in the postnatal period is associated with an increased risk of behavioural problems in their offspring, particularly for boys. The aim of this study was to examine for differential effects of depression in fathers on children's subsequent psychological functioning via a natural experiment comparing prenatal and postnatal exposure. Methods:In a longitudinal population cohort study (the Avon Longitudinal Study of Parents and Children (ALSPAC)) we examined the associations between depression in fathers measured in the prenatal and postnatal period (measured using the Edinburgh Postnatal Depression Scale), and later behavioural/emotional and psychiatric problems in their children, assessed at ages 31/2 and 7 years. Results: Children whose fathers were depressed in both the prenatal and postnatal periods had the highest risks of subsequent psychopathology, measured by total problems at age 31/2 years (Odds Ratio 3.55; 95% confidence interval 2.07, 6.08) and psychiatric diagnosis at age 7 years (OR 2.54; 1.19, 5.41). Few differences emerged when prenatal and postnatal depression exposure were directly compared, but when compared to fathers who were not depressed, boys whose fathers had postnatal depression only had higher rates of conduct problems aged 31/2 years (OR 2.14; 1.22, 3.72) whereas sons of the prenatal group did not (OR 1.41; .75, 2.65). These associations changed little when controlling for maternal depression and other potential confounding factors. Conclusions: The findings of this study suggest that the increased risk of later conduct problems, seen particularly in the sons of depressed fathers, maybe partly mediated through environmental means. In addition, children whose fathers are more chronically depressed appear to be at a higher risk of emotional and behavioural problems. Efforts to identify the precise mechanisms by which transmission of risk may occur should be encouraged to enable the development of focused interventions to mitigate risks for young children.

Scenarios as the basis for assessment of mitigation and adaptation

The possibilities and need for adaptation and mitigation depends on uncertain future developments with respect to socio-economic factors and the climate system. Scenarios are used to explore the impacts of different strategies under uncertainty. In this chapter, some scenarios are presented that are used in the ADAM project for this purpose. One scenario explores developments with no mitigation, and thus with high temperature increase and high reliance on adaptation (leading to 4oC increase by 2100 compared to pre-industrial levels). A second scenario explores an ambitious mitigation strategy (leading to 2oC increase by 2100 compared to pre-industrial levels). In the latter scenario, stringent mitigation strategies effectively reduces the risks of climate change, but based on uncertainties in the climate system a temperature increase of 3oC or more cannot be excluded. The analysis shows that, in many cases, adaptation and mitigation are not trade-offs but supplements. For example, the number of people exposed to increased water resource stress due to climate change can be substantially reduced in the mitigation scenario, but even then adaptation will be required for the remaining large numbers of people exposed to increased stress. Another example is sea level rise, for which adaptation is more cost-effective than mitigation, but mitigation can help reduce damages and the cost of adaptation. For agriculture, finally, only the scenario based on a combination of adaptation and mitigation is able to avoid serious climate change impacts.

A review of climate risk information for adaptation and development planning

Although the use of climate scenarios for impact assessment has grown steadily since the 1990s, uptake of such information for adaptation is lagging by nearly a decade in terms of scientific output. Nonetheless, integration of climate risk information in development planning is now a priority for donor agencies because of the need to prepare for climate change impacts across different sectors and countries. This urgency stems from concerns that progress made against Millennium Development Goals (MDGs) could be threatened by anthropogenic climate change beyond 2015. Up to this time the human signal, though detectable and growing, will be a relatively small component of climate variability and change. This implies the need for a twin-track approach: on the one hand, vulnerability assessments of social and economic strategies for coping with present climate extremes and variability, and, on the other hand, development of climate forecast tools and scenarios to evaluate sector-specific, incremental changes in risk over the next few decades. This review starts by describing the climate outlook for the next couple of decades and the implications for adaptation assessments. We then review ways in which climate risk information is already being used in adaptation assessments and evaluate the strengths and weaknesses of three groups of techniques. Next we identify knowledge gaps and opportunities for improving the production and uptake of climate risk information for the 2020s. We assert that climate change scenarios can meet some, but not all, of the needs of adaptation planning. Even then, the choice of scenario technique must be matched to the intended application, taking into account local constraints of time, resources, human capacity and supporting infrastructure. We also show that much greater attention should be given to improving and critiquing models used for climate impact assessment, as standard practice. Finally, we highlight the over-arching need for the scientific community to provide more information and guidance on adapting to the risks of climate variability and change over nearer time horizons (i.e. the 2020s). Although the focus of the review is on information provision and uptake in developing regions, it is clear that many developed countries are facing the same challenges. Copyright © 2009 Royal Meteorological Society

Gauging the investment potential of international real estate markets

Investing in real estate markets overseas means venturing into the unknown, where you meet unfamiliar political and economic environments, unstable currencies, strange cultures and languages, and so although the advantages of international diversification might appear attractive, the risks of international investment must not be overlooked. However, capital markets are becoming global markets, and commercial real estate markets are no exception, accordingly despite the difficulties posed by venturing overseas no investor can overlook the potential international investment holds out. Thus, what strategies are appropriate for capitalising on this potential? Three issues must be considered: (1) the potential of the countries real estate market in general; (2) the potential of the individual market sectors; and (3) the investment process itself. Although each step in foreign real estate investment is critical, the initial assessment of opportunities is especially important. Various methods can be used to achieve this but a formal and systematic analysis of aggregate market potential should prove particularly fruitful. The work reported here, therefore, develops and illustrates such a methodology for the over 50 international real estate markets.

Transfers, contracts and regulation: a new institutional economics perspective on the changing provision of social housing in Britain

Social housing policy in the UK mirrors wider processes Associated with shifts in broad welfare regimes. Social housing has moved from dominance by state housing provision to the funding of new investment through voluntary sector housing associations to what is now a greater focus on the regulation and private financing of these not-for-profit bodies. If these trends run their course, we are likely to see a range of not-for-profit bodies providing non-market housing in a highly regulated quasi-market. This paper examines these issues through the lens of new institutional economics, which it is believed can provide important insights into the fundamental contractual and regulatory relationships that are coming to dominate social housing from the perspective of the key actors in the sector (not-for-profit housing organisations, their tenants, private lenders and the regulatory state). The paper draws on evidence recently collected from a study evaluating more than 100 stock transfer organisations that inherited ex-public housing in Scotland, including 12 detailed case studies. The paper concludes that social housing stakeholders need to be aware of the risks (and their management) faced across the sector and that the state needs to have clear objectives for social housing and coherent policy instruments to achieve those ends.

Assessing continental-scale risks for generalist and specialist pollinating bee species under climate change

Increased risks of extinction to populations of animals and plants under changing climate have now been demonstrated for many taxa. This study assesses the extinction risks to species within an important genus of pollinating bees (Colletes: Apidae) by estimating the expected changes in the area and isolation of suitable habitat under predicted climatic condition for 2050. Suitable habitat was defined on the basis of the presence of known forage plants as well as climatic suitability. To investigate whether ecological specialisation was linked to extinction risk we compared three species which were generalist pollen foragers on several plant families with three species which specialised on pollen from a single plant species. Both specialist and generalist species showed an increased risk of extinction with shifting climate, and this was particularly high for the most specialised species (Colletes anchusae and C. wolfi). The forage generalist C. impunctatus, which is associated with Boreo-Alpine environments, is potentially threatened through significant reduction in available climatic niche space. Including the distribution of the principal or sole pollen forage plant, when modelling the distribution of monolectic or narrowly oligolectic species, did not improve the predictive accuracy of our models as the plant species were considerably more widespread than the specialised bees associated with them.

Discovery of novel GPVI receptor antagonists by structure-based repurposing.

Inappropriate platelet aggregation creates a cardiovascular risk that is largely managed with thienopyridines and aspirin. Although effective, these drugs carry risks of increased bleeding and drug 'resistance', underpinning a drive for new antiplatelet agents. To discover such drugs, one strategy is to identify a suitable druggable target and then find small molecules that modulate it. A good and unexploited target is the platelet collagen receptor, GPVI, which promotes thrombus formation. To identify inhibitors of GPVI that are safe and bioavailable, we docked a FDA-approved drug library into the GPVI collagen-binding site in silico. We now report that losartan and cinanserin inhibit GPVI-mediated platelet activation in a selective, competitive and dose-dependent manner. This mechanism of action likely underpins the cardioprotective effects of losartan that could not be ascribed to its antihypertensive effects. We have, therefore, identified small molecule inhibitors of GPVI-mediated platelet activation, and also demonstrated the utility of structure-based repurposing.

A multilevel model for the study of breastfeeding determinants in Brazil

The benefits of breastfeeding for the children`s health have been highlighted in many studies. The innovative aspect of the present study lies in its use of a multilevel model, a technique that has rarely been applied to studies on breastfeeding. The data reported were collected from a larger study, the Family Budget Survey-Pesquisa de Orcamentos Familiares, carried out between 2002 and 2003 in Brazil that involved a sample of 48 470 households. A representative national sample of 1477 infants aged 0-6 months was used. The statistical analysis was performed using a multilevel model, with two levels grouped by region. In Brazil, breastfeeding prevalence was 58%. The factors that bore a negative influence on breastfeeding were over four residents living in the same household [odds ratio (OR) = 0.68, 90% confidence interval (CI) = 0.51-0.89] and mothers aged 30 years or more (OR = 0.68, 90% CI = 0.53-0.89). The factors that positively influenced breastfeeding were the following: higher socio-economic levels (OR = 1.37, 90% CI = 1.01-1.88), families with over two infants under 5 years (OR = 1.25, 90% CI = 1.00-1.58) and being a resident in rural areas (OR = 1.25, 90% CI = 1.00-1.58). Although majority of the mothers was aware of the value of maternal milk and breastfed their babies, the prevalence of breastfeeding remains lower than the rate advised by the World Health Organization, and the number of residents living in the same household along with mothers aged 30 years or older were both factors associated with early cessation of infant breastfeeding before 6 months.

Clues to the presence of pathogenic fungi in certain environments

The presence of various pathogenic fungi in rather unsuspected hosts and environments has always attracted the attention of the scientific community. Reports on the putative role of animals in fungal infections of humans bear important consequences on public health as well as on the understanding of fungal ecology. Fungi are ubiquitous in nature and their great capacity for adaptation allows them to survive and indeed, to thrive, in plants, trees and other natural substrata. Nonetheless, we are just beginning to learn the significance that these diverse fungal habitats have on the increasing number of immunosuppressed individuals. The accidental or permanent presence of fungi in animals, plants, soils and watercourses should not be taken too lightly because they constitute the source where potential pathogens will be contracted. If those fungal habitats that carry the largest risks of exposure could be defined, if seasonal variations in the production of infectious propagules could be determined, and if their mode of transmission were to be assessed, it would be possible to develop protective measures in order to avoid human infection. Additionally, unsuspected avenues for the exploration of fungal survival strategies would be opened, thus enhancing our capacity to react properly to their advancing limits. This paper explores several ecological connections between human pathogenic fungi and certain animals, trees, waterways and degraded organic materials. The occurrence of such connections in highly endemic areas will hopefully furnish more precise clues to fungal habitats and allow the design of control programs aimed at avoiding human infection.

Study of Descendent Course of Buccal Nerve in Adults Individuals

The knowledge of the buccal nerve anatomy is of fundamental importance not only for the anesthesia but also for a safe intervention in the retromolar area. The aim of this work was to study its trajectory, in the area where it is related to the anterior margin of the ramus of the mandible, therefore providing important data for a safe intervention in the region. In this study we used 10 hemi-heads from male and female adults, from different ethnic groups. They were fixed in formol, and belong to the Anatomy Laboratory at the Faculty of Dentistry in Araraquara UNESP. These hemi-heads were dissected by lateral access, preserving the buccal nerve in its trajectory related to the anterior margin of the ramus of the mandible until its penetration in the buccinator muscle. Next, we desinserted the masseter muscle so that all the ramus of the mandible were exposed. Then, the following measurements were carried out: from the base of the mandible until the buccal nerve and from the base of the mandible until the apices of the mandibular coronoid process. These measurements were accomplished with a Mitutoyo CD-6'' CS digital paquimeter. The following average values were obtained: 32.26 mm (to the left side) and 32.04 mm (to the right side), from the base of the mandible until the buccal nerve and 59.09 mm (to the left side) and 58.95 mm (to the right side) from the base of the mandible until the apices of the coronoid process. We have concluded that normally, the buccal nerve crosses the anterior margin of the ramus of the mandible in an area which is above the superior half of the ramus of the mandible and also that the interventions in the retromolar region do not offer great risks of injury in the buccal nerve.

Acute and subchronic exposure of diplopods to substrate containing sewage mud: Tissular responses of the midgut

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of sedation on morbidity and mortality in the intensive care unit

CONTEXTO: Embora cerca de 30% a 50% dos pacientes hospitalizados em unidades de terapia intensiva (UTI) recebam algum tipo de sedativo, existe escassez de informações sobre efeitos adversos desta prática, especialmente no Brasil. Estes efeitos podem ser significantes e o uso de sedativos é associado a elevação de infecção e mortalidade, mesmo sendo difícil avaliar o impacto clínico deste procedimento. OBJETIVO: Avaliar o impacto da sedação sobre incidência de complicações e mortalidade em doentes graves durante internação em unidade de terapia intensiva. TIPO DE ESTUDO: Estudo prospectivo. LOCAL: Unidade de Terapia Intensiva Cirúrgica da Universidade Federal de São Paulo (UNIFESP) - Escola Paulista de Medicina. PARTICIPANTES: Após excluídos pacientes que permaneceram menos de 24 horas ou sem exames indispensáveis para o cálculo do índice de gravidade (APACHE II), restaram 307 pacientes. Estes foram divididos em dois grupos: Grupo Sedado e Grupo Não Sedado. Constatada heterogeneidade com relação ao APACHE II, foram pareados 97 sedados e 97 não sedados com idênticos índices de gravidade. VARIÁVEIS ESTUDADAS: Impacto da sedação e das técnicas sobre a mortalidade, tempo de internação, além da incidência de escara de decúbito ou pressão, trombose venosa profunda e infecção. RESULTADOS: Não houve diferença na incidência de trombose venosa profunda, entre os grupos Sedado e Não Sedado, enquanto que escara de decúbito foi significativamente maior nos sedados (p = 0,03). Infecção foi detectada em 45,4% dos pacientes com sedação e em 21,6% dos pacientes sem sedação (p = 0,006). A mortalidade para os pacientes que não receberam qualquer tipo de sedativo foi de 20,6% e, para aqueles que foram sedados durante a internação, foi de 52,6% (p < 0,0001). CONCLUSÕES: Conclui-se que a sedação está associada a maior duração da internação, morbidade e mortalidade significativas. Apesar da intensidade das associações encontradas, não é possível estabelecer relação causal entre sedação e mortalidade.