Noninvasive integrative approach applied to children in the context of recent air pollution exposure demonstrates association between fractional exhaled nitric oxide (FeNO) and urinary CC16.

Exposure to the air pollutant particulate matter (PM) is associated with increased risks of respiratory diseases and enhancement of airway inflammation in children. In the context of large scale air pollution studies, it can be challenging to measure fractional exhaled nitric oxide (FeNO) as indicator of lung inflammation. Urinary CC16 (U-CC16) is a potential biomarker of increased lung permeability and toxicity, increasing following short-term PM2.5 exposure. The single nucleotide polymorphism (SNP) CC16 G38A (rs3741240) affects CC16 levels and respiratory health. Our study aimed at assessing the use of U-CC16 (incl. CC16 G38A from saliva) as potential alternative for FeNO by investigating their mutual correlation in children exposed to PM. Samples from a small-scale study conducted in 42 children from urban (n = 19) and rural (n = 23) schools examined at two time points, were analysed. When considering recent (lag1) low level exposure to PM2.5 as air pollution measurement, we found that U-CC16 was positively associated with FeNO (ß = 0.23; 95% CI [-0.01; 0.47]; p = 0.06) in an adjusted analysis using a linear mixed effects model. Further, we observed a positive association between PM2.5 and FeNO (ß = 0.56; 95% CI [0.02; 1.09]; p = 0.04) and higher FeNO in urban school children as compared to rural school children (ß = 0.72; 95% CI [0.12; 1.31]; p = 0.02). Although more investigations are needed, our results suggest that inflammatory responses evidenced by increased FeNO are accompanied by potential increased lung epithelium permeability and injury, evidenced by increased U-CC16. In future large scale studies, where FeNO measurement is less feasible, the integrated analysis of U-CC16 and CC16 G38A, using noninvasive samples, might be a suitable alternative to assess the impact of air pollution exposure on the respiratory health of children, which is critical for policy development at population level.

Assessment of the Feasibility of a Future Integrated Larger-Scale Epidemiological Study to Evaluate Health Risks of Air Pollution Episodes in Children.

Air pollution exposure can lead to exacerbation of respiratory disorders in children. Using sensitive biomarkers helps to assess the impact of air pollution on children's respiratory health and combining protein, genetic and epigenetic biomarkers gives insights on their interrelatedness. Most studies do not contain such an integrated approach and investigate these biomarkers individually in blood, although its collection in children is challenging. Our study aimed at assessing the feasibility of conducting future integrated larger-scale studies evaluating respiratory health risks of air pollution episodes in children, based on a qualitative analysis of the technical and logistic aspects of a small-scale field study involving 42 children. This included the preparation, collection and storage of non-invasive samples (urine, saliva), the measurement of general and respiratory health parameters and the measurement of specific biomarkers (genetic, protein, epigenetic) of respiratory health and air pollution exposure. Bottlenecks were identified and modifications were proposed to expand this integrated study to a higher number of children, time points and locations. This would allow for non-invasive assessment of the impact of air pollution exposure on the respiratory health of children in future larger-scale studies, which is critical for the development of policies or measures at the population level.

Urinary CC16, a potential indicator of lung integrity and inflammation, increases in children after short-term exposure to PM2.5/PM10 and is driven by the CC16 38GG genotype.

Particular matter (PM) exposure is a big hazard for public health, especially for children. Serum CC16 is a well-known biomarker of respiratory health. Urinary CC16 (U-CC16) can be a noninvasive alternative, albeit requiring adequate adjustment for renal handling. Moreover, the SNP CC16 G38A influences CC16 levels. This study aimed to monitor the effect of short-term PM exposure on CC16 levels, measured noninvasively in schoolchildren, using an integrative approach. We used a selection of urine and buccal DNA samples from 86 children stored in an existing biobank. Using a multiple reaction monitoring method, we measured U-CC16, as well as RBP4 (retinol binding protein 4) and ß2M (beta-2-microglobulin), required for adjustment. Buccal DNA samples were used for CC16 G38A genotyping. Linear mixed-effects models were used to find relevant associations between U-CC16 and previously obtained data from recent daily PM ≤ 2.5 or 10 µm exposure (PM2.5, PM10) modeled at the child's residence. Our study showed that exposure to low PM at the child's residence (median levels 18.9 µg/m³ (PM2.5) and 23.6 µg/m³ (PM10)) one day before sampling had an effect on the covariates-adjusted U-CC16 levels. This effect was dependent on the CC16 G38A genotype, due to its strong interaction with the association between PM levels and covariates-adjusted U-CC16 (P = 0.024 (PM2.5); P = 0.061 (PM10)). Only children carrying the 38GG genotype showed an increase of covariates-adjusted U-CC16, measured 24h after exposure, with increasing PM2.5 and PM10 (ß = 0.332; 95% CI: 0.110 to 0.554 and ß = 0.372; 95% CI: 0.101 to 0.643, respectively). To the best of our knowledge, this is the first study using an integrative approach to investigate short-term PM exposure of children, using urine to detect early signs of pulmonary damage, and taking into account important determinants such as the genetic background and adequate adjustment of the measured biomarker in urine.

Development of a multiplex mass spectrometry method for simultaneous quantification of urinary proteins related to respiratory health.

Respiratory health of children is a health priority. Club cell protein (CC16) is an interesting biomarker of lung diseases and adverse effects towards the airway epithelium integrity. Osteopontin (OPN) and nuclear factor-kappa B (NF-κB) also play a role in respiratory health. The use of urine as biomarker source is useful in studies involving children but necessitates proper adjustment for physiological confounders influencing the urinary excretion, potentially characterized with beta-2-microglobulin (ß2M), retinol binding protein 4 (RBP4) or myoglobin (MYO), as well as adjustment for possible renal dysfunction, characterized by human serum albumin (HSA). The simultaneous quantification of all these proteins in urine could facilitate children's health monitoring. A multiple reaction monitoring method (MRM) was developed and validated for the relative quantification of the seven mentioned urinary proteins. A total of nine proteotypic peptides were selected and used for the relative quantification of the seven proteins. The MRM method was completely validated for all proteins and partially for OPN. LOQ's ranged from 0.3 to 42.8 ng/ml, a good reproducibility and a good linearity were obtained across the analytical measurement range (r2 > 0.98). The method yielded varying correlations (r2 of 0.78, 0.71, 0.34 and 0.15 for CC16, ß2M, RBP4 and HSA respectively) with available immunoassay data. It also allowed the identification and successful quantification of ß2M and RBP4 as a protein candidate for adjustment of renal handling and dysfunction. All proteins were detected in the urine samples except for MYO and NF-κB. Our validated MRM-method is able to simultaneously quantify in urine biomarkers of airway epithelium integrity and biomarkers of variation in renal function and urinary dilution. This will allow to investigate further in future studies if urine can be used as a good surrogate source for biomarkers of airway epithelium integrity, and to understand the complex relationship between cause and effect in children's respiratory health monitoring.

Gender-dependent association between exhaled nitric oxide and the CC16 38AA genotype in young school children.

BACKGROUND: Studies that investigated the association between the CC16 A38G polymorphism and the risk of asthma yielded conflicting results. The aim of this study among schoolchildren was to assess the relationships of CC16 A38G polymorphism with aeroallergen sensitization and fractional exhaled nitric oxide (FeNO), two outcomes predicting asthma later in life. METHODS: The study included 139 children (72 boys), median age of 7.7. Information on each child's health, lifestyle, and environment was collected through a questionnaire completed by their parents. CC16 genotypes were determined using urinary DNA. We measured FeNO, the CC16 protein in urine and nasal lavage fluid and aeroallergen-specific immunoglobulin E in nasal mucosa fluid. RESULTS: Children with the homozygous mutant CC16 38AA genotype had higher odds of increased FeNO (>30 ppb) compared with their peers with the wild-type genotype 38GG (OR, 9.85; 95% CI, 2.09-46.4; P = .004). This association was female gender specific (P = .002) not being observed in boys (P = .40). It was also independent of allergic sensitization, which yet emerged as the strongest predictor of FeNO along with the use of bleach for house cleaning. Children with the CC16 38AA genotype had lower covariates-adjusted urinary CC16 levels than those with 38GG (median, µg/L, 1.17 vs 2.08, P = .02). CONCLUSION: Our study suggests that the CC16 38AA allele promotes airway inflammation as measured by FeNO through a gender-dependent association. Deficient levels of CC16 in the deep lung, measured noninvasively in urine, as a possible proxy for serum CC16, might underlie this promoting effect.

Weight and head circumference at birth in function of placental paraben load in Belgium: an ENVIRONAGE birth cohort study.

BACKGROUND: Parabens are a group of esters of para-hydroxybenzoic acid utilized as antimicrobial preservatives in many personal care products. Epidemiological studies regarding the adverse effects of parabens on fetuses are limited. The aim of this study was to determine the association between placental paraben exposure and birth outcomes. We assessed paraben concentrations in placental tissue, which potentially gives a better understanding of fetal exposure than the maternal urinary concentrations which are the current golden standard. METHODS: Placental tissue was collected immediately after birth from 142 mother-child pairs from the ENVIRONAGE birth cohort. The placental concentrations of four parabens (methyl (MeP), ethyl (EtP), propyl (PrP), and butyl (BuP)) were determined by ultra-performance liquid chromatography coupled with tandem mass-spectrometry. Generalized linear regression models were used to determine the association between paraben exposure levels and birth outcomes. RESULTS: The geometric means of placental MeP, EtP, PrP, and BuP were 1.84, 2.16, 1.68 and 0.05 ng/g tissue, respectively. The sum of parabens (∑ parabens, including MeP, EtP and PrP) was negatively associated with birth weight in newborn girls (- 166 g, 95% CI: - 322, - 8.6, p = 0.04) after adjustment for a priori selected covariates. The sum of parabens was negatively associated with head circumference (- 0.6 cm, 95% CI: - 1.1, - 0.2, p = 0.008) and borderline associated with birth length (- 0.6 cm, 95% CI:-1.3, 0.1, p = 0.08). In newborn girls the placental concentration of EtP was negatively associated with head circumference (- 0.6 cm, 95% CI:-1.1, - 0.1, p = 0.01) and borderline significantly associated with birth weight and birth length. Lastly, placental EtP and ∑parabens were negatively associated with placental weight in newborn girls but not in newborn boys (- 45.3 g, 95% CI:-86.2, - 4.4, p = 0.03). CONCLUSION: The negative association between maternal paraben exposure and birth outcomes warrants further research and follow-up over time to determine long term effects of gestational exposure to parabens.

Selection of a Noninvasive Source of Human DNA Envisaging Genotyping Assays in Epidemiological Studies: Urine or Saliva?

Genetic epidemiology requires an appropriate approach to measure genetic variation within the population. The aim of this study was to evaluate the characteristics and genotyping results of DNA extracted from 2 human DNA sources, selected for their rapid and noninvasive sampling, and the use of simple and standardized protocols that are essential for large-scale epidemiologic studies. Saliva and urine samples were collected at the same day from 20 subjects aged 9-10 yr. Genomic DNA was extracted using commercial kits. Quantitative and qualitative evaluation was done by assessing the yield, the purity, and integrity of the extracted DNA. As a proof-of-concept, genotyping was performed targeting CC16 A38G and uteroglobin-related protein 1 (UGRP1)-112G/A. Saliva was found to provide the highest yield and concentration of total DNA extracted. Salivary DNA showed higher purity and a significantly less degraded state compared to urinary DNA. Consequently, the salivary DNA gave better genotyping results than urinary DNA. Therefore, if the choice exists, saliva is the preferred noninvasive matrix for genotyping purposes in large-scale genetic epidemiologic studies. Only in particular cases using urine could nevertheless be considered useful, although specific limitations need to be taken into account.

Personal exposure to traffic-related air pollutants and relationships with respiratory symptoms and oxidative stress: A pilot cross-sectional study among urban green space workers.

Exposure to ambient air pollution has been associated with various adverse health effects including respiratory, cardiovascular and neurological diseases. Exposure data for some specific pollutants and settings are however still insufficient and mechanisms underlying negative health outcomes are not fully elucidated. This pilot study aimed to assess individual exposure to three traffic-related air pollutants, black carbon (BC), polycyclic aromatic hydrocarbons (PAHs) and benzene, and the relationship with respiratory and oxidative stress outcomes in a cross-sectional sample of 48 green space workers in Brussels, Belgium. Participants were followed during four consecutive working days in 2016-2017 during which their individual exposure to BC, PAHs, benzene and more generally air pollution was measured using aethalometers, urinary biomarkers (1-hydroxypyrene, 1-naphthol, 2-naphthol, S-phenylmercapturic acid) and questionnaires. Data on respiratory health and oxidative stress were collected using questionnaires and respiratory/urinary biomarkers (exhaled nitric oxide [NO], 8-hydroxydeoxyguanosine [8-OHdG]). Associations between exposure and health outcomes were investigated using comparison tests and linear regression models, after stratification by present-day smoking status. Spatial variation in BC exposure was high, with concentrations varying between 0.26 and 5.69 µg/m3. The highest levels were recorded during transport and, to a lesser extent, in green spaces located in the vicinity of roads with high traffic intensity. Concentrations of PAHs and benzene biomarkers did not systematically exceed the limits of detection. Among smokers, respiratory inflammation increased linearly with exposure to BC measured over the four days of follow-up (ß = 8.73, 95% CI: 4.04, 13.41). Among non-smokers, oxidative stress increased linearly with BC measured on the fourth day (ß = 2.88, 95% CI: 1.52, 4.24). Despite some limitations, this work supports the hypothesis that BC induces respiratory inflammation and oxidative stress. It also highlights the value of this compound as well as exhaled NO and urinary 8-OHdG biomarkers to detect early/mild effects of air pollution.

Relationships between aeroallergen levels and hospital admissions for asthma in the Brussels-Capital Region: a daily time series analysis.

BACKGROUND: Outdoor pollen grain and fungal spore concentrations have been associated with severe asthma exacerbations at the population level. The specific impact of each taxon and the concomitant effect of air pollution on these symptoms have, however, still to be better characterized. This study aimed to investigate the short-term associations between ambient concentrations of various aeroallergens and hospitalizations related to asthma in the Brussels-Capital Region (Belgium), an area recording especially high rates of admissions. METHODS: Based on administrative records of asthma hospitalizations and regular monitoring of 11 tree/herbaceous pollen taxa and 2 fungal spore taxa, daily time series analyses covering the 2008-2013 period were performed. Effects up to 6 days after exposure were captured by combining quasi-Poisson regression with distributed lag models, adjusting for seasonal and long-term trends, day of the week, public holidays, mean temperature and relative humidity. Effect modification by age and air pollution (PM, NO2, O3) was tested. RESULTS: A significant increase in asthma hospitalizations was observed for an interquartile range increase in grass (5.9%, 95% CI: 0.0, 12.0), birch (3.2%, 95% CI: 1.1, 5.3) and hornbeam (0.7%, 95% CI: 0.2, 1.3) pollen concentrations. For several taxa including grasses, an age modification effect was notable, the hospitalization risk tending to be higher in individuals younger than 60 years. Air pollutants impacted the relationships too: the risk appeared to be stronger for grass and birch pollen concentrations in case of high PM10 and O3 concentrations respectively. CONCLUSIONS: These findings suggest that airborne grass, birch and hornbeam pollen are associated with severe asthma exacerbations in the Brussels region. These compounds appear to act in synergy with air pollution and to more specifically affect young and intermediate age groups. Most of these life-threatening events could theoretically be prevented with improved disease diagnosis/management and targeted communication actions.

Silicone Wristband Passive Samplers Yield Highly Individualized Pesticide Residue Exposure Profiles.

Monitoring human exposure to pesticides and pesticide residues (PRs) remains crucial for informing public health policies, despite strict regulation of plant protection product and biocide use. We used 72 low-cost silicone wristbands as noninvasive passive samplers to assess cumulative 5-day exposure of 30 individuals to polar PRs. Ethyl acetate extraction and LC-MS/MS analysis were used for the identification of PRs. Thirty-one PRs were detected of which 15 PRs (48%) were detected only in worn wristbands, not in environmental controls. The PRs included 16 fungicides (52%), 8 insecticides (26%), 2 herbicides (6%), 3 pesticide derivatives (10%), 1 insect repellent (3%), and 1 pesticide synergist (3%). Five detected pesticides were not approved for plant protection use in the EU. Smoking and dietary habits that favor vegetable consumption were associated with higher numbers and higher cumulative concentrations of PRs in wristbands. Wristbands featured unique PR combinations. Our results suggest both environment and diet contributed to PR exposure in our study group. Silicone wristbands could serve as sensitive passive samplers to screen population-wide cumulative dietary and environmental exposure to authorized, unauthorized and banned pesticides.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria.

Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n = 39), and clarithromycin and rifabutin (NTM, n = 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve [AUC] of >0.015, relative growth [RG] of <0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.

Unique Phylogenetic Lineage Found in the Fusarium-like Clade after Re-examining BCCM/IHEM Fungal Culture Collection Material.

Recently, the Fusarium genus has been narrowed based upon phylogenetic analyses and a Fusarium-like clade was adopted. The few species of the Fusarium-like clade were moved to new, re-installed or existing genera or provisionally retained as "Fusarium." Only a limited number of reference strains and DNA marker sequences are available for this clade and not much is known about its actual species diversity. Here, we report six strains, preserved by the Belgian fungal culture collection BCCM/IHEM as a Fusarium species, that belong to the Fusarium-like clade. They showed a slow growth and produced pionnotes, typical morphological characteristics of many Fusarium-like species. Multilocus sequencing with comparative sequence analyses in GenBank and phylogenetic analyses, using reference sequences of type material, confirmed that they were indeed member of the Fusarium-like clade. One strain was identified as "Fusarium" ciliatum whereas another strain was identified as Fusicolla merismoides. The four remaining strains were shown to represent a unique phylogenetic lineage in the Fusarium-like clade and were also found morphologically distinct from other members of the Fusarium-like clade. Based upon phylogenetic considerations, a new genus, Pseudofusicolla gen. nov., and a new species, Pseudofusicolla belgica sp. nov., were installed for this lineage. A formal description is provided in this study. Additional sampling will be required to gather isolates other than the historical strains presented in the present study as well as to further reveal the actual species diversity in the Fusarium-like clade.

Short-Term Effect of Pollen and Spore Exposure on Allergy Morbidity in the Brussels-Capital Region.

Belgium is among the European countries that are the most affected by allergic rhinitis. Pollen grains and fungal spores represent important triggers of symptoms. However, few studies have investigated their real link with disease morbidity over several years. Based on aeroallergen counts and health insurance datasets, the relationship between daily changes in pollen, fungal spore concentrations and daily changes in reimbursable systemic antihistamine sales has been investigated between 2005 and 2011 in the Brussels-Capital Region. A Generalized Linear Model was used and adjusted for air pollution, meteorological conditions, flu, seasonal component and day of the week. We observed an augmentation in drug sales despite no significant increase in allergen levels in the long term. The relative risk of buying allergy medications associated with an interquartile augmentation in pollen distributions increased significantly for Poaceae, Betula, Carpinus, Fraxinus and Quercus. Poaceae affected the widest age group and led to the highest increase of risk which reached 1.13 (95% CI [1.11-1.14]) among the 19- to 39-year-old men. Betula showed the second most consistent relationship across age groups. Clear identification of the provoking agents may improve disease management by customizing prevention programmes. This work also opens several research perspectives related to impact of climate modification or subpopulation sensitivity.

Fusarium musae infected banana fruits as potential source of human fusariosis: May occur more frequently than we might think and hypotheses about infection.

The banana fruit infecting fungus Fusarium musae was originally known as a distinct population within Fusarium verticillioides. However, recently, Fusarium musae was installed as a separate species and the first cases of human infection associated with Fusarium musae were found. In this article, we report an additional survey indicating that human pathogenic Fusarium musae infections may occur more frequently than we might think. Moreover, we evaluate the hypotheses on how infection can be acquired. A first hypothesis is that banana fruits act as carriers of Fusarium musae spores and thereby be the source of human infection with Fusarium musae. Acquisition is likely to be caused through contact with Fusarium musae contaminated banana fruits, either being imported or after traveling of the patient to a banana-producing country. An alternative hypothesis is that Fusarium musae is not only present on banana fruits, but also on other plant hosts or environmental sources.

Application of LC-MS/MS MRM to Determine Staphylococcal Enterotoxins (SEB and SEA) in Milk.

Staphylococcus aureus is one of the important aetiological agents of food intoxications in Europe and can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Due to their stability and ease of production and dissemination, some SEs have also been studied as potential agents for bioterrorism. Therefore, specific and accurate analytical tools are required to detect and quantify SEs. Online solid-phase extraction liquid chromatography electrospray ionization tandem mass spectrometry (online SPE-LC-ESI-MS/MS) based on multiple reaction monitoring (MRM) was used to detect and quantify two types of SE (A and B) spiked in milk and buffer solution. SE extraction and concentration was performed according to the European Screening Method developed by the European Reference Laboratory for Coagulase Positive Staphylococci. Trypsin digests were screened for the presence of SEs using selected proteotypic heavy-labeled peptides as internal standards. SEA and SEB were successfully detected in milk samples using LC-MS/MS in MRM mode. The selected SE peptides were proteotypic for each toxin, allowing the discrimination of SEA and SEB in a single run. The detection limit of SEA and SEB was approximately 8 and 4 ng/g, respectively.

Nicotine Dependence and Urinary Nicotine, Cotinine and Hydroxycotinine Levels in Daily Smokers.

INTRODUCTION: Nicotine dependence and smoking frequency are critical factors for smoking cessation. The aims of this study are (1) to determine if nicotine dependence Fagerström Test for Nicotine Dependence (FTND) scores are associated with urinary levels of nicotine metabolites, (2) to assess the relationship of hydroxycotinine/cotinine ratio with FTND score and cigarettes smoked per day (CPD), and (3) to identify significant predictors of cigarettes per day among biomarker concentrations and individual FTND items. METHODS: Urine samples and questionnaire data of 239 daily smokers were obtained. Nicotine, cotinine and hydroxycotinine urinary levels were determined by UPLC MS/MS.Multiple linear regression models were developed to explore the relationship between nicotine, cotinine, hydroxycotinine levels and separate FTND scores (for all six items). RESULTS: We found significant correlations between the different urinary biomarker concentrations, and the FTND score. The time before the first cigarette after waking (TTFC) was significantly associated with the nicotine, cotinine and hydroxycotinine concentrations. No association was found between the ratio of hydroxycotinine to cotinine and either the FTND or the CPD. A model including four FTND questions, sex, age, and the cotinine concentration, accounted for 45% of the variance of CPD. CONCLUSIONS: There are significant relationships between urinary levels of nicotine, cotinine, and hydroxycotinine and the FTND score. Especially the FTND question about TTFC is relevant for explaining the biomarker concentrations. CPD (below 15) was significantly explained by four FTND dependence items and urinary cotinine levels in a regression model. IMPLICATIONS: We investigated associations between urinary levels of nicotine, cotinine, and hydroxycotinine in daily smokers and the FTND scores for nicotine dependence. We did not find association between the hydroxycotinine/cotinine ratio and CPD. We developed a model that explains the cigarettes smoked daily (CPD) in a group of light smokers by combining FTND items, urinary cotinine levels, sex, and age. Our results might be of importance for clinical use or future studies on larger smoking populations.

Exposure determinants of cadmium in European mothers and their children.

The metal cadmium (Cd) is a widespread environmental pollutant with documented adverse effects on the kidneys and bones from long-term environmental exposure, but with insufficiently elucidated public health consequences such as risk of cardiovascular disease, hormone-related cancer in adults and developmental effects in children. This study is the first pan-European human biomonitoring project that succeeded in performing harmonized measurements of Cd in urine in a comparable way in mother-child couples from 16 European countries. The aim of the study was to evaluate the overall Cd exposure and significant determinants of Cd exposure. A study population of 1632 women (24-52 years of age), and 1689 children (5-12 years of age), from 32 rural and urban areas, was examined within a core period of 6 months in 2011-2012. Women were stratified as smokers and non-smokers. As expected, smoking mothers had higher geometric mean (gm) urinary cadmium (UCd; 0.24 µg/g crea; n=360) than non-smoking mothers (gm 0.18 µg/g crea; n=1272; p<0.0001), and children had lower UCd (gm 0.065 µg/g crea; n=1689) than their mothers at the country level. Non-smoking women exposed to environmental tobacco smoke (ETS) at home had 14% (95% CI 1-28%) higher UCd than those who were not exposed to ETS at home (p=0.04). No influence of ETS at home or other places on UCd levels was detected in children. Smoking women with primary education as the highest educational level of the household had 48% (95% CI 18-86%) higher UCd than those with tertiary education (p=0.0008). The same observation was seen in non-smoking women and in children; however they were not statistically significant. In children, living in a rural area was associated with 7% (95% CI 1-13%) higher UCd (p=0.03) compared to living in an urban area. Children, 9-12 years had 7% (95% CI 1-13%) higher UCd (p=0.04) than children 5-8 years. About 1% of the mothers, and 0.06% of the children, exceeded the tolerable weekly intake (TWI) appointed by EFSA, corresponding to 1.0 µg Cd/g crea in urine. Poland had the highest UCd in comparison between the 16 countries, while Denmark had the lowest. Whether the differences between countries are related to differences in the degree of environmental Cd contamination or to differences in lifestyle, socioeconomic status or dietary patterns is not clear.

Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of molds of the Fusarium genus.

The rates of infection with Fusarium molds are increasing, and a diverse number of Fusarium spp. belonging to different species complexes can cause infection. Conventional species identification in the clinical laboratory is time-consuming and prone to errors. We therefore evaluated whether matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a useful alternative. The 289 Fusarium strains from the Belgian Coordinated Collections of Microorganisms (BCCM)/Institute of Hygiene and Epidemiology Mycology (IHEM) culture collection with validated sequence-based identities and comprising 40 species were used in this study. An identification strategy was developed, applying a standardized MALDI-TOF MS assay and an in-house reference spectrum database. In vitro antifungal testing was performed to assess important differences in susceptibility between clinically relevant species/species complexes. We observed that no incorrect species complex identifications were made by MALDI-TOF MS, and 82.8% of the identifications were correct to the species level. This success rate was increased to 91% by lowering the cutoff for identification. Although the identification of the correct species complex member was not always guaranteed, antifungal susceptibility testing showed that discriminating between Fusarium species complexes can be important for treatment but is not necessarily required between members of a species complex. With this perspective, some Fusarium species complexes with closely related members can be considered as a whole, increasing the success rate of correct identifications to 97%. The application of our user-friendly MALDI-TOF MS identification approach resulted in a dramatic improvement in both time and accuracy compared to identification with the conventional method. A proof of principle of our MALDI-TOF MS approach in the clinical setting using recently isolated Fusarium strains demonstrated its validity.

Banana infecting fungus, Fusarium musae, is also an opportunistic human pathogen: are bananas potential carriers and source of fusariosis?

During re-identification of Fusarium strains in the BCCM™/IHEM fungal collection by multilocus sequence-analysis we observed that five strains, previously identified as Fusarium verticillioides, were Fusarium musae, a species described in 2011 from banana fruits. Four strains were isolated from blood samples or biopsies of immune-suppressed patients and one was isolated from the clinical environment, all originating from different hospitals in Belgium or France, 2001-2008. The F. musae identity of our isolates was confirmed by phylogenetic analysis using reference sequences of type material. Absence of the gene cluster necessary for fumonisin biosynthesis, characteristic to F. musae, was also the case for our isolates. In vitro antifungal susceptibility testing revealed no important differences in their susceptibility compared to clinical F. verticillioides strains and terbinafine was the most effective drug. Additional clinical F. musae strains were searched by performing BLAST queries in GenBank. Eight strains were found, of which six were keratitis cases from the U.S. multistate contact lens-associated outbreak in 2005 and 2006. The two other strains were also from the U.S., causing either a skin infection or sinusitis. This report is the first to describe F. musae as causative agent of superficial and opportunistic, disseminated infections in humans. Imported bananas might act as carriers of F. musae spores and be a potential source of infection with F. musae in humans. An alternative hypothesis is that the natural distribution of F. musae is geographically a lot broader than originally suspected and F. musae is present on different plant hosts.

The European COPHES/DEMOCOPHES project: towards transnational comparability and reliability of human biomonitoring results.

COPHES/DEMOCOPHES has its origins in the European Environment and Health Action Plan of 2004 to "develop a coherent approach on human biomonitoring (HBM) in Europe". Within this twin-project it was targeted to collect specimens from 120 mother-child-pairs in each of the 17 participating European countries. These specimens were investigated for six biomarkers (mercury in hair; creatinine, cotinine, cadmium, phthalate metabolites and bisphenol A in urine). The results for mercury in hair are described in a separate paper. Each participating member state was requested to contract laboratories, for capacity building reasons ideally within its borders, carrying out the chemical analyses. To ensure comparability of analytical data a Quality Assurance Unit (QAU) was established which provided the participating laboratories with standard operating procedures (SOP) and with control material. This material was specially prepared from native, non-spiked, pooled urine samples and was tested for homogeneity and stability. Four external quality assessment exercises were carried out. Highly esteemed laboratories from all over the world served as reference laboratories. Web conferences after each external quality assessment exercise functioned as a new and effective tool to improve analytical performance, to build capacity and to educate less experienced laboratories. Of the 38 laboratories participating in the quality assurance exercises 14 laboratories qualified for cadmium, 14 for creatinine, 9 for cotinine, 7 for phthalate metabolites and 5 for bisphenol A in urine. In the last of the four external quality assessment exercises the laboratories that qualified for DEMOCOPHES performed the determinations in urine with relative standard deviations (low/high concentration) of 18.0/2.1% for cotinine, 14.8/5.1% for cadmium, 4.7/3.4% for creatinine. Relative standard deviations for the newly emerging biomarkers were higher, with values between 13.5 and 20.5% for bisphenol A and between 18.9 and 45.3% for the phthalate metabolites. Plausibility control of the HBM results of all participating countries disclosed analytical shortcomings in the determination of Cd when using certain ICP/MS methods. Results were corrected by reanalyzes. The COPHES/DEMOCOPHES project for the first time succeeded in performing a harmonized pan-European HBM project. All data raised have to be regarded as utmost reliable according to the highest international state of the art, since highly renowned laboratories functioned as reference laboratories. The procedure described here, that has shown its success, can be used as a blueprint for future transnational, multicentre HBM projects.