Dynamics of the bacterial gut microbiota during controlled human infection with Necator americanus larvae.

Hookworms are soil-transmitted helminths that use immune-evasive strategies to persist in the human duodenum where they are responsible for anemia and protein loss. Given their location and immune regulatory effects, hookworms likely impact the bacterial microbiota. However, microbiota studies struggle to deconvolute the effect of hookworms from confounders such as coinfections and malnutrition. We thus used an experimental human hookworm infection model to explore temporal changes in the gut microbiota before and during hookworm infection. Volunteers were dermally exposed to cumulative dosages of 50, 100 or 150 L3 Necator americanus larvae. Fecal samples were collected for microbiota profiling through 16S rRNA gene amplicon sequencing at weeks zero, four, eight, fourteen and twenty. During the acute infection phase (trial week zero to eight) no changes in bacterial diversity were detected. During the established infection phase (trial week eight to twenty), bacterial richness (Chao1, p = .0174) increased significantly over all volunteers. No relation was found between larval dosage and diversity, stability or relative abundance of individual bacterial taxa. GI symptoms were associated with an unstable microbiota during the first eight weeks and rapid recovery at week twenty. Barnesiella, amongst other taxa, was more abundant in volunteers with more GI symptoms throughout the study. In conclusion, this study showed that clinical GI symptoms following N. americanus infection are associated with temporary microbiota instability and relative abundance of specific bacterial taxa. These results suggest a possible role of hookworm-induced enteritis on microbiota stability.

Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines.

When studying the microbiome using next-generation sequencing, the DNA extraction method, sequencing procedures, and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity, and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with a maximum 3-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for observed richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs, and variation induced by DNA extraction methods was lower than intersubject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally, with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we again demonstrate the importance of including negative controls when analyzing low-bacterial-biomass samples.IMPORTANCE Method choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiota studies. Our methods were applied not only to commonly studied samples for microbiota analysis, e.g., feces, but also to more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g., urine and tumor biopsy specimens) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiota studies, especially when low bacterial biomass is expected.

Preconception lifestyle intervention reduces long term energy intake in women with obesity and infertility: a randomised controlled trial.

BACKGROUND: The preconceptional period may be an optimal window of opportunity to improve lifestyle. We previously showed that a 6 month preconception lifestyle intervention among women with obesity and infertility was successful in decreasing the intake of high caloric snacks and beverages, increasing physical activity and in reducing weight in the short term. We now report the effects of the preconception lifestyle intervention on diet, physical activity and body mass index (BMI) at 5.5 years (range = 3.7-7.0 years) after the intervention. METHODS: We followed women who participated in the LIFEstyle study, a multicentre RCT in which women with obesity and infertility were assigned to a six-month lifestyle intervention program or prompt infertility treatment (N = 577). Diet and physical activity 5.5 years later were assessed with an 173-item food frequency questionnaire (N = 175) and Actigraph triaxial accelerometers (N = 155), respectively. BMI was calculated from self-reported weight and previously measured height (N = 179). Dietary intake, physical activity, and BMI in the intervention and control group were compared using multivariate regression models. Additionally, dietary intake, physical activity and BMI of women allocated to the intervention arm with successful weight loss during the intervention (i.e. BMI < 29 kg/m2 or ≥ 5% weight loss), unsuccessful weight loss and the control group were compared with ANCOVA. RESULTS: Although BMI did not differ between the intervention and control group 5.5 years after the intervention (- 0.5 kg/m2 [- 2.0;1.1]; P = 0.56), the intervention group did report a lower energy intake (- 216 kcal/day [- 417;-16]; P = 0.04). Women in the intervention arm who successfully lost weight during the intervention had a significantly lower BMI at follow-up compared to women in the intervention arm who did not lose weight successfully (- 3.4 kg/m2 [- 6.3;-0.6]; P = 0.01), and they reported a significantly lower energy intake compared to the control group (- 301 kcal [- 589;-14]; P = 0.04). Macronutrient intake, diet quality, and physical activity did not differ between the intervention and control group, irrespective of successful weight loss during the intervention. CONCLUSIONS: In our study population, a preconception lifestyle intervention led to reduced energy intake 5.5 years later. Additionally, women allocated to the intervention group who were successful in losing weight during the intervention also had a lower BMI at follow-up. This shows the potential sustainable effect of a preconception lifestyle intervention. TRIAL REGISTRATION: This trial was registered on 16 November 2008 in the Dutch trial register; clinical trial registry number NTR1530 .

Diet and physical activity in pregnancy and offspring's cardiovascular health: a systematic review.

There is increasing evidence linking maternal diet and physical activity before and during pregnancy with offspring's cardiovascular health. Although many studies examined this association, the evidence has not been reviewed systematically. We therefore undertook a systematic review to synthesize evidence examining the association of maternal diet and physical activity before and during pregnancy with offspring's blood pressure and vascular health. We systematically searched the databases MEDLINE and EMBASE from inception to June 30, 2017. Eligibility screening, data extraction and quality assessment were performed by two independent reviewers. A total of 19 articles were included comprising three randomized controlled trials and 16 observational studies. Of the studies that examined the association of interest, 60% (three out of five studies) showed that high maternal carbohydrate intake was associated with higher offspring's blood pressure. Maternal protein intake during pregnancy was negatively associated with offspring carotid intima-media thickness in two out of two studies. No consistent findings for maternal fatty acid intake were found. There were too few studies to draw conclusions on energy intake, fibre intake, protein/carbohydrate ratio, specific foods, dietary patterns and maternal physical activity. Heterogeneity in exposure and outcome assessment hampered pooling. Also, owing to the observational nature of most studies, causality cannot be established. Harmonization of valid exposure and outcome measurements, and the development of core outcome sets are needed to enable more robust conclusions.

Associations between company at dinner and daily diet quality in Dutch men and women from the NQplus study.

BACKGROUND/OBJECTIVES: Consuming the evening meal in the company of others has been associated with overall diet quality. Nevertheless, studies on the association between type of company at dinner and diet quality in adults are scarce. SUBJECTS/METHODS: Dutch men (n=895) and women (n=845) aged between 20 and 70 years, included in a population-based observational study, were studied. Dietary intake was assessed by multiple 24-h recalls (6013 recalls) to estimate the Dutch Healthy Diet index (0-80 points) representing daily diet quality. Sex-specific linear mixed models adjusting for covariates were calculated. Out-of-home dinners and company at dinner were strongly associated (r=0.66), and hence in additional analyses, out-of-home dinners were excluded to avoid multicollinearity. RESULTS: Among men, daily diet quality was similar when dinners were consumed in company or consumed alone, but higher when dinner was accompanied by family (mean 46.0, s.e. 0.3) than when dinner was accompanied by others (mean 42.3, s.e. 0.7; P=0.001). Adjustment for dinner location attenuated this association, but it remained significant when excluding out-of-home dinners. Among women, daily diet quality was lower when dinner was consumed in company (mean 48.9, s.e. 0.3) than when consumed alone (mean 51.1, s.e. 0.6; P<0.001). Dinners consumed in the company of family were associated with higher daily diet quality (mean 49.3, s.e. 0.4) than dinners consumed with others (mean 45.7, s.e. 0.6; P=0.001). These associations persisted when excluding out-of-home dinners. CONCLUSIONS: Only among women, dinners consumed alone as compared with dinners in company were associated with higher diet quality. In both men and women, dinners consumed with family were associated with higher diet quality as compared with dinners with others.

Adherence to the Dutch dietary guidelines is inversely associated with 20-year mortality in a large prospective cohort study.

BACKGROUND/OBJECTIVES: The Dutch guidelines for a healthy diet aim to reduce major chronic diseases. However, supporting evidence on their overall association with all-cause and cause-specific mortality is limited. Recently, the Dutch Healthy Diet-index (DHD-index) has been developed to assess adherence to these guidelines. The aim was to examine the association between the DHD-index and all-cause mortality and deaths from cardiovascular disease (CVD), coronary heart disease (CHD), stroke and cancer. SUBJECTS/METHODS: We followed 3593 men and women aged 55 years and older enrolled in the Rotterdam Study, a population-based prospective cohort study, from baseline in 1990-1993 to 2011. A validated 170-item food frequency questionnaire at baseline was used to calculate the DHD-index score (maximum 90 points). Cox proportional hazard models were used to estimate hazard ratios (HRs) adjusting for age, sex, total energy intake, smoking and educational level. RESULTS: During the 20-year follow-up, 1831 (51%) deaths were reported. Mean DHD-index score was 60.6 (s.d. 10.6). The score was inversely associated with all-cause mortality (highest vs lowest quartile HR 0.77; 95% confidence interval (CI) 0.67, 0.89). Inverse but non-significant associations were observed for mortality due to CVD (HR 0.74; 95% CI 0.55, 1.01), CHD (HR 0.60; 95% CI 0.34, 1.06) and stroke (HR 0.67; 95% CI 0.36, 1.22), whereas no association was observed with cancer mortality (HR 0.99; 95% CI 0.90, 1.11). CONCLUSIONS: A higher level of adherence to the Dutch dietary guidelines, as assessed with the DHD-index, was associated with a lower risk of all-cause mortality, probably due to an inverse association with cardiovascular causes of death.

Large inter-individual variation in isoflavone plasma concentration limits use of isoflavone intake data for risk assessment.

BACKGROUND/OBJECTIVES: Isoflavones are present in soy foods and soy-based supplements. Despite low plasma isoflavone concentrations in the general Western population, concentrations in supplement users exceed those suggested to be beneficial for health in Asian populations, raising concerns for adverse effects. To aid risk assessment, quantification of the relation between isoflavone intake and plasma concentrations is essential. SUBJECTS/METHODS: Plasma samples were collected from postmenopausal women in three placebo-controlled crossover studies with 8-week periods for supplements (two studies, ~100 mg isoflavones/day, n=88) or 4-week periods for soy foods (one study, ~48 mg isoflavones/day, n=15). Plasma isoflavone concentrations (daidzein, equol, genistein and glycitein) were quantified using high-performance liquid chromatography and electrochemical detection. The association between plasma concentrations and isoflavone intake, equol producer status, intake-producer interaction and background dietary intake was assessed based on the assumption of a log-linear relation. RESULTS: Median plasma total isoflavone concentrations after the soy food and supplement interventions were respectively 2.16 and 3.47 µmol/l for equol producers and 1.30 and 2.39 µmol/l for non-producers. Regression analysis showed that doubling isoflavone intake increased plasma concentrations by 55-62% (±s.e. 1-2%, R(2)>0.87) for daidzein, genistein, equol (only for producers) and total isoflavones; for glycitein the association was weaker (15±1%, R(2)=0.48). Adjustments for energy, carbohydrate and fat intake did not affect these estimates. Inter-individual variation, estimated based on repeated measures in one of the studies, was 30-96%. CONCLUSIONS: Although the relation between isoflavone intake and plasma concentrations was adequately quantified, the use of isoflavone intake data for risk assessment needs caution due to large inter-individual variation in plasma concentrations.

Nutrient-rich foods, cardiovascular diseases and all-cause mortality: the Rotterdam study.

BACKGROUND/OBJECTIVES: The nutrient-rich food (NRF) index assesses nutrient quality of individual food items by ranking them according to their nutrient composition. The index reflects the nutrient density of the overall diet. We examined the associations between the NRF9.3 index-a score on the basis of nine beneficial nutrients (protein, fiber, vitamins and minerals) and three nutrients to limit (saturated fat, sugar and sodium)-incidence of cardiovascular disease (CVD) events and all-cause mortality. SUBJECTS/METHODS: A total of 4969 persons aged 55 and older from the Rotterdam Study, a prospective cohort study in the Netherlands, were studied. First, all foods were scored on the basis of their nutrient composition, resulting in an NRF9.3 score on food item level. Subsequently, they were converted into individual weighted scores on the basis of the amount of calories of each food item consumed by the subjects and the total energy intake. The hazard ratios (HRs) of the NRF9.3 index score were adjusted for age, gender, body mass index, smoking history, doctor-prescribed diet, alcohol consumption and education. RESULTS: Food groups that contributed most to the NRF9.3 index score were vegetables, milk and milk products, fruit, bread and potatoes. A high NRF9.3 index score was inversely associated with all-cause mortality (HR Q4 versus Q1: 0.84 (95% confidence interval: 0.74, 0.96)). Associations were stronger in women than in men. The NRF9.3 index score was not associated with incidence of CVD. CONCLUSION: Elderly with a higher NRF9.3 index score, indicating more beneficial components and/or less limiting components, had a lower risk of all-cause mortality. Consuming a nutrient-dense diet may improve survival.

Alcoholic beverage preference and diet in a representative Dutch population: the Dutch national food consumption survey 2007-2010.

BACKGROUND/OBJECTIVES: The habitual consumption of a specific type of alcoholic beverage may be related to the overall dietary pattern. The objective of this cross-sectional study was to investigate associations between alcoholic beverage preference and dietary intake in The Netherlands. SUBJECTS/METHODS: A total of 2100 men and women from the Dutch National Food Consumption Survey 2007-2010 were studied. A general questionnaire assessed alcoholic beverage preference and two non-consecutive 24-h dietary recalls assessed overall diet. Mean nutrient and food group intakes, and adherence to the 2006 Dutch dietary guidelines across categories of alcoholic beverage preference were compared and adjusted for age, sex, body mass index (BMI), education, smoking, physical activity, energy intake and frequency and absolute alcohol consumption. RESULTS: Largest differences in dietary habits were detected between persons who preferred wine and those who preferred beer. Persons with a beer preference had a higher absolute intake of meat, soft drinks, margarine and snacks. In contrast, persons with a wine preference had a higher absolute consumption of healthy foods. However, after multiple adjustments, wine consumers still consumed less energy and more vegetables and fruit juices compared with beer consumers. Adherence to the Dutch dietary guidelines did not differ between preference categories after multiple adjustments. CONCLUSIONS: In this cross-sectional analysis in a representative sample of the Dutch population, a beer preference was associated with less healthy dietary behaviour, especially compared with wine preference. However, these differences were largely explained by other socio-demographic and lifestyle factors. These results suggest that alcoholic beverage preference may not be independently related to diet.

Evaluation of food and nutrient intake assessment using concentration biomarkers in European adolescents from the Healthy Lifestyle in Europe by Nutrition in Adolescence study.

Accurate food and nutrient intake assessment is essential for investigating diet-disease relationships. In the present study, food and nutrient intake assessment among European adolescents using 24 h recalls (mean of two recalls) and a FFQ (separately and the combination of both) were evaluated using concentration biomarkers. Biomarkers included were vitamin C, ß-carotene, DHA+EPA, vitamin B12 (cobalamin and holo-transcobalamin) and folate (erythrocyte folate and plasma folate). For the evaluation of the food intake assessment 390 adolescents were included, while 697 were included for the nutrient intake assessment evaluation. Spearman rank and Pearson correlations, and validity coefficients, which are correlations between intake estimated and habitual true intake, were calculated. Correlations were higher between frequency of food consumption (from the FFQ) and concentration biomarkers than between mean food intake (from the recalls) and concentration biomarkers, especially for DHA+EPA (r 0·35 v. r 0·27). Most correlations were higher among girls than boys. For boys, the highest validity coefficients were found for frequency of fruit consumption (0·88) and for DHA+EPA biomarker (0·71). In girls, the highest validity coefficients were found for fruit consumption frequency (0·76), vegetable consumption frequency (0·74), mean fruit intake (0·90) and DHA+EPA biomarker (0·69). After exclusion of underreporters, correlations slightly improved. Correlations between usual food intakes, adjusted for food consumption frequency, and concentration biomarkers were higher than correlations between mean food intakes and concentration biomarkers. In conclusion, two non-consecutive 24 h recalls in combination with a FFQ seem to be appropriate to rank subjects according to their usual food intake.

Rationale and methods of the European Food Consumption Validation (EFCOVAL) Project.

BACKGROUND/OBJECTIVES: The overall objective of the European Food Consumption Validation (EFCOVAL) Project was to further develop and validate a trans-European food consumption method to be used for the evaluation of the intake of foods, nutrients and potentially hazardous chemicals within the European population. SUBJECTS/METHODS: The EFCOVAL Project was carried out by 13 institutes from 11 European countries. The main activities were centered on the three main objectives of the project organized in different sub-projects. RESULTS: In EFCOVAL, EPIC-Soft (the software developed to conduct 24-h dietary recalls (24-HDRs) in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study) was reprogrammed and adapted according to prioritized specifications, resulting in a software program working under the Windows operating system. In parallel of the EPIC-Soft development, the repeated 24-HDR method using EPIC-Soft and a food propensity questionnaire was evaluated against biomarkers in 24-h urine collections and in blood samples among adults from Belgium, the Czech Republic, (the South of) France, the Netherlands and Norway. As a result from an expert workshop on a proposed dietary assessment method for children (4-12 years), the suggested method was tested in a feasibility study in Denmark and Spain among children of 4-5, 7-8 and 12-13 years. To ensure that collected data had sufficient detail in food description for the assessment of additives and contaminants to foods the EPIC-Soft databases were adapted. Finally, the EFCOVAL Consortium developed a statistical tool (Multiple Source Method) for estimating the usual intake and distribution, which has been tested using real food consumption data and compared with three other statistical methods through a simulation study. In addition, a methodology was developed to quantify uncertainty due to portion-size estimation in usual intake distributions. CONCLUSION: The findings of EFCOVAL provide sufficient evidence to conclude that the repeated 24-HDR using EPIC-Soft for standardization in combination with a food propensity questionnaire and modeling of usual intake is a suitable method for pan-European surveillance of nutritional adequacy and food safety among healthy adults and maybe in children aged 7 years and older.

The European Food Consumption Validation Project: conclusions and recommendations.

BACKGROUND/OBJECTIVES: To outline and discuss the main results and conclusions of the European Food Consumption Validation (EFCOVAL) Project. SUBJECTS/METHODS: The EFCOVAL Project was carried out within the EU Sixth Framework Program by researchers in 11 EU countries. The activities focused on (1) the further development of the EPIC-Soft software (the software developed to conduct 24-h dietary recalls (24-HDRs) in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study) and the validation of the 2-day non-consecutive 24-HDR method using EPIC-Soft, (2) defining and investigating the applicability of the most appropriate dietary assessment method to younger age groups and expanding the applicability of the software for use in exposure assessment of some potentially hazardous chemicals and (3) to improve the methodology and statistical methods that estimate usual intake distributions from short-term dietary intake information and develop a methodology to quantify uncertainty in usual intake distributions. RESULTS: The preexisting EPIC-Soft application was reprogrammed into a Windows environment and more than 60 new specifications were implemented in the software. A validation study showed that two non-consecutive EPIC-Soft 24-HDRs are suitable to estimate the usual intake distributions of protein and potassium of European adult populations. The 2-day non-consecutive 24-HDRs in combination with a food propensity questionnaire also appeared to be appropriate to rank individuals according to their fish and fruit and vegetable intake in a comparable way in five European centers. Dietary intake of (young) children can be assessed by the combination of EPIC-Soft 24-HDRs and food recording booklets. The EPIC-Soft-standardized method of describing foods is useful to estimate dietary exposure to potentially hazardous chemicals such as specific flavoring substances. With the developed Multiple Source Method, repeated non-consecutive 24-HDR data in combination with food propensity data can be used to estimate the population distribution of the usual intake by estimating the individual usual intakes. CONCLUSIONS: The findings provide sufficient evidence to conclude that the repeated 24-HDR using EPIC-Soft for standardization in combination with a food propensity questionnaire and modeling of usual intake is a suitable method for pan-European surveillance of nutritional adequacy and food safety among healthy adults and maybe in children aged 7 years and older. To facilitate this methodology in other European countries, the next step is to provide and standardize an implementation plan that accounts for maintenance and updates, sampling designs, national surveillance programs, tailored capacity building and training, and linkage to food composition and occurrence databases.

Inventory of experiences from national/regional dietary monitoring surveys using EPIC-Soft.

BACKGROUND/OBJECTIVES: The EPIC-Soft 24-h recall (the software developed to conduct 24-h dietary recalls (24-HDRs) in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study) has been used in several regional/national dietary monitoring surveys. The main objective of the study was to present and discuss design, settings, logistics, data management and quality controls of dietary monitoring surveys that used EPIC-Soft for the collection of food consumption data. SUBJECTS/METHODS: Within European Food Consumption Validation (EFCOVAL), a questionnaire including questions on current/past EPIC-Soft experiences and requirements for the future was developed and sent to all institutes that used EPIC-Soft in their food consumption survey(s) (five surveys in four different countries). RESULTS: EPIC-Soft was used in the national food consumption survey in Belgium (≥ 15-97 years), Germany (14-80 years), the Netherlands (19-30 years and 2-6 years) and Spain (regional only; 4-18 years). Participation rates in these surveys were 46% (Belgium), 42% (Germany), 42% (Dutch survey in adults), 79% (Dutch survey in children) and 77% (Basque survey). Two 24-HDRs were collected by conducting face-to-face interviews in Belgium and Spain, and through telephone interviews in Germany and the Netherlands. Except the Netherlands (19-30 years), where the study was conducted only in autumn, in all other countries the study was conducted throughout the four seasons, including all days of the week. Interviews were conducted by dietitians, except in Germany and Spain. Mean EPIC-Soft interview time was 20-34 min. The dropout rate between the first and second interviews was low (<7.5%) in all surveys. CONCLUSION: EPIC-Soft has been used in different study settings and populations for nutritional exposure assessments. To guarantee the comparability of data across countries, recommendations for the design of future pan-European dietary monitoring surveys using EPIC-Soft should be drawn.

Respondents' evaluation of the 24-h dietary recall method (EPIC-Soft) in the EFCOVAL Project.

BACKGROUND: To improve participation rate, accuracy and respondents' compliance, it is important to know the respondents' viewpoint. OBJECTIVE: To evaluate respondents' preferences and perception about the EPIC-Soft (the software developed to conduct 24-h dietary recalls (24-HDRs) in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study) 24-HDR interviews and to compare these preferences and perception between population groups (for example, between genders). DESIGN: Data were collected in Belgium, Czech Republic, France, the Netherlands and Norway in 2007. Two 24-HDRs (face-to-face and telephone administered) were conducted using EPIC-Soft. An evaluation questionnaire on different study aspects was completed by the respondents. SETTING: Data were collected in the European Food Consumption Validation Study. SUBJECTS: A convenience sample of 600 apparently healthy men and women, 45-65 years old and including all educational levels, were recruited (120 subjects per country). Differences among population groups were compared by means of the χ (2)-test. RESULTS: A total of 585 respondents completed the evaluation questionnaire. In all, 88% experienced problems only to a low degree when answering face-to-face and telephone-administered 24-HDR using EPIC-Soft. A total of 15% would have preferred help of another person during the face-to-face interview in the study center (mainly men: P < 0.001). Significantly, more subjects in the Netherlands and in Norway preferred two telephone (instead of face-to-face) interviews compared with the other countries (P<0.001). CONCLUSION: Most subjects only experienced problems to a low degree during the EPIC-Soft interviews. Differences in preferences and capabilities to answer the EPIC-Soft interviews were identified between population groups (for example, gender differences). Therefore, the methods and the design to be used in a survey should be adapted according to the study population, so as to optimize response rate and compliance.

Biomarker-based evaluation of two 24-h recalls for comparing usual fish, fruit and vegetable intakes across European centers in the EFCOVAL Study.

BACKGROUND/OBJECTIVES: A standardized methodology is important to enable consistent monitoring of dietary intake across European countries. For this reason, we evaluated the comparability of the assessment of usual food intake collected with two non-consecutive computerized 24-h dietary recalls (24-HDRs) and a food propensity questionnaire (FPQ) among five European centers. SUBJECTS/METHODS: Two 24-HDRs using EPIC-Soft (the software developed to conduct 24-HDRs in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study) were performed to determine fish, fruit and vegetable (FV) consumed by 600 adults in Belgium (BE), the Czech Republic (CZ), France (FR), the Netherlands (NL) and Norway (NO) in a validation study. An FPQ was used to identify non-consumers. Information from the 24-HDRs and FPQ were used to estimate individual usual food intake by the Multiple Source Method (MSM). Blood samples were drawn to determine fatty acids in phospholipids and serum carotenoids as biomarkers of fish, and FV intake, respectively. RESULTS: The pooled correlation between usual fish intake and eicosapentaenoic acid plus docosahexaenoic acid in phospholipids was 0.19 in men and 0.31 in women (P for heterogeneity >0.50) and center-specific correlations ranged between 0.08 (CZ) and 0.28 (BE and NO) in men, and between 0.19 (BE) and 0.55 (FR) in women. For usual FV intake, the pooled correlation with serum carotenoids was 0.31 in men and 0.40 in women (P for heterogeneity >0.10); the center-specific correlations varied between 0.07 (NO) and 0.52 (FR) in men, and between 0.25 (NL) and 0.45 (NO) in women. CONCLUSION: Two standardized 24-HDRs using EPIC-Soft and an FPQ appeared to be appropriate to rank individuals according to their fish and FV intake in a comparable way among five European centers.

Potential and requirements for a standardized pan-European food consumption survey using the EPIC-Soft software.

BACKGROUND/OBJECTIVES: To describe the strengths, limitations and requirements of using EPIC-Soft software (the software developed to conduct 24-h dietary recalls in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study) in pan-European food consumption surveys, and to discuss potentials and barriers for a harmonized pan-European food consumption survey. SUBJECTS/METHODS: The paper is based on the experiences in the 'European Food Consumption and Validation' Project, which included updating six existing and preparing one new country-specific EPIC-Soft version, applying EPIC-Soft in validation and feasibility studies, and estimating the intake of nutrients and flavoring substances. The experiences were discussed in the September 2009 workshop 'Pan-European Food Consumption Surveys--for Standardized and Comparable Transnational Data Collection'. RESULTS: EPIC-Soft is suitable for detailed and standardized food consumption data collection in pan-European food consumption surveys. A thorough preparation of all aspects of the food consumption survey is important for the quality and efficiency during data collection and processing. The preparation and data-handling phase of working with EPIC-Soft is labor intensive and requires trained, motivated and qualified personnel. CONCLUSIONS: Given the suitability of EPIC-Soft as standardized dietary assessment tool in European dietary monitoring, the proposed strategy toward a pan-European food consumption survey is to prepare well, to allow flexibility in national extensions and to start with a limited number of countries that are interested.

The standardized computerized 24-h dietary recall method EPIC-Soft adapted for pan-European dietary monitoring.

BACKGROUND/OBJECTIVES: The EPIC-Soft program (the software initially developed to conduct 24-h dietary recalls (24-HDRs) in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study) was recommended as the best way to standardize 24-HDRs for future pan-European dietary monitoring. Within European Food Consumption Validation (EFCOVAL), EPIC-Soft was adapted and further developed on various aspects that were required to optimize its use. In this paper, we present the structure and main interview steps of the EPIC-Soft program, after implementation of a series of new specifications deemed to satisfy specific requirements of pan-European monitoring surveys and other international studies. SUBJECTS/METHODS: Updates to optimize the EPIC-Soft program were ascertained according to the following stepwise approach: (1) identification of requested specifications to be potentially implemented through an ad hoc 'EPIC-Soft specifications questionnaire' sent to past, current and possible future users of the software; (2) evaluation of the specifications in collaboration with two ad hoc task force groups and through a workshop; (3) development of a technical solution for each retained specification; (4) implementation of the specifications by software developers; (5) testing and amendment of bugs. RESULTS: A number of new specifications and facilities were implemented to EPIC-Soft program. In addition, the software underwent a full reprogramming and migration to a modern Windows environment, including changes in its internal architecture and user interface. Although the overall concept and structure of the initial software were not changed substantially, these improvements ease the current and future use of EPIC-Soft and increase further its adaptation to other countries and study contexts. CONCLUSIONS: EPIC-Soft is enriched with further functions and facilities expected to fulfil specific needs of pan-European dietary monitoring and risk assessment purposes. The validity, feasibility and relevance of this software for different national and international study designs, and the logistical aspects related to its implementation are reported elsewhere.

Comparing four methods to estimate usual intake distributions.

BACKGROUND/OBJECTIVES: The aim of this paper was to compare methods to estimate usual intake distributions of nutrients and foods. As 'true' usual intake distributions are not known in practice, the comparison was carried out through a simulation study, as well as empirically, by application to data from the European Food Consumption Validation (EFCOVAL) Study in which two 24-h dietary recalls (24-HDRs) and food frequency data were collected. The methods being compared were the Iowa State University Method (ISU), National Cancer Institute Method (NCI), Multiple Source Method (MSM) and Statistical Program for Age-adjusted Dietary Assessment (SPADE). SUBJECTS/METHODS: Simulation data were constructed with varying numbers of subjects (n), different values for the Box-Cox transformation parameter (λ(BC)) and different values for the ratio of the within- and between-person variance (r(var)). All data were analyzed with the four different methods and the estimated usual mean intake and selected percentiles were obtained. Moreover, the 2-day within-person mean was estimated as an additional 'method'. These five methods were compared in terms of the mean bias, which was calculated as the mean of the differences between the estimated value and the known true value. The application of data from the EFCOVAL Project included calculations of nutrients (that is, protein, potassium, protein density) and foods (that is, vegetables, fruit and fish). RESULTS: Overall, the mean bias of the ISU, NCI, MSM and SPADE Methods was small. However, for all methods, the mean bias and the variation of the bias increased with smaller sample size, higher variance ratios and with more pronounced departures from normality. Serious mean bias (especially in the 95th percentile) was seen using the NCI Method when r(var) = 9, λ(BC) = 0 and n = 1000. The ISU Method and MSM showed a somewhat higher s.d. of the bias compared with NCI and SPADE Methods, indicating a larger method uncertainty. Furthermore, whereas the ISU, NCI and SPADE Methods produced unimodal density functions by definition, MSM produced distributions with 'peaks', when sample size was small, because of the fact that the population's usual intake distribution was based on estimated individual usual intakes. The application to the EFCOVAL data showed that all estimates of the percentiles and mean were within 5% of each other for the three nutrients analyzed. For vegetables, fruit and fish, the differences were larger than that for nutrients, but overall the sample mean was estimated reasonably. CONCLUSIONS: The four methods that were compared seem to provide good estimates of the usual intake distribution of nutrients. Nevertheless, care needs to be taken when a nutrient has a high within-person variation or has a highly skewed distribution, and when the sample size is small. As the methods offer different features, practical reasons may exist to prefer one method over the other.

Dietary exposure to flavouring substances: from screening methods to detailed assessments using food consumption data collected with EPIC-Soft software.

This study aimed to compare different methods of assessing dietary exposure to flavourings in the context of a stepwise approach. The dietary exposure to four flavourings--raspberry ketone, glycyrrhizinic acid, coumarin, and caffeine--was determined. When dietary exposure exceeded the safety limits, the need for more detailed assessment using less aggregated data was judged necessary. First, screening methods--maximized survey-derived daily intake (MSDI), single-portion exposure technique (SPET), and modified theoretical added maximum daily intake (mTAMDI)--were applied. Next, individual food consumption data were used for creating models with different levels of detail to identify the foods: a model based on food groups and models based on food items. These were collected from 121 Dutch adults using a standardized 2 x 24-h dietary recall (EPIC-Soft) in the European Food Consumption Validation (EFCOVAL) study. Three food item models were developed: without improvements of the flavouring descriptor built in the software; with improvements; and with use of non-specified flavour descriptors. Based on the results of at least one of the three screening methods, refined assessment was necessary for raspberry ketone, glycyrrhizinic acid, and caffeine. When applying the food group model, the need for refinement was indicated for the four flavourings. When applying the food item models, only glycyrrhizinic acid and caffeine presented dietary exposure above the safety limits. In the raspberry ketone case, dietary exposure increased when improvements in food description were considered. The use of non-specified flavour descriptors hardly changed the results. The collection of detailed food consumption data at the individual level is useful in the dietary exposure assessment of these flavourings.

SPUD qPCR assay confirms PREXCEL-Q softwares ability to avoid qPCR inhibition.

Real-time quantitative polymerase chain reaction is subject to inhibition by substances that co-purify with nucleic acids during isolation and preparation of samples. Such materials alter the activity of reverse transcriptase (RT) and thermostable DNA polymerase enzymes on which the assay depends. When removal of inhibitory substances by column or reagent-based methods fails or is incomplete, the remaining option of appropriately, precisely and differentially diluting samples and standards to non-inhibitory concentrations is often avoided due to the logistic problem it poses. To address this, we invented the PREXCEL-Q software program to automate the process of calculating the non-inhibitory dilutions for all samples and standards after a preliminary test plate has been performed on an experimental sample mixture. The SPUD assay was used to check for inhibition in each PREXCEL-Q-designed qPCR reaction. When SPUD amplicons or SPUD amplicon-containing plasmids were spiked equally into each qPCR reaction, all reactions demonstrated complete absence of qPCR inhibition. Reactions spiked with about 15,500 SPUD amplicons yielded a Cq of 27.39 plus/minus 0.28 (at about 80.8% efficiency), while reactions spiked with about 7,750 SPUD plasmids yielded a Cq of 23.82 plus/minus 0.15 (at about 97.85% efficiency). This work demonstrates that PREXCEL-Q sample and standard dilution calculations ensure avoidance of qPCR inhibition.