Refining animal research: The Animal Study Registry.

The Animal Study Registry (ASR; www.animalstudyregistry.org) was launched in January 2019 for preregistration of animal studies in order to increase transparency and reproducibility of bioscience research and to promote animal welfare. The registry is free of charge and is designed for exploratory and confirmatory studies within applied science as well as basic and preclinical research. The registration form helps scientists plan their study thoroughly by asking detailed questions concerning study design, methods, and statistics. With registration, the study automatically receives a digital object identifier (DOI) that marks it as intellectual property of the researcher. To accommodate the researchers concerns about theft of ideas, users can restrict the visibility of their registered studies for up to 5 years. The full content of the study becomes publicly accessible at the end of the embargo period. Because the platform is embedded in the infrastructure of the German Federal Government, continuity and data security are provided. By registering a study in the ASR, researchers can show their commitment to transparency and data quality to reviewers and editors, to third-party donors, and to the general public.

Use of meat juice and blood serum with a miniaturised protein microarray assay to develop a multi-parameter IgG screening test with high sample throughput potential for slaughtering pigs.

BACKGROUND: Serological screening of pig herds at the abattoir is considered a potential tool to improve meat inspection procedures and herd health management. Therefore, we previously reported the feasibility of a miniaturised protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs. The present study investigates whether the protein microarray-based assay is applicable for high sample throughput using either blood serum or meat juice. MATERIAL AND METHODS: Microarrays with 12 different antigens were produced by Abbott (formerly Alere Technologies GmbH) Jena, Germany in a previously offered 'ArrayTube' platform and in an 'ArrayStrip' platform for large-scale use. A test protocol for the use of meat juice on both microarray platforms was developed. Agreement between serum and meat juice was analysed with 88 paired samples from three German abattoirs. Serum was diluted 1:50 and meat juice 1:2. ELISA results for all tested antigens from a preceding study were used as reference test to perform Receiver Operating Characteristic analysis for both test specimens on both microarray platforms. RESULTS: High area under curve values (AUC > 0.7) were calculated for the analysis of T. gondii (0.87), Y. enterocolitica (0.97), Mycoplasma hyopneumoniae (0.84) and Actinobacillus pleuropneumoniae (0.71) with serum as the test specimen and for T. gondii (0.99), Y. enterocolitica (0.94), PRRSV (0.88), A. pleuropneumoniae (0.78) and Salmonella spp. (0.72) with meat juice as the test specimen on the ArrayStrip platform. Cohens kappa values of 0.92 for T. gondii and 0.82 for Y. enterocolitica were obtained for the comparison between serum and meat juice. When applying the new method in two further laboratories, kappa values between 0.63 and 0.94 were achieved between the laboratories for these two pathogens. CONCLUSION: Further development of a miniaturised pig-specific IgG protein microarray assay showed that meat juice can be used on microarray platforms. Two out of twelve tested antigens (T. gondii, Y. enterocolitica) showed high test accuracy on the ArrayTube and the ArrayStrip platform with both sample materials.

Comparison of Bayesian and frequentist methods for prevalence estimation under misclassification.

BACKGROUND: Various methods exist for statistical inference about a prevalence that consider misclassifications due to an imperfect diagnostic test. However, traditional methods are known to suffer from truncation of the prevalence estimate and the confidence intervals constructed around the point estimate, as well as from under-performance of the confidence intervals' coverage. METHODS: In this study, we used simulated data sets to validate a Bayesian prevalence estimation method and compare its performance to frequentist methods, i.e. the Rogan-Gladen estimate for prevalence, RGE, in combination with several methods of confidence interval construction. Our performance measures are (i) error distribution of the point estimate against the simulated true prevalence and (ii) coverage and length of the confidence interval, or credible interval in the case of the Bayesian method. RESULTS: Across all data sets, the Bayesian point estimate and the RGE produced similar error distributions with slight advantages of the former over the latter. In addition, the Bayesian estimate did not suffer from the RGE's truncation problem at zero or unity. With respect to coverage performance of the confidence and credible intervals, all of the traditional frequentist methods exhibited strong under-coverage, whereas the Bayesian credible interval as well as a newly developed frequentist method by Lang and Reiczigel performed as desired, with the Bayesian method having a very slight advantage in terms of interval length. CONCLUSION: The Bayesian prevalence estimation method should be prefered over traditional frequentist methods. An acceptable alternative is to combine the Rogan-Gladen point estimate with the Lang-Reiczigel confidence interval.

Aggregated aluminium exposure: risk assessment for the general population.

Aluminium is one of the most abundant elements in earth's crust and its manifold uses result in an exposure of the population from many sources. Developmental toxicity, effects on the urinary tract and neurotoxicity are known effects of aluminium and its compounds. Here, we assessed the health risks resulting from total consumer exposure towards aluminium and various aluminium compounds, including contributions from foodstuffs, food additives, food contact materials (FCM), and cosmetic products. For the estimation of aluminium contents in foodstuff, data from the German "Pilot-Total-Diet-Study" were used, which was conducted as part of the European TDS-Exposure project. These were combined with consumption data from the German National Consumption Survey II to yield aluminium exposure via food for adults. It was found that the average weekly aluminium exposure resulting from food intake amounts to approx. 50% of the tolerable weekly intake (TWI) of 1 mg/kg body weight (bw)/week, derived by the European Food Safety Authority (EFSA). For children, data from the French "Infant Total Diet Study" and the "Second French Total Diet Study" were used to estimate aluminium exposure via food. As a result, the TWI can be exhausted or slightly exceeded-particularly for infants who are not exclusively breastfed and young children relying on specially adapted diets (e.g. soy-based, lactose free, hypoallergenic). When taking into account the overall aluminium exposure from foods, cosmetic products (cosmetics), pharmaceuticals and FCM from uncoated aluminium, a significant exceedance of the EFSA-derived TWI and even the PTWI of 2 mg/kg bw/week, derived by the Joint FAO/WHO Expert Committee on Food Additives, may occur. Specifically, high exposure levels were found for adolescents aged 11-14 years. Although exposure data were collected with special regard to the German population, it is also representative for European and comparable to international consumers. From a toxicological point of view, regular exceedance of the lifetime tolerable aluminium intake (TWI/PTWI) is undesirable, since this results in an increased risk for health impairments. Consequently, recommendations on how to reduce overall aluminium exposure are given.

[Systematic Review of the Burden of Campylobacter-associated Disease]. / Systematischer Review zur Krankheitslast durch Campylobacter spp.

Bacteria of the genus Campylobacter spp. are one of the most common causes of gastroenteritis and can lead to serious sequelae. Several studies have estimated the disease burden of Campylobacter spp. with the quantitative metric of disability-adjusted life years (DALY). The aim of this systematic review is to give an overview of the information available about different countries and periods for which DALYs were calculated and how the different results are comparable. One of the most important transmission pathways for Campylobacter spp. is food. Therefore, special attention was given to studies that only estimated the foodborne disease burden of Campylobacter bacteria. With a systematic search for the period 1/1996-6/2016, one worldwide and 21 country-specific publications of the WHO were identified. Because of the different methods and the quality of the different data sets, the estimated results of all Campylobacter health outcomes of the country-specific studies vary from 0.4 DALYs per 100000 people in France to 109 DALY per population in Poland. The calculation of the attributable foodborne disease burden was based on the estimations of the incidences of all Campylobacter health outcomes with the associated uncertainty for each result. So the estimations of the foodborne disease burden show a large range from 0.5 DALYs per 100000 people in Greek to 21.2 DALYs per 100000 people in New Zealand. This span can only be partially explained by the country-specific variability in the food production, the consumption behavior and the incidence of Campylobacter bacteria.

[Health reporting of poisoning in Germany-a scientific investigation into the establishment of national poisoning monitoring in Germany : Background, preliminary work, requirements]. / Gesundheitsberichterstattung über Vergiftungen in Deutschland ­ Wissenschaftliche Untersuchung zur Einrichtung eines nationalen Monitorings von Vergiftungen in Deutschland : Hintergründe, Vorarbeiten, Anforderungen.

As a result of decentralized and non-harmonized documentation of poisoning cases within Germany, in comparison to other countries (e.g. the USA, Switzerland, Sweden, and the Netherlands), there is a substantial lack of harmonized national data, in particular for the early detection and risk assessment at the German Federal Institute for Risk Assessment (BfR).The establishment of a national monitoring system for poisonings in Germany is therefore an important step towards enabling faster and more efficient technical access to consolidated accumulated national poisoning data. Additionally, the monitoring would consolidate the publication of annual reports (modelled on the USA National Poison Data System reports) of the German Poison Information Centres and the BfR. Timely assessment of poisoning cases enables fast recognition and confirmation of acute as well as previously unrecognized poisoning hazards. Also relevant here is the safety of products and the associated protection, especially of children, against poisoning accidents.The continuous monitoring and reporting requirements of such a system necessitate additional and challenging work, suitable human resources and IT infrastructure and an annual budget that must be taken into consideration for overall as well as financial planning.

[Dietary exposure assessment of substances in foods : The BfR MEAL study - the first German total diet study]. / Expositionsschätzung von Stoffen in Lebensmitteln : Die BfR-MEAL-Studie ­ die erste Total-Diet-Studie in Deutschland.

The dietary exposure of a population to chemical substances is estimated based on representative consumption data and data on the occurrence of substances in foods. Consumption data in Germany for different age groups are provided by the National Nutrition Survey ll, VELS, EsKiMo and the ongoing KiESEL study. The data for the levels of substances in foods is currently obtained from the German food monitoring programme (Lebensmittel-Monitoring) and the German Food Composition Table (Bundeslebensmittelschlüssel) and is not sufficient for a comprehensive evaluation of dietary exposure in Germany. More data is needed and, in particular, the range of analysed substances should be broadened to reduce the uncertainties of the exposure assessment. A representative data basis for concentrations of substances in foods will be established with the first German total diet study, called the BfR MEAL study. The study will markedly reduce major uncertainties in the dietary exposure assessment through its wide coverage of substances in food groups, the inclusion of prepared foods, the orientation on low measurement limits as well as the analysis of substances without a reliable data basis.

Statistical approach for optimization of external quality assurance (EQA) studies of molecular and serological viral diagnostics.

Management of viral diagnostic quality is based on external quality assurance (EQA), where laboratories involved in diagnostics of a targeted virus are offered to analyze a panel of blinded samples. The utility of EQAs is compromised because of the absence of an approach to EQA design which upfront defines acceptance criteria and associated statistical analysis ensuring fair and consistent interpretation. We offer a rigorous statistically based approach for EQA planning. Instead of a conventional performance characteristic (the score) which is calculated as the sum of the points for correctly identified samples in a blinded test panel, Youden index is used as the performance measure. Unlike the score, Youden index requires an estimate of sensitivity and specificity and incorporates the relationship of these performance parameters. Based on the assumption that the coordinator is a reputable expert of viral diagnostics, the performance of the coordinator's laboratory is defined as a proficiency standard for performance evaluation. The immediate goal of EQA is defined as to obtain a statistically reliable estimation for every laboratory whether its performance meets the proficiency standard, while the overall goal is to match every laboratory to its specific performance level. Dependence of informational capacities of test panel from the panel size and content is quantitatively analyzed and the optimal design and informational capacities of both idealized panels (whose size is not restricted by financial factors) and currently feasible panels are considered. Our approach provides the basis both for rational design of currently feasible EQA test panels and for an increased panel size.

Estimation of the sensitivity and specificity of a Leptospira spp. in-house ELISA through Bayesian modelling.

The microscopic agglutination test (MAT) is still considered the gold standard for the diagnosis of leptospirosis, although studies have shown that the test is an imperfect gold standard for clinical samples and unsuitable for epidemiological studies. Here, test characteristics of an in-house ELISA were identified for both subclinical and clinical populations by Bayesian latent class models. A conditional dependence model for two diagnostic tests and two populations was adapted to analyse a clinical and a subclinical scenario, respectively. These Bayesian models were used to estimate the sensitivity and specificity of the in-house ELISA and the MAT as well as the prevalences. The Bayesian estimates of the in-house ELISA were: clinical sensitivity=83.0%, clinical specificity=98.5%, subclinical sensitivity=85.7% and subclinical specificity=99.1%. In contrast, the estimates of the MAT were: clinical sensitivity=65.6%, clinical specificity=97.7%, subclinical sensitivity 54.9% and subclinical specificity=97.3%. The results show the suitability of the in-house ELISA for both clinical investigations and epidemiological studies in mildly endemic areas.

Regulatory toxicology in the twenty-first century: challenges, perspectives and possible solutions.

The advent of new testing systems and "omics"-technologies has left regulatory toxicology facing one of the biggest challenges for decades. That is the question whether and how these methods can be used for regulatory purposes. The new methods undoubtedly enable regulators to address important open questions of toxicology such as species-specific toxicity, mixture toxicity, low-dose effects, endocrine effects or nanotoxicology, while promising faster and more efficient toxicity testing with the use of less animals. Consequently, the respective assays, methods and testing strategies are subject of several research programs worldwide. On the other hand, the practical application of such tests for regulatory purposes is a matter of ongoing debate. This document summarizes key aspects of this debate in the light of the European "regulatory status quo", while elucidating new perspectives for regulatory toxicity testing.

German outbreak of Escherichia coli O104:H4 associated with sprouts.

BACKGROUND: A large outbreak of the hemolytic-uremic syndrome caused by Shiga-toxin-producing Escherichia coli O104:H4 occurred in Germany in May 2011. The source of infection was undetermined. METHODS: We conducted a matched case-control study and a recipe-based restaurant cohort study, along with environmental, trace-back, and trace-forward investigations, to determine the source of infection. RESULTS: The case-control study included 26 case subjects with the hemolytic-uremic syndrome and 81 control subjects. The outbreak of illness was associated with sprout consumption in univariable analysis (matched odds ratio, 5.8; 95% confidence interval [CI], 1.2 to 29) and with sprout and cucumber consumption in multivariable analysis. Among case subjects, 25% reported having eaten sprouts, and 88% reported having eaten cucumbers. The recipe-based study among 10 groups of visitors to restaurant K included 152 persons, among whom bloody diarrhea or diarrhea confirmed to be associated with Shiga-toxin-producing E. coli developed in 31 (20%). Visitors who were served sprouts were significantly more likely to become ill (relative risk, 14.2; 95% CI, 2.6 to ∞). Sprout consumption explained 100% of cases. Trace-back investigation of sprouts from the distributor that supplied restaurant K led to producer A. All 41 case clusters with known trading connections could be explained by producer A. The outbreak strain could not be identified on seeds from the implicated lot. CONCLUSIONS: Our investigations identified sprouts as the most likely outbreak vehicle, underlining the need to take into account food items that may be overlooked during subjects' recall of consumption.

How long can you store vitamins? Stability of tocopherols and tocotrienol during different storage conditions in broccoli and blueberries.

Differences between the stability of α-, ß-, γ-, and δ-tocopherol as well α-tocotrienol stored at -20 °C and -80 °C were studied in broccoli and blueberry samples. Before storage up to 28 days, they underwent different initializing processes such as freezing quickly with liquid nitrogen and freeze-drying, followed by homogenization. While α-tocopherol levels in blueberries did not significantly differ, levels in broccoli were substantially higher after homogenization of freeze-dried samples compared to fresh broccoli samples. This might be caused by higher extractability of α-tocopherol from the changed cell structure. Storage of fresh broccoli samples at -20 °C led to decreasing α-tocopherol levels. Nevertheless, the deviation between freeze-dried samples to the initial fresh samples and fresh samples frozen with liquid nitrogen stored at -20 °C for 7 days were in the same order of magnitude. In conclusion, storage up to 7 days for vitamin relevant samples before analysis seemed to be justifiable.

Scientific Committee guidance on appraising and integrating evidence from epidemiological studies for use in EFSA's scientific assessments.

EFSA requested its Scientific Committee to prepare a guidance document on appraising and integrating evidence from epidemiological studies for use in EFSA's scientific assessments. The guidance document provides an introduction to epidemiological studies and illustrates the typical biases, which may be present in different epidemiological study designs. It then describes key epidemiological concepts relevant for evidence appraisal. This includes brief explanations for measures of association, exposure assessment, statistical inference, systematic error and effect modification. The guidance then describes the concept of external validity and the principles of appraising epidemiological studies. The customisation of the study appraisal process is explained including tailoring of tools for assessing the risk of bias (RoB). Several examples of appraising experimental and observational studies using a RoB tool are annexed to the document to illustrate the application of the approach. The latter part of this guidance focuses on different steps of evidence integration, first within and then across different streams of evidence. With respect to risk characterisation, the guidance considers how evidence from human epidemiological studies can be used in dose-response modelling with several different options being presented. Finally, the guidance addresses the application of uncertainty factors in risk characterisation when using evidence from human epidemiological studies.

Trace-back and trace-forward tools developed ad hoc and used during the STEC O104:H4 outbreak 2011 in Germany and generic concepts for future outbreak situations.

The Shiga toxin-producing Escherichia coli O104:H4 outbreak in Germany in 2011 required the development of appropriate tools in real-time for tracing suspicious foods along the supply chain, namely salad ingredients, sprouts, and seeds. Food commodities consumed at locations identified as most probable site of infection (outbreak clusters) were traced back in order to identify connections between different disease clusters via the supply chain of the foods. A newly developed relational database with integrated consistency and plausibility checks was used to collate these data for further analysis. Connections between suppliers, distributors, and producers were visualized in network graphs and geographic projections. Finally, this trace-back and trace-forward analysis led to the identification of sprouts produced by a horticultural farm in Lower Saxony as vehicle for the pathogen, and a specific lot of fenugreek seeds imported from Egypt as the most likely source of contamination. Network graphs have proven to be a powerful tool for summarizing and communicating complex trade relationships to various stake holders. The present article gives a detailed description of the newly developed tracing tools and recommendations for necessary requirements and improvements for future foodborne outbreak investigations.

Collection of occurrence data in foods - The value of the BfR MEAL study in addition to the national monitoring for dietary exposure assessment.

Two different data sets of occurrence data are available in Germany at present: the German National Food Monitoring and the BfR MEAL Study. To determine the suitability of each data set for exposure assessment and to develop concepts for a target-oriented selection and application of data, possibilities, limitations and scope of substance as well as food selection is quantitatively compared. The National Monitoring data provides comprehensive information on the variability of substance levels. This enables short- and long-term exposure assessment and consumer-loyal scenarios. The BfR MEAL Study supplements the monitoring data set with > 100 substances or by complementing the food spectrum for substances already included in the National Monitoring. The study design benefits especially the long-term dietary exposure assessment for the German population including the total diet. Using both programmes enables case-dependent selection of the appropriate dataset and in combination both sets can contribute to enhanced consumer safety.

Results of the BfR MEAL Study: The food type has a stronger impact on calcium, potassium and phosphorus levels than factors such as seasonality, regionality and type of production.

The BfR MEAL Study aims to provide representative levels of chemical substances in foods consumed by the population in Germany for dietary exposure assessment. Calcium, potassium and phosphorus (Ca, K, P) are essential to obtain physiological functions in humans. Levels were investigated in 356 foods. Foods were purchased representatively, prepared as typically consumed and pooled before analysis. High mean levels were found in milk, dairy products, legumes, nuts, oilseeds and spices as well as chia seeds (Ca, K, P), chewing gum (Ca) and cocoa powder (K). Different levels comparing organically and conventionally produced foods were determined among others in cereal cracker (puffed), olives and tofu. Higher K levels were found in fried compared to boiled potatoes. Similar P levels were mainly found in regionally and seasonally sampled foods. These data provide a substantially improved basis to address dietary exposure assessment of the population in Germany for Ca, K and P.

Exposure assessment of methylmercury in samples of the BfR MEAL Study.

The BfR MEAL Study is the first German total diet study and will establish a representative and comprehensive database for dietary exposure assessment in Germany. The present study reports first results of the BfR MEAL Study regarding methylmercury in fish, seafood and mushrooms. In total, 34 MEAL foods were purchased nationally or regionally according to a defined sampling plan, prepared in a representative way for German households, pooled into 49 samples, homogenized and subjected to ICP-MS analysis. Dogfish, tuna, ocean perch, halibut and eel were the fish species with highest MeHg concentrations, while levels in mushrooms and mushroom products had markedly lower MeHg levels. Exposure was estimated by matching the present results with consumption data at appropriate levels of food group aggregation. MeHg exposure for adult high consumers (P 95) exceeded the tolerable weekly intake recommended by the European Food Safety Authority in two age groups (14-17 and 18-24 years). In children, no age group exceeded the recommended tolerable weekly intake. Regional samples differed only slightly in MeHg levels. The differences in exposure found in four regions of Germany were influenced by consumption habits rather than MeHg level in the investigated food.

Analysis of German BSE Surveillance Data: Estimation of the Prevalence of Confirmed Cases versus the Number of Infected, but Non-Detected, Cattle to Assess Confidence in Freedom from Infection.

Quantitative risk assessments for Bovine spongiform encephalopathy (BSE) necessitate estimates for key parameters such as the prevalence of infection, the probability of absence of infection in defined birth cohorts, and the numbers of BSE-infected, but non-detected cattle entering the food chain. We estimated three key parameters with adjustment for misclassification using the German BSE surveillance data using a Gompertz model for latent (i.e., unobserved) age-dependent detection probabilities and a Poisson response model for the number of BSE cases for birth cohorts 1999 to 2015. The models were combined in a Bayesian framework. We estimated the median true BSE prevalence between 3.74 and 0.216 cases per 100,000 animals for the birth cohorts 1990 to 2001 and observed a peak for the 1996 birth cohort with a point estimate of 16.41 cases per 100,000 cattle. For birth cohorts ranging from 2002 to 2013, the estimated median prevalence was below one case per 100,000 heads. The calculated confidence in freedom from disease (design prevalence 1 in 100,000) was above 99.5% for the birth cohorts 2002 to 2006. In conclusion, BSE surveillance in the healthy slaughtered cattle chain was extremely sensitive at the time, when BSE repeatedly occurred in Germany (2000-2009), because the entry of BSE-infected cattle into the food chain could virtually be prevented by the extensive surveillance program during these years and until 2015 (estimated non-detected cases/100.000 [95% credible interval] in 2000, 2009, and 2015 are 0.64 [0.5,0.8], 0.05 [0.01,0.14], and 0.19 [0.05,0.61], respectively).

Refinement of dietary exposure assessment using origin-related scenarios.

Global sourcing of food may lead to variability in concentrations of contaminants or pesticide residues. It would be important to incorporate origin influences in dietary exposure assessment. To characterise uncertainties, substance concentrations from GFM (German Food Monitoring), chosen based on the highest CV (coefficient of variation), and food consumption from NVS II (German National Nutrition Survey II) were combined in standard scenarios. Averages or higher percentiles of non-grouped concentrations were used. Additional origin-related scenarios used concentrations grouped by origin. For bromide in tomatoes the most conservative origin-related scenario for Italian tomatoes resulted in the highest exposure of 0.015 mg/d/kg BW. The impact of origin was not covered by the conservative standard scenario (0.006 mg/d/kg BW). For ethephon in pineapples and aluminium in kiwifruits, the highest intake estimates were obtained with the conservative standard scenario resulting in 0.895 µg/d/kg BW and 0.023 mg/week/kg BW, respectively. In these two cases, standard scenarios cover origin influences but the conservative origin-related scenario based on origins with higher concentrations identifies lower exposures of 0.835 µg/d/kg BW for ethephon from African pineapples and 0.014 mg/week/kg BW for aluminium from non-EU kiwifruits. Hence, the inclusion of origin information can refine exposure assessment.