PBTK model-based analysis of CYP3A4 induction and the toxicokinetics of the pyrrolizidine alkaloid retrorsine in man.

Cytochrome P450 (CYP)3A4 induction by drugs and pesticides plays a critical role in the enhancement of pyrrolizidine alkaloid (PA) toxicity as it leads to increased formation of hepatotoxic dehydro-PA metabolites. Addressing the need for a quantitative analysis of this interaction, we developed a physiologically-based toxicokinetic (PBTK) model. Specifically, the model describes the impact of the well-characterized CYP3A4 inducer rifampicin on the kinetics of retrorsine, which is a prototypic PA and contaminant in herbal teas. Based on consumption data, the kinetics after daily intake of retrorsine were simulated with concomitant rifampicin treatment. Strongest impact on retrorsine kinetics (plasma AUC 24 and C max reduced to 67% and 74% compared to the rifampicin-free reference) was predicted directly after withdrawal of rifampicin. At this time point, the competitive inhibitory effect of rifampicin stopped, while CYP3A4 induction was still near its maximum. Due to the impacted metabolism kinetics, the cumulative formation of intestinal retrorsine CYP3A4 metabolites increased to 254% (from 10 to 25 nmol), while the cumulative formation of hepatic CYP3A4 metabolites was not affected (57 nmol). Return to baseline PA toxicokinetics was predicted 14 days after stop of a 14-day rifampicin treatment. In conclusion, the PBTK model showed to be a promising tool to assess the dynamic interplay of enzyme induction and toxification pathways.

PBTK modeling of the pyrrolizidine alkaloid retrorsine to predict liver toxicity in mouse and rat.

Retrorsine is a hepatotoxic pyrrolizidine alkaloid (PA) found in herbal supplements and medicines, food and livestock feed. Dose-response studies enabling the derivation of a point of departure including a benchmark dose for risk assessment of retrorsine in humans and animals are not available. Addressing this need, a physiologically based toxicokinetic (PBTK) model of retrorsine was developed for mouse and rat. Comprehensive characterization of retrorsine toxicokinetics revealed: both the fraction absorbed from the intestine (78%) and the fraction unbound in plasma (60%) are high, hepatic membrane permeation is dominated by active uptake and not by passive diffusion, liver metabolic clearance is 4-fold higher in rat compared to mouse and renal excretion contributes to 20% of the total clearance. The PBTK model was calibrated with kinetic data from available mouse and rat studies using maximum likelihood estimation. PBTK model evaluation showed convincing goodness-of-fit for hepatic retrorsine and retrorsine-derived DNA adducts. Furthermore, the developed model allowed to translate in vitro liver toxicity data of retrorsine to in vivo dose-response data. Resulting benchmark dose confidence intervals (mg/kg bodyweight) are 24.1-88.5 in mice and 79.9-104 in rats for acute liver toxicity after oral retrorsine intake. As the PBTK model was built to enable extrapolation to different species and other PA congeners, this integrative framework constitutes a flexible tool to address gaps in the risk assessment of PA.

Influence of motivational placebo-related factors on the effects of exercise treatment in depressive adolescents.

Recent meta-analyses reveal a moderate effect of physical activity (PA) in the treatment of adolescent depression. However, not only the underlying neurobiological mechanisms, also the influences of placebo-related motivational factors (beliefs and expectancies in sporting, enjoyment and prior sports experiences), are still unclear. Based on the data of our prior study "Mood Vibes", we hypothesized that placebo-inherent factors like positive prior sports experiences and motivational factors, (positive beliefs, expectancies, and enjoyment related to PA), would increase the effects of an add-on exercise-therapy in juvenile depression. From 64 included depressed adolescents, 41 underwent an intensive add-on PA-therapy. Motivational factors were assessed using sport-specific scales. The changes in depression scores under treatment were rated by self-rating scale (German "Childhood Depression Inventory", (DIKJ)). A mixed model for repeated measures (MMRM) was used to analyze the effects of the different motivational variates on DIKJ. While prior sports experiences had no impact, motivational factors showed a significant effect on PA-induced changes in DIKJ scores (p = 0.002). The demotivated participants improved less, whereas it was sufficient to be neutral towards sporting to benefit significantly more. Motivational placebo-related factors (beliefs, expectancies and enjoyment regarding PA) affected the outcomes of an exercise treatment in depressed adolescents. Yet, a neutral mindset was sufficient to profit more from PA. Prior sporting in the sense of positive conditioning and as a protective factor did not play a role. Knowledge about these influences could in a second step help to develop tailored therapies.

[Evaluation of the Language-related Domain of the Pediatric Developmental Screening Instrument for Preschool Health Examinations (SOPESS): Results from the Prospective Child Health Survey ikidS]. / Evaluation der Sprachaufgaben des sozialpädiatrischen Entwicklungsscreenings für Schuleingangsuntersuchungen (SOPESS): Ergebnisse der prospektiven Kindergesundheitsstudie ikidS.

PURPOSE OF THE STUDY: Developmental delays at school enrollment can affect early educational achievement. Thus, diagnosis and treatment prior to school entry are important. In Germany, SOPESS - a pediatric developmental screening instrument for preschool health examinations (PHE) - was introduced in several federal states. We investigated the relationships between the results of the language-related domain of the SOPESS and later academic language competencies at the end of first grade. PROCEDURES: Data of the population-based cohort study ikidS (Rhineland Palatinate; Germany) and the PHE provided by public health authorities were linked at individual level. The relationship between the SOPESS-language risk score (range: 0 to 6, higher values indicate more developmental delay) and class teacher-based ratings of academic language competencies (range: -4 to+4, higher values indicate better competencies) was investigated with linear regression models and adjusted for several socio-economic and medical language predictors (e. g. age at school entry, gender, parental education, migration background, hearing disorder). The additional benefits of the SOPESS-language risk score was analyzed using analysis of variance. RESULTS: In total, 1357 children (48% girls, age at PHE 4.9-7.2 years) were included. A clear negative relationship between the SOPESS-language risk score and competencies in spoken and written language was found. Mean academic language competencies decreased with increasing SOPESS-language risk level from 0.8 (SD=1.7) at a risk score of 0 to-3.2 (SD=0.9) at a risk score of 6. The relationship persisted after adjusting for other language predictors. Analysis of variance showed significant benefits of the SOPESS-language risk score, even when further language predictors were considered. CONCLUSIONS: There was a clear relationship between results of the SOPESS-language screening and later academic language competencies. Hence, the language-related domain of the SOPESS may be useful for further recommendations of tailored special educational and medical support services, especially if additional socio-economic and medical factors are considered.

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.

Mental health problems and school performance in first graders: results of the prospective cohort study ikidS.

We aimed to estimate unbiased effects of mental health problems (MHPs) on school performance in first graders, with an emphasis on rigorous adjustment for potential confounders. A population-based prospective cohort study was performed in the area of Mainz-Bingen (Germany). In 2015, all preschoolers were approached, and the presence and type of MHP (externalising/internalising) and other physical chronic health conditions were identified by the preschool health examination and study-specific questionnaires. At the end of the first grade, school performance (reading, writing, numeracy, and science) was assessed by the class teacher and rated on a four-item scale ranging from - 8 to + 8. Of 3683 children approached, 2003 (54%) were enrolled. School performance was available for 1462 children (51% boys, mean age 7.3 years). Of these, 41% had signs of at least one MHP. Compared to children without indications of mental and physical chronic health conditions, children with MHPs had lower school performance scores [adjusted mean difference - 0.98, 95% CI (- 1.35; - 0.61); P < 0.001]. Regarding the type of MHP, externalising MHPs were associated with poor school performance [adjusted mean difference - 1.44, 95% CI (- 1.83; - 1.05); P < 0.001], while internalising MHPs were not. Children with hyperactivity inattention problems were most affected [adjusted mean difference - 1.96, 95% CI (- 2.36; - 1.56); P < 0.001]. Externalising MHPs and in particular hyperactivity inattention problems may already affect school performance in early primary school. Identification of children with externalising MHPs prior to school entry may help to prevent impaired academic achievement in affected children.

Bubble Formation in Children and Adolescents after Two Standardised Shallow Dives.

Circulating venous bubbles after dives are associated with symptoms of decompression sickness in adults. Up to now it is not known to what extent children and adolescents are subjected to a bubble formation during their shallow dives and if there are possible indications for that. The aim of this pilot study is to investigate whether bubbles and/or symptoms occur after standardised repeated dives performed by young divers. 28 children and adolescents (13.5±1.1 years) carried out two 25 min dives to a depth of 10 m with a 90 min surface interval. Before and after, echocardiographic data were recorded and evaluated with regard to circulating bubbles with an extended Eftedal-Brubakk-Scale by 2 different examiners. Bubbles were observed for a total of 6 subjects, Grade I (n=5) and Grade III (n=1). None of them showed any symptoms of decompression sickness. No differences were established regarding potential influencing factors on bubble formation between the groups with and without bubbles. The results indicate that even relatively shallow and short dives can generate venous bubbles in children and adolescents. To what extent this relates to the decompression sickness or clinical symptoms cannot be validated at this point.

[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.

Metabolic Health-The Role of Adipo-Myokines.

Obesity is now a worldwide epidemic. In recent years, different phenotypes of obesity, ranging from metabolically healthy normal weight to metabolically unhealthy obese, were described. Although there is no standardized definition for these phenotypes or for metabolic health, the influence of lifestyle and early-life factors is undisputed. In this context, the ratio of muscle-to-fat tissue seems to play a crucial role. Both adipose tissue and skeletal muscle are highly heterogeneous endocrine organs secreting several hormones, with myokines and adipokines being involved in local autocrine/paracrine interactions and crosstalk with other tissues. Some of these endocrine factors are secreted by both tissues and are, therefore, termed adipo-myokines. High (cardiorespiratory) fitness as a surrogate parameter for an active lifestyle is epidemiologically linked to "better" metabolic health, even in the obese; this may be partly due to the role of adipo-myokines and the crosstalk between adipose and muscle tissue. Therefore, it is essential to consider (cardiovascular) fitness in the definition of metabolically healthy obese/metabolic health and to perform longitudinal studies in this regard. A better understanding of both the (early-life) lifestyle factors and the underlying mechanisms that mediate different phenotypes is necessary for the tailored prevention and personalized treatment of obesity.

Whole body vibration added to treatment as usual is effective in adolescents with depression: a partly randomized, three-armed clinical trial in inpatients.

There is growing evidence for the effectiveness of exercise in the treatment of adult major depression. With regard to adolescents, clinical trials are scarce. Due to the inherent symptoms of depression (lack of energy, low motivation to exercise), endurance training forms could be too demanding especially in the first weeks of treatment. We hypothesized that an easy-to-perform passive muscular training on a whole body vibration (WBV) device has equal anti-depressive effects compared to a cardiovascular training, both administered as add-ons to treatment as usual (TAU). Secondly, we presumed that both exercise interventions would be superior in their response, compared to TAU. In 2 years 64 medication-naïve depressed inpatients aged 13-18, were included. Both exercise groups fulfilled a supervised vigorous training for 6 weeks. Depressive symptoms were assessed by self-report ("Depressions Inventar für Kinder und Jugendliche"-DIKJ) before intervention and after weeks 6, 14 and 26. Compared to TAU, both groups responded earlier and more strongly measured by DIKJ scores, showing a trend for the WBV group after week 6 (p = 0.082). The decrease became statistically significant for both intervention groups after week 26 (p = 0.037 for ergometer and p = 0.042 for WBV). Remission rates amounted to 39.7% after week 6 and 66% after week 26, compared to 25% after week 26 in TAU. These results provide qualified support for the effectiveness of exercise as add-on treatment for medication-naïve depressed adolescents. The present results are limited by the not randomized control group.

[Recommendations for Physical Activity During and After Pregnancy]. / Bewegungsempfehlungen für Frauen während und nach der Schwangerschaft.

Regular physical activity during and after pregnancy has beneficial effects for mother and child. German recommendations for physical activity during pregnancy are still missing.We searched PubMed (MedLine) for guidelines published between January 2010 and September 2015 on physical activity during and after pregnancy.A systematic literature research identified 29 articles. Recommendations in the context of specific diseases (gestational diabetes, preeclampsia, obesity, hypertension, PCO, incontinence) or intervention programs were excluded. Only 2 articles met the criteria. 2 separate articles summarizing all international recommendations for physical activity during and after pregnancy were included.In order for mother and child to benefit from physical activity, pregnant women without contraindications should be encouraged to participate in regular physical activity or to remain active. Healthy pregnant and postpartum women should exercise for at least 150 min per week (analogically 20-30 min per day on most or all days of the week) in moderate to vigorous aerobic intensity.

[Identification of Good-Practice Projects in Promoting Physical Activity - Methods, Pitfalls and Sampled Outcomes]. / Identifikation von Beispielen guter Praxis der Bewegungsförderung ­ Methoden, Fallstricke und ausgewählte Ergebnisse.

The aim of this paper is to identify and show examples of good practice of public health promotion. For this, uniform quality criteria were worked out under consideration of national and international scientific literature.For the identification of examples of good practice, a comparison of different quality criteria was carried out and combined with each other in a first step. In the following step, examples of good practice were identified after a comprehensive search. The choice of the "good-practice" projects is exemplary and lays no claim to completeness.6 main quality criteria (QC) of programs promoting physical activity could be identified in the national and international context. The analysis showed altogether 10 projects which can exemplarily be classified as examples of good practice of the target groups of children and teenagers, adults, older people and people with pre-existing illnesses. These projects, however, show major differences in their (methodological) quality.The analysis reports a lack of "Good-Practice" examples. Deficits lie mainly in documentation and sustainability. Because of incomplete documentation, an assessment as a "Good-Practice" example is only possible to a limited extent; a lot of information, particularly in the evaluation, is missing.

[Recommendations for Physical Activity and Sedentary Behaviour for Children and Adolescents: Methods, Database and Rationale]. / Empfehlungen für körperliche Aktivität und Inaktivität von Kindern und Jugendlichen ­ Methodisches Vorgehen, Datenbasis und Begründung.

The benefits of physical activity and a reduction of sedentary behaviour in childhood and adolescence are well established.Based on a quality-assured literature review, the German recommendations were updated. Guidelines for infants, toddlers, children of kindergarten and primary school age and youth are clearly depictedIn general, a higher amount of physical activity is associated with more health benefits. Preschool age children should have 180 min of physical activity daily, and from primary school age on, at least 90 min daily are recommended. Sedentary behaviour, especially time in front of screens, is to be reduced to a minimum.

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.

Secular trends in pregnancy weight gain in German women and their influences on foetal outcome: a hospital-based study.

BACKGROUND: Increasing rates of overweight have been reported. In Germany, women of childbearing age are especially affected. Those women are at increased risks of several peri- and postnatal complications. The purpose of this study was to carry out Germany's first study in terms of secular trends of overweight and weight gain during pregnancy related to foetal clinical outcomes (birth weight, Apgar score and umbilical blood pH). METHODS: A database maintained by a large regional university hospital in Cologne, Germany was used to evaluate clinical routine data from 1996 to 2012. 11771 women (23.5 ± 5.4 years; 18-48 years), who gave birth to a live singleton child (>2000 gram) were included. Recommended weight gain during pregnancy was based on IOM guidelines: Total weight gain range for underweight (initial BMI < 18.5 kg/m2) is 12.5-18 kg/ 28-40 lbs respectively, for normal-weight (initial BMI 18.5 -24.9 kg/m2) is 11.5-16 kg/ 25-35 lbs respectively, for overweight (initial BMI 25.0-29.9 kg/m2) is 7-11.5 kg/ 15-25 lbs respectively and for obese (initial BMI ≥ 30.0 kg/m2) is 5-9 kg/ 11-20 lbs respectively.A one-way variance analysis was employed to test for differences in particular factors in various groups. Multiple linear regression analysis was used to model impact factors. RESULTS: Over the second analysed period (2005-2012), the number of women with high weight gain increased from 33.8% to 42.9% (p <0.001). 54.5% overweight and 57.7% obese women were affected (p <0.001). Women with high weight gain were 54.5% significantly more likely to give birth to an infant ≥ 4000 grams than women with normal (31.7%) or low weight gain (13.8%, p < 0.001). Women with normal weight gain had significantly better foetal outcomes in terms of the Apgar score at 5 min and umbilical cord blood pH. CONCLUSION: These data confirm an increase in maternal weight gain before and during pregnancy. An excessive weight gain is accompanied by macrosomia, lower Apgar scores and pH-value. Women should therefore be advised about the risks of obesity before and during pregnancy as well as excessive maternal weight gain during pregnancy.

Strength training alters MCT1-protein expression and exercise-induced translocation in erythrocytes of men with non-insulin-dependent type-2 diabetes.

We investigated the cellular distribution of lactate transporter (MCT1) and its chaperone CD147 (using immunohistochemistry and fluorescence-activated cell sorting) in the erythrocytes of men with non-insulin-dependent type-2 diabetes (NIDDM, n = 11, 61 ± 8 years of age) under acute exercise (ergometer cycling test, World Health Organisation scheme) performed before and after a 3-month strength training program. Cytosolic MCT1 distribution and membraneous CD147 density did not change after acute exercise (ergometer). After the 3-month strength training, MCT1-density was increased and the reaction of MCT1 (but not that of CD147) towards acute exercise (ergometer) was altered. MCT1 localisation was shifted from the centre to the cellular membrane. This resulted in a decrease in the immunohistochemically measured cytosolic MCT1-density. We conclude that strength training alters the acute exercise reaction of MCT1 but not that of CD147 in erythrocytes in patients with NIDDM. This reaction may contribute to long-term normalisation and stabilisation of the regulation of lactate plasma concentration in NIDDM.

Identification and X-ray co-crystal structure of a small-molecule activator of LFA-1-ICAM-1 binding.

Stabilization of protein-protein interactions by small molecules is a concept with few examples reported to date. Herein we describe the identification and X-ray co-crystal structure determination of IBE-667, an ICAM-1 binding enhancer for LFA-1. IBE-667 was designed based on the SAR information obtained from an on-bead screen of tagged one-bead one-compound combinatorial libraries by confocal nanoscanning and bead picking (CONA). Cellular assays demonstrate the activity of IBE-667 in promoting the binding of LFA-1 on activated immune cells to ICAM-1.

Meta-Analysis of Integrated Proteomic and Transcriptomic Data Discerns Structure-Activity Relationship of Carbon Materials with Different Morphologies.

The Fiber Pathogenicity Paradigm (FPP) establishes connections between fiber structure, durability, and disease-causing potential observed in materials like asbestos and synthetic fibers. While emerging nanofibers are anticipated to exhibit pathogenic traits according to the FPP, their nanoscale diameter limits rigidity, leading to tangling and loss of fiber characteristics. The absence of validated rigidity measurement methods complicates nanofiber toxicity assessment. By comprehensively analyzing 89 transcriptomics and 37 proteomics studies, this study aims to enhance carbon material toxicity understanding and proposes an alternative strategy to assess morphology-driven toxicity. Carbon materials are categorized as non-fibrous, high aspect ratio with shorter lengths, tangled, and rigid fibers. Mitsui-7 serves as a benchmark for pathogenic fibers. The meta-analysis reveals distinct cellular changes for each category, effectively distinguishing rigid fibers from other carbon materials. Subsequently, a robust random forest model is developed to predict morphology, unveiling the pathogenicity of previously deemed non-pathogenic NM-400 due to its secondary structures. This study fills a crucial gap in nanosafety by linking toxicological effects to material morphology, in particular regarding fibers. It demonstrates the significant impact of morphology on toxicological behavior and the necessity of integrating morphological considerations into regulatory frameworks.

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.