We provide evidence that there is little “stability” within resident clonal populations of the common gut microbiome bacterial family, Enterobacteriaceae. Given that clones can vary substantially in genome content and that evolutionary processes operate on the population level, these results question the biological relevance of apparent stability at higher taxonomic levels.Longitudinal human gut microbiome datasets generated using community-level, sequence-based approaches often report a sub-set of long-lived “resident” taxa that rarely, if ever, are lost. This result contrasts with population-level turnover of resident clones on the order of months to years. We hypothesized that the disconnect between these results is due to a relative lack of simultaneous discrimination of the human gut microbiome at both the community and population-levels. Here, we present results of a small, longitudinal cohort study ( Fundamental aspects of the adult human gut ecosystem remain understudied, including why and/or how some microbes colonize and persist while others do not. Estimates of residency (the length of time an organism occupies a niche) in the gut are scarce even for bacteria easily isolated on selective and/or differential media. The few cultivation-based, longitudinal studies of human adults suggest that strain-level turnover in the gut is common over relatively short time scales  –4. Given6 CFU/gram feces for 16S sequencing [At least some of the above discrepancy is likely due to the scarcity of published studies. However, the discriminatory power of sequencing-based versus cultivation-based approaches is also likely to lead to different conclusions. For example, 16S sequencing is notoriously inconsistent or, depending on one’s view, inaccurate at identifying bacterial species , and is quencing ), makingEnterobacteriaceae is the most taxonomically diverse bacterial family [Enterobacteriaceae in humans has narrowly focused on the epidemiology, pathogenesis, virulence, and/or antibiotic resistance of pathogenic strains. Similarly, despite the ability to cultivate all  of the currently named Enterobacteriaceae species on a single selective, differential medium (MacConkey agar), there are few longitudinal studies from healthy human adults. As a result, it is difficult to extrapolate fundamental aspects of non-pathogenic Enterobacteriaceae ecology  from studies of pathogenic and/or antibiotic resistant strains from hospitalized patients [l family  recognizl family . Most repatients .Enterobacteriaceae isolates  from MacConkey agar plates and identified residents at the individual clone level. The dynamics observed with cultured Enterobacteriaceae isolates are at odds with community-level stability and suggest that the adult human gut microbiome is not as stable as previously reported. Finally, with respect to longitudinal studies of healthy human adults, this is the first study to report statistical evidence that certain Enterobacteriaceae clones are more likely to be resident compared to others.For this study, we collected semi-weekly stool samples from a cohort of eight healthy human adults and quantified the abundance and distribution of resident and transient operational taxonomic units (OTUs) and amplified sequence variants (ASVs) using 16S rRNA sequencing. From the same samples, we generated a large collection of TM Commode Specimen Collection System, Thermo Fisher Scientific, Inc.) into sterile 50 mL conical screw-cap tubes using a sterile tongue depressor. If samples could not be processed immediately, they were refrigerated at 4 °C for no longer than 2 hours prior to processing. Samples were processed inside an anaerobic chamber  to help preserve anaerobic bacteria for future investigations. Each sample was mixed by shaking with ~30 mL of pre-reduced, sterile phosphate buffered saline. Aliquots (1 mL) were then mixed with 0.2 mL of 80% glycerol in sterile cryogenic vials containing gaskets  at a final glycerol concentration of 15%. In parallel, additional aliquots (0.2 mL of stool) were made directly into DNeasy Power Soil DNA extraction tubes  for bulk DNA extraction prior to 16S rRNA sequencing (see below). All aliquots were immediately frozen at −80 °C.This study was approved by the institutional review board of Montana State University. All participants were enrolled with informed consent, and a total of 9 volunteers  were recruited, ranging in age from 25 to 40 years of age. No information beyond sex and age range was recorded, as correlating host factors with temporal dynamics was not the focus of this study. Although not asked directly, none of the participants co-habitated to our knowledge. Stool samples were requested once every 2 weeks from March 2016 to January 2018 (Table Enterobacteriaceae species as opposed to precisely estimating true abundances. LB cultures were incubated overnight (37 °C), and freezer stocks were made in 15% glycerol and immediately frozen at −80 °C.Fecal slurries were thawed to room temperature, serially diluted in sterile PBS, and plated onto MacConkey agar plates, followed by overnight incubation at 37 °C. A target of 95 single colonies per sample were picked from plates using sterile toothpicks and transferred into lysogeny broth (LB) in a 96 deep-well format. One well served as an uninoculated control. If lactose fermenting and non-fermenting colonies were present, each type was picked at roughly the ratio present on the plate. In addition, care was taken to select colonies that differed in morphology, such that rare, odd-looking colonies were present in downstream characterizations. This approach was used to maximize the probability of observing diverse E. coli phylogrouping algorithm of Clermont et al. [E. coli. Residents were defined as described in the Results section. Resident clones were identified as having identical GTG5 rep-PCR fingerprints [E. coli residents were characterized biochemically (API 20E test strips) to identify their likely species designation. In one case, a cryptic Escherichia clade was confirmed by multilocus sequence typing (Sanger sequencing) following a published protocol [Diluted 1:10) cultures were used for PCR-based genotyping; first using the t et al.   and the V4 region of 16S rRNA encoding gene was amplified and sequenced as described  at the URaw reads were processed using mothur v.1.39.5 . Low-quaReads from each sample were rarefied to 10,000 and all downstream analyses were conducted using R version 3.5.1 as follows: PERMANOVA was conducted using the Adonis function in vegan 2.5-3 and 1 000 permutations , partitiNine healthy volunteers between 24 and 40 years of age  were enrolled with informed consent. No other information was collected , but all were well enough to participate and presumed to represented healthy adults in this geographic location. Only a single enrollee did not participate beyond three sampling dates and was excluded. From the remaining eight, a total of 392 stool samples were collected, averaging 49 samples per individual (range = 25–97), spanning an average of 512 days (range = 245–849 days), and a median of 8 days apart  supported statistically different groups . However, 150 of the 1 247 (12%) between-participant comparisons yielded lower beta diversity than the median within-participant diversity, highlighting the high degree of similarity between these microbiomes.Stool samples from eight participants were sequenced using dual-index barcoding and paired-end Illumina MiSeq sequencing V4 region), resulting in 293 OTUs among all eight participants. Non-metric multidimensional scaling (NMDS) grouped samples according to participant, and PERMANOVA , and PAM clustering  for a dietary residue in healthy human adults [Residency was defined based on human gut transit time. We reasoned that bacteria entering the gut would be lost at the rate of gut transit n adults , we set p = 0.1976, 0.2502, 0.4157 for Min, Ave, and Max, respectively). Thus, there was little support for a “core resident microbiome”.The most stable sub-set of the microbiome was those OTUs/ASVs that were resident for the entire study period (Always Resident), corresponding to an average of 34% (Min), 39% (Ave), or 48% (Max) of OTUs and 27% (Min), 32% (Ave), or 41% (Max) of ASVs observed within participants Table . In suchWe next generated stacked bar charts of OTUs and ASVs according to percentile residency rank Fig. . These pn = 324) used for 16S sequencing and the additional 68 stool samples that were available . An average of 84 colonies per stool sample per participant (SD = 4.7) were characterized by PCR-based assays, and the clear majority  belonged to one of ten E. coli sensu stricto phylogroups. The percentage of non-E. coli varied between participants from <1 to 37% . Since all members of the Enterobacteriaceae known to colonize the human gut should be cultivable on MacConkey agar and since we attempted to isolate all colony morphology types, these results suggest that E. coli is by far the most dominant Enterobacteriaceae in healthy humans. Interestingly, a significant number of E. coli  did not appear to ferment lactose when grown on MacConkey agar, suggesting that this commonly used biochemical marker of E. coli sensu stricto requires re-evaluation.A total of 32 470 single colonies were picked from MacConkey agar plates from the same stool samples  and a reasonable random sub-set (22–68% of isolates) in the remaining participants  of these were resident. Note that because rep-PCR was not performed on all isolates, the number of transient clones was likely to be somewhat greater than 86. Of the 37 resident clones, only three were non-E. coli, and each belonged to a different “species” , suggesting that with respect to residency, E. coli was again the dominant species. Within E. coli sensu stricto, all phylogroups were observed with the single exception of phylogroup C. The most commonly observed phylogroup was B1 , followed by A.1 , A , B2.3 , and F . All other clones  comprised ≤ 5% each and together accounted for only 15%. Residence time (log 10 transformed number of days observed) was significantly different between Enterobacteriaceae clones  and approached significance using the Min estimate . Phylogroups A and B2.3 had the longest residence times, and following correction for multiple comparisons, both phylogroups resided significantly longer than phylogroup A.1 using both Ave  using the Ave estimate  stool samples that were evaluated by 16S sequencing. Only two OTUs classified as Enterobacteriaceae, and one of these classified to the Escherichia/Shigella genus. Likewise, five ASVs classified as Enterobacteriaceae and one of these classified to the Escherichia/Shigella genus. By comparing OTU/ASV presence-absence to culture-based presence-absence across samples, we evaluated the diagnostic potential of 16S to identify Enterobacteriaceae or Escherichia/Shigella in stool samples. Counts for false positives, true positives, false negatives, and true negatives as well as positive and negative predictive values and accuracy were generated . OTU-based analysis was slightly more accurate than ASV-based analysis, but neither performed remarkably well . Thus, with respect to Enterobacteriaceae and Escherichia/Shigella, 16S sequencing and culture results did not correlate well.E. coli [Few longitudinal studies have been conducted to identify short and long-lived members of the human gut microbiome, estimate rates of migration into and out of the gut ecosystem, or understand the functional impact of taxonomic turnover. Temporal variability is an important attribute of ecosystems affecting colonization dynamics . In macrE. coli , and as n = 8 participants for an of ~17 months) more than doubles the size of current datasets. Other longitudinal 16S datasets have been published, but their comparatively short sampling periods  [E. coli populations in the gut are dynamic and turn over on the order of months to years. Indeed, clones observed in only two of our eight participants resided for the entire sampling period.Two previous longitudinal 16S rRNA sequencing studies of healthy human adults  both con months) , 11, eva months)  and/or l months) , 38 make months) . Such st months) . Our res months) –41 have E. coli clone dynamics scale to the rest of the microbiome, but culture-based evidence suggests that it does. For example, Faith et al. cultured and genome sequenced resident (up to 1.3 years) “strains” of the Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria phyla from longitudinally collected stool samples [E. coli resident turnover in our study. Another study cultured Bacteroides species from longitudinal stool samples of 15 adults and found that some individuals harbored different strains over the study period [E. coli shared <40% of their enzyme coding loci [Our results do not address whether  samples . They dey period . So, howE. coli residency in human adults have been limited to some degree by both sampling depth (within samples) and breadth . According to rare events calculation, the average of 84 isolates per sample that we achieved allowed for confident detection of representative clones at ~2% relative abundance , which is by far the most comprehensive study to date. It is important to recognize, however, that our approach was meant to capture diversity and so while our identification of resident clones  requireE. coli is so prevalent among humans, why is it often missing from gut microbiome datasets? A principle factor seems to be that the accuracy of 16S sequencing, regardless of analytical approach (OTU or ASV), is poor for Enterobacteriaceae and Escherichia/Shigella, which is consistent with analyses of simple mock communities (n ≤ 20 members) where E. coli cells or genomic DNA were mixed at relatively equal abundances with that of other gut bacteria [Enterobacteriaceae was E. coli in our cohort, which is not the case for other human gut microbiome families, such as the Bacteroidaceae, that can be comprised of at least 20 different species [Bacteroidaceae vs. Enterobacteriaceae) should be avoided, as they could mean very little at the individual population or species levels.If bacteria , 44. In Enterobacteriaceae and Escherichia/Shigella. That said, given the widespread use of both analyses, more diagnostic testing against the gold-standard of culture is needed to benchmark their biological relevance.Regarding the use of OTU versus ASV-based analysis, we found these two approaches yielded nearly identical ordinations (a common proxy for community diversity) for five of eight participants but produced different results for the remaining three. This suggests that ASV-analysis differs in discriminatory power from sample-to-sample and that comparisons are sometimes (and perhaps unpredictably) made at different taxonomic levels . In contrast, the 97% 16S OTU cut-off is a reasonable approximation of both the 95% average nucleotide identity of genomes and 70% DNA-DNA hybridization cut-offs that are commonly used to differentiate bacterial species , 46, thuE. coli were more prevalent among people from industrialized compared to developing countries [E. coli were remarkably stable in the face of enterotoxigenic E. coli infection and antibiotic treatment [E. coli residency, and especially factors associated with uropathogenesis . Thountries , 40. Simreatment . Microbireatment  seem to molysins , 53). Comolysins   lac- resident clones belonging to three different phylogroups were observed among half (n = 4) of our cohort; lac- clones belonging to six of the nine observed phylogroups were identified; and lac- clones comprised 17% of the 120 E. coli clones observed. These results are not entirely without precedent as Sears et al. [E. coli also appear to be associated with uropathogenesis [E. coli, regardless of lactose utilization.Few studies have considered lactose non-fermenting (lac-) s et al.  and a pus et al.  reportedogenesis , 60, whiSupplementary Material


Language Complaints” (LC) or “No Language Complaints” (NLC). They also performed a neuropsychological assessment tackling attention/processing speed, memory, executive functioning, and language at baseline. From these, 275 (68.4%) participated in a follow-up evaluation 4.9 (±0.6) years later. At re-evaluation, subjects had a mean age of 70.4 (±8.3) years, 7.5 (±4.4) years of education, and 63.3% were female. Multivariate linear regression analysis was used to investigate whether language complaints at baseline predicted poorer language performance at follow-up or increased the risk of cognitive decline, with correction for sex, depressive symptoms, living status, baseline performance, and composite memory and executive performance. Results indicated that LC subjects had significantly worse performances than NLC subjects on semantic fluency 5 years later, but with a similar rate of decline overtime that was not associated with a follow-up outcome of cognitive decline/dementia. Language difficulties may represent a specific type of age-related cognitive complaints. Longer follow-ups are necessary to understand if they are associated with an increased risk of language or cognitive decline.Subjective cognitive complaints are rather prevalent in the elderly population and are associated with an increased risk of cognitive impairment and dementia. However, the predictive role of specific types of cognitive complaints has been less systematically assessed. The aim of the present study is to examine the predictive value of language complaints for cognitive and language decline in a cohort of community-dwelling healthy older adults, followed longitudinally over a 5-year period. A total of 402 subjects were enrolled in a prospective longitudinal study on aging and cognition. Participants answered a cognitive complaints questionnaire including two questions directed to language and were classified at baseline as having “ Subjective cognitive complaints (SCCs) are prevalent in the elderly populations . They arβ deposition (Subjective language complaints (SLCs) are frequently reported by elderly subjects, who typically complain of difficulty in remembering proper names and word-finding problems. Cross-sectional studies indicate that SLC are associated with worse objective cognitive performance in healthy, community-dwelling subjects ,b and shposition , which fThe role of SCC in the prediction of future cognitive decline has been a matter of debate. While several studies support the relationship between SCC and poorer cognitive performance  and the The aim of the present study is to examine the predictive value of language complaints for cognitive and language decline in a cohort of community-dwelling healthy older adults, followed longitudinally over a 5-year period. We hypothesize that (1) having significant language complaints at baseline is associated with increased risk of cognitive/language decline at follow-up; (2) subjects with language complaints show significantly more decline over time on language measures when compared to subjects without complaints; and (3) having language complaints is associated with a diagnosis of cognitive decline (cognitive impairment or dementia) at follow-up.via their general practitioner if (1) Portuguese was their native language; (2) they were aged 50 years old or more; (3) they presented normal education-adjusted score on the MMSE the MMSE ,b. BriefA total of 127 subjects previously enrolled in the baseline evaluation were lost to follow-up due to several reasons  . This haThe study was carried out in accordance with the recommendations of the Ethics Committee of the Lisbon Medical Academic Center with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committees of the Lisbon Medical Academic Center, Portuguese Health Authority, and local primary care centers.z-scores according to the existing norms (ZTMTA + ZTMTB)/2] and Memory [/2], respectively.Neuropsychological evaluations were performed by fully licensed neuropsychologists. It comprised the MMSE , tests ong norms . A memorIn addition, the 15-item Geriatric Depression Scale  and the Do you ever forget names of family member or friends”) and 6 (referred to word finding difficulties—“Do you ever have difficulties in finding particular words?”). For item 3, scores range from 0 – No to 3 – Yes, with problems, whereas item 6 presents is scores 0 – No and 1 – Yes. In order to avoid scaling bias, responses to item 3 were dichotomized into the absence (score of 0 or 1) or presence of problematic complaints (scores of 2 or 3). We opted for this method as suggested by the original author  if both SMCQ items had a score of 1 or “No Language Complaints” (NLC) if a score of 0 was obtained in at least one of those items (NLC).Language outcome was evaluated with the The diagnosis of cognitive impairment (MCI/dementia) was done in accordance to published criteria  and has t-test for continuous variables or Chi-Square (χ2) test for categorical variables. To investigate whether the presence of language complaints at baseline was a predictor of poorer language performance at follow-up, multivariate linear regression analysis was used, with correction for sex, depressive symptoms, living status and baseline performance, executive and memory composite scores. Whenever statistical differences existed between groups on baseline measures, change between baseline and follow-up performances were used as an independent variable in the regression models. Due to the multiplicity of tests, Bonferroni correction was used and adjusted p for each comparison were computed by multiplying uncorrected p with the number of tests conducted in each analysis. Results were considered statistically significant at p < 0.05.Data were analyzed using IBM SPSS computer software . Descriptive statistics was used for continuous variables and counts (percent) for categorical variables. Group differences were tested using the Student’s Item 6 and 31.7% on SMCQItem 3. Altogether 25 participants were classified as LC. The LC group had significantly lower baseline performance on semantic verbal fluency  and had more depressive symptoms and overall cognitive complaints (p = 0.0014). At follow-up, LC group showed significantly lower scores on semantic  and phonemic fluencies (p = 0.0014).At follow-up, participants had a mean age of 70.4 (±8.3) years, 7.5 (±4.4) years of education, 63.6% were female, and 13.6% were living alone . No statF = 11.382, p < 0.0001; F = 14.617, p < 0.0001; F = 19.969, p < 0.0001, respectively]. Overall, baseline performance predicted follow-up performance (p < 0.01). After controlling for possible confounders  (p = 0.005). Analyses were repeated using change (between baseline and follow-up) in fluency scores as independent variables, although models were not statistically significant . A trend toward more pronounced decline from baseline to follow-up is depicted in To investigate whether the presence of language complaints at baseline was a predictor of worse language performance at follow-up, a multivariate linear regression analysis was used, with correction for sex, depressive symptoms, living status, baseline performance, executive, and memory composite scores. Models for semantic  and phonemic fluency were statistically significant [founders  and adjup = 0.004), executive (p < 0.0001), and memory baseline performances (p = 0.007) were also significant predictors of semantic (food) fluency.In addition, gender .The presence of language complaints was not significantly associated with a clinical diagnosis of cognitive impairment (MCI or dementia) at follow-up  as well as high executive processing  , thus taAlthough having language complaints at baseline was not significantly predictive of a higher decline nor the diagnosis of MCI/dementia 5 years later, these individuals showed worse objective performances at this time corroborating that their complaints had an objective translation. Future studies should address whether this single domain is declining at a faster pace than global cognitive decline in subjects presenting with significant language complaints.In line with previous work from our group ,b, livinWe acknowledge some limitations to this study. The first is the high attrition rate. As previously reported , individThe 5-year follow-up, the use of a comprehensive neuropsychological at baseline, and the clinical data provided by general practitioners at the primary care centers were strengths of this study.Subjective language difficulties may represent a domain-specific cognitive complaint, that is associated to objects poor performance 5 years later in tests highly depending on language abilities, when compared to subjects not reporting such difficulties. Although more studies are necessary to disentangle the contribution of different cognitive processes to these complaints, and their long-term impact, they must be valued since they may allow the identification of subjects that have an objective worse performance in language tasks.isabel_martins@medicina.ulisboa.pt.The datasets for this manuscript are not publicly available because Datasets belong to Language Research Laboratory (Faculty of Medicine of Lisbon) and are currently still being analyzed. Requests to access the datasets should be directed to Initial concepts and framework were developed by CM and IM. Data analysis and interpretation were performed by CM and IM. Manuscript was prepared by CM. IM helped with revision and final comments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mercury is toxic to the developing brain, but the lowest concentration associated with the development of behavior problems is unclear. The purpose of this study was to examine the association between very low-level mercury exposure during fetal development and behavior problems in children.We used data from 389 mothers and children in a prospective pregnancy and birth cohort study. We defined mean prenatal mercury concentration as the mean of total whole blood mercury concentrations in maternal samples collected at 16- and 26-weeks of gestation, delivery, and neonatal cord blood samples. We assessed parent-reported child behavior up to five times from two to 8 years of age using the Behavioral Assessment System for Children (BASC-2). At 8 years of age, we assessed self-reported child anxiety using the Spence Children’s Anxiety Scale (SCAS). We used multiple linear mixed models and linear regression models to estimate the association between mean prenatal mercury concentrations and child behavior and anxiety, respectively.The median prenatal total blood mercury concentrations was 0.67 μg/L. Overall, we did not find statistically significant associations between mean prenatal mercury concentrations and behavior problems scores, but a 2-fold increase in mercury concentrations at 16-weeks gestation was associated with 0.83 point  higher BASC-2 anxiety scores. Maternal and cord blood mercury concentrations at delivery were associated with parent-reported anxiety at 8 years.We found limited evidence of an association between very-low level prenatal mercury exposure and behaviors in children, with an exception of anxiety. The neurotoxicity of mercury at high doses was established in congenital mercury poisonings in Japan and Iraq –4. MethyWe hypothesized that very low-level prenatal whole blood mercury concentrations (< 4–5 μg/L) would be associated with behavior problems among children from 2 to 8 years of age. In general, fetuses may be more susceptible to the adverse effects of exposure to environmental toxicants because they have rapidly developing organ system, different pharmacokinetics and higher exposure dose per body surface area compared to adults. Any disruption during pregnancy can significantly result in adverse outcomes in growth and development. The magnitude of adverse outcomes also depends on the timing of the exposure [early vs late pregnancy exposure] and these periods of heightened sensitivity are toxicant-specific. This makes it necessary for researchers to study and understand these periods of heightened vulnerability/window of vulnerability . For merWe used data from a prospective pregnancy and birth cohort study, the Health Outcomes and Measures of the Environment (HOME) study, in Cincinnati, Ohio. The original study was designed to test and quantify the association between low-level prenatal and postnatal exposure to various environmental toxicants with the development of cognitive and behavioral problems in children. We enrolled pregnant women in the study from March 2003 to January 2006. Eligibility criteria at enrollment were: a) age ≥ 18 years, b) between 13 to 19 weeks of gestation, c) residing in a house built before 1978, d) not on a medication for seizure or thyroid disorders, and e) negative HIV status. Of 1263 eligible pregnant women, 468 (37%) agreed to participate in the study and 389 women remained in the study until delivery of a liveborn singleton infant. The details about the study cohort are described elsewhere .We collected whole blood samples from mothers at approximately 16 - and 26 weeks of gestation, and at the time of delivery. We also collected whole blood samples from children at birth (cord blood) and at the 2, 3, 4, 5, and 8 year visits. The whole blood samples were collected in EDTA vacutainer tubes that were lot tested for metal contamination, and stored at − 80 °C until shipment to the United States Centers for Disease Control and Prevention (CDC) for analysis. Total whole blood mercury was quantified using inductively coupled plasma mass spectrometry (ICP-MS) . This mu> 60 is considered “at-risk behavior,” with the exception for adaptive skills and corresponding subscales, in which a score < 40 is non-optimal. We were primarily focused on examining the relationship of prenatal mercury with composite behaviors , but we also examined the associations with ADHD  and anxiety subscales as prior studies found that prenatal mercury was associated with these specific behaviors [Children’s behavior was assessed using the second edition of the Behavioral Assessment System for Children (BASC-2) Parent Rating Scale during follow-up visits when children were 2, 3, 4, 5, and 8 years. The BASC-2 is a reliable and valid assessment of a child’s adaptive and problem behaviors in community and home settings , 28. Theehaviors , 16, 18.We also assessed anxiety in children using the self-reported Spence Children’s Anxiety Scale (SCAS) when the children were about 8 years of age. The SCAS assesses six domains including generalized anxiety, panic/agoraphobia, social phobia, separation anxiety, obsessive-compulsive disorder, and physical injury fears . A trainAll assessments for the study were conducted in a standardized clinic setting to minimize distractions. The examiners were blinded to maternal and child mercury concentrations.Our selection of potential confounders was guided by other research studies , 18, 30.We examined other variables as potential confounders, including maternal age at delivery (≤30 years and > 30 years), maternal ethnicity (white and nonwhite), annual household income , maternal education (completed and not completed bachelor’s degree), marital status , and child sex. Maternal age at delivery and annual household income were categorized based on the frequency distribution of the variables. We also adjusted for the caregiving environment using the Home Observation for Measurement of the Environment (HOME) Inventory conducted during a 12-month home visit . We cate2-transformed mercury concentrations to reduce the influence of outliers. We compared 389 women and their children’s  demographic characteristics with 320 dyads who had at least one blood mercury and BASC-2 data. We compared the 320 dyads with at least one mercury and behavior assessment with 69 dyads not included in the analyses because of missing either mercury and behavior assessment. We also stratified mean prenatal mercury concentrations and child BASC-2 scores of 226 dyads who were followed at the 8-year visit by demographics and by maternal and child characteristics.We defined mean prenatal mercury concentrations as the mean of mercury measured in whole blood collected at 16 and 26 weeks gestation, at delivery from the mothers, and in cord blood from the infants. We used average of the logWe examined the associations of prenatal mercury with BASC-2 composite externalizing problems, internalizing problems, and BSI, and adaptive skills using linear mixed models with an unstructured covariance matrix. The linear mixed models account for the correlated nature of the repeated observations. Thus, the coefficient from a linear mixed model would represent an average increase in mean BASC-2 scores for every 2-fold increase in mercury concentrations.We constructed linear mixed models for composite scores. First, we constructed unadjusted models. Then we added fish intake in the unadjusted models to test for confounding with whole blood mercury. Next, we added participants’ demographics and characteristics, child blood lead concentrations, maternal serum cotinine concentrations with and without child mercury concentrations to the unadjusted models and called this multivariable model 1. We added fish intake to the multivariable model 1 and call this model multivariable model 2. As mentioned before, the choice of the inclusion of the variables in the models was based on the criteria of confounders  and also guided by previous research , 18, 30.We conducted secondary analyses to test for evidence of a developmental window of vulnerability for behaviors that were associated with mean prenatal mercury concentrations. We constructed a series of regression models for 16- and 26 weeks gestation, at delivery, cord and mean prenatal mercury concentrations with and without including cord blood concentrations. Not only anxiety, we also examined the association of prenatal mercury concentrations and other composite scores/subscales at 8-years of age. We examined anxiety at 8-years of age in more depth because there is some evidence suggesting an association between prenatal mercury exposure and anxiety . AlthougAdditionally, we examined models stratified by child sex and maternal race/ethnicity. We checked for two-way interactions by adding interaction terms for child age, child sex, maternal race/ethnicity, child blood lead concentrations, maternal cotinine concentrations, and PCBs with mercury at each prenatal visit. Finally, we examined the association between mean of post-natal child blood mercury concentrations and behavior scores in children.Out of 468 enrolled pregnant women in the study, 67 14.3%) dropped out of the study before delivery, 12 (2.6%) delivered twins or stillborn children, and 389 (83.1%) delivered live singleton infants. The 320 (82.2%) dyads who had at least one mercury and one behavior assessment were similar to the original sample of 389 mothers and their singleton children (Table 4.3% dropThe arithmetic mean (±SD) and median prenatal mercury concentrations were 0.95 ± 1.02 and 0.67 μg/L, respectively. The median 16- weeks, 26-weeks, at delivery and cord blood mercury concentrations were 0.68, 0.60, 0.63, and 0.70 μg/L, respectively , 26 weeks (Pearson r = 0.77), and delivery (Pearson r = 0.82) with p-value of < 0.001 for all correlation coefficients (data not shown).We found that cord blood mercury concentrations were strongly correlated with maternal mercury concentrations at 16 weeks   Fig. .Fig. 4Adp = 0.81).In secondary analysis, we did not find any statistically significant associations between maternal mercury concentrations at 16- or 26 weeks of gestation, delivery and cord blood mercury concentrations and composite BASC-2 scores, attention problems, aggression, or hyperactivity subscales (data not shown). In contrast, we found a significant positive association between mercury concentrations at 16 weeks of gestation and BASC-2 anxiety subscales through 8-years   between maternal mercury concentrations at delivery and BASC-2 anxiety subscales through 8-years in girls, but the association was not significant in boys . The In secondary analyses, inclusion of other interaction terms for maternal race/ethnicity, child age, child blood lead concentrations, and maternal cotinine and PCB concentrations with mercury at each prenatal visit did not substantially alter our results.inverse associations between prenatal mercury concentrations and self-reported SCAS total scores; the relationship with 16-week gestation mercury was significant  . We did not find any statistically significant association between prenatal mercury concentrations and other BASC composite scores/subscales at 8 years of age.We observed a pattern of positive associations between maternal mercury concentrations and parent-reported BASC-2 anxiety subscale at 8 years. The association was statistically significant for cord blood  and maternal  mercury concentrations at delivery  anxiety scores from 2 to 5 years and at 8 years of age. However, we did not observe the same pattern of associations between prenatal mercury concentrations and child-reported anxiety scores (SCAS) at 8 years. Indeed, we found that maternal mercury concentrations during pregnancy were We compared our findings for the prenatal mercury exposure and attention or externalizing  behaviors with other prospective cohort studies. In the Faroe Islands cohort, prenatal mercury exposure was significantly associated with impaired performance in attention at 7 and 14 years of age , 13. HowWe also compared the findings of our study with other studies for children’s internalizing behaviors , 15, 16.R > 0.73).The United States Environmental Protection Agency derived a blood reference dose (RfD) for mercury of 5.8 μg/L , 18, 36.r = 0.04, P = 0.60). The lack of a correlation between parent and child reported anxiety in our study was consistent with reports by other investigators [r = 0.30) [Paradoxically, we found that maternal prenatal mercury concentrations were associated with higher parent reported BASC-2 anxiety subscales, but lower self-reported SCAS anxiety subscales in 8-year old children. Moreover, parent reported BASC-2 anxiety subscale at 8-year and self-reported SCAS total score at 8-year were not correlated with each other (Pearson tigators –39. For  = 0.30) . These sSeveral limitations should be considered while interpreting the findings of the study. First, we measured total blood mercury concentrations. Total blood mercury concentrations in our study participants, which were below 4 μg/L (mean = 0.95 μg/L), are a measure of both inorganic mercury and methylmercury –42. SecoThis study also has several strengths. First, the low blood mercury concentrations in the HOME Study allowed us to examine the effect of prenatal mercury exposure at levels below a previously reported apparent threshold levels for children’s behavior. This is the first study to examine the effect of such a low-level prenatal mercury concentrations on a full spectrum of child behaviors. Second, mercury concentrations of our study cohort are comparable with the pregnant women in the NHANES study, a nationally representative sample of the U.S. population . The geoWe did not find a consistent association between very low-level prenatal mercury exposure and behavior problem scores in children, but we did find some evidence of an association between very low-level mercury exposure during early pregnancy and parent-reported anxiety scores in children. The association of very low-level mercury exposures during early brain development with the development of anxiety and other behavior problems in children deserves further scrutiny.


Over-aggressive intravenous fluid therapy with crystalloids has adverse effects in trauma patients. We assessed the role of large-volume (≥5l) administration of crystalloids within 24h of injury as an independent risk-factor for mortality, in-hospital complications, and prolonged mechanical ventilation.A retrospective cohort analysis of adult trauma patients admitted to a level 1-trauma center between December 2011 and December 2012. Patient demographics, clinical and laboratory values, and total resuscitation fluid administered within the first 24h of injury were obtained. Outcomes included mortality, in-hospital complications and ventilator-days. Multivariable logistic regression and Poisson regression analyses were performed to investigate any association between the administration of ≥5L crystalloids with the aforementioned outcomes while controlling for selected clinical variables.n = 708) were males. The median injury severity score (ISS) was 17 (IQR 9–25). Overall mortality rate was 7% (n = 67). Multivariable logistic regression analysis showed several variables independently associated with mortality (p < 0.05), including resuscitation with ≥5L crystalloid in the first 24h (adjusted odds ratio (aOR) 2.55), older age (aOR 1.03), higher ISS (aOR 1.09), and lower temperature (aOR 0.68). The variables independently associated with in-hospital complications (p < 0.05) were older age, longer ICU stay, and platelet transfusion within 24h of the injury. Need for mechanical ventilation was more common in patients who received ≥5L crystalloids (RR 2.31) had higher ISS (RR 1.02), developed in-hospital complications (RR 1.91) and had lower presenting temperature (RR 0.87).A total of 970 patients were included in the analysis. 264 (27%) received ≥5L of crystalloids in the first 24h of injury. 118 (12%) had in-hospital complications and 337 (35%) required mechanical ventilation. The median age was 46 years (interquartile range (IQR) 27–65) years and 73% (Large-volume crystalloid resuscitation is associated with increased mortality and longer time ventilated, but not with in-hospital complications such as pneumonia and sepsis. Based on this data, we recommend judicious use of crystalloids in the resuscitation of trauma patients. Trauma is a major cause of mortality, with traumatic injures estimated to result in over five million deaths annually worldwide . Severe Injudicious use of crystalloid during resuscitation has been shown to exacerbate the systemic insult in response to hemorrhagic shock, including worsened cardiac function, pulmonary complications, gastric dysmotility, abdominal compartment syndrome, coagulopathy, immunological and inflammatory dysfunction –14. ExceA retrospective cohort analysis of all adult trauma patients admitted to St. Michael’s Hospital (SMH) between December 2011 and December 2012 was performed. Patients with traumatic hanging injuries, and those who died within 1h of hospital admission were excluded from data analysis. The study was approved by the research ethics board (REB) of St. Michael’s Hospital, Toronto, Canada.The trauma registry of our institution captures data on individual trauma patients admitted to the hospital’s Emergency Department (ED) up to the day of discharge. The following data were obtained from the registry: demographic information, Injury Severity Score (ISS), initial laboratory parameters , partial thromboplastin time (PTT), lactate, pH, fibrinogen), total volume of crystalloid and blood products administered during the first 24h of injury , intensive care unit (ICU) stay and hospital length of stay (LOS), ventilator days, in-hospital complications, and in-hospital mortality. Data from pre-hospital fluid infusion data were extracted from ambulance report forms. Crystalloid infusion was calculated as the total volume of normal saline (NS) and/or lactated Ringer’s (LR) received during the first 24h, starting from the time of injury. Quality of data was assessed for accuracy and completeness. Any outliers were verified by revisiting patient records. Missing values from the trauma registry were obtained from the hospital charts when available. Due to a high percentage of missing data for pH values (258/903 missing) and lactate values (475/903 missing), these variables were not included in the analysis. Furthermore, blood pressure on arrival was not collected and missing values of base excess (608/903 missing) precluded their inclusion in the analysis.The primary outcome was in-hospital mortality. This was recorded in the registry as either death during hospital stay or alive at discharge. Secondary outcomes included: development of in-hospital complications (IHC) defined by pneumonia and/or sepsis) and total number of ventilator-days . In our study, large-volume resuscitation was defined as infusion of more than 5l of crystalloids in the first 24h.Complications were defined by the Ontario Trauma Registry Comprehensive Data Set (OTR CDS) data dictionary (May 2014) . The OTRDescriptive statistics were performed for all variables. Continuous variables were reported as median and interquartile range (IQR), as the variables did not follow a normal distribution. Categorical variables were reported as absolute numbers and percentages. Univariate in-hospital mortality (non-survivors versus survivors) and complications (complications versus no-complications) were compared using Wilcoxon Rank-Sum test for continuous variables and Chi-square test for categorical variables. To assess the independent relationship of large-volume crystalloid transfusion to mortality and complications, we performed a multivariable logistic regression analysis using a backward selection process while controlling for variables selected a priori. The results were reported as adjusted odds ratio (aOR) with 95% confidence interval (CI).p-value of < 0.05 was considered statistically significant. All data analyses were carried out using Statistical Software SAS .A multivariable Poisson regression analysis was performed to assess the independent association between administration of ≥5L crystalloid/24h with respect to number of ventilator-days while controlling for variables selected a priori. Prior to multivariable modeling, the variables were assessed for multicollinearity using tolerance statistics. Appropriate numbers of variables were used so as not to overfit our model. Presence of variance inflation factor (VIF) of > 0.4 was considered to be multicollinear. Model diagnostics were performed, and a p < 0.05) between the patients according to the volume of crystalloid received. Patients who received ≥5L of crystalloids within 24h were, for the most part, more severely injured (median ISS17 (IQR 9–25)), had worse physiologic parameters during admission, received more blood products (except for cryoprecipitate), had higher complication rate and ultimately higher mortality  versus 2.2 L (IQR 1–5) in the survivor group. Larger volumes of blood products were also administered in the first 24 h to the non-survivor group as compared to the survivor group ; severely injured (26 (IQR 19–35) vs. 16 [p < 0.0001) and received more blood products . Total duration of ICU stay (15 (IQR 9–24) vs 0 (IQR 0–1) p < 0.0001); duration of mechanical ventilation ((13.5 (8–21)vs0 (0–1), p < 0.0001) and total hospital stay ((26 (IQR 17–45)vs. 4 (IQR 1–9), p < 0.0001) were significantly longer in patients who experienced complications compared to the patients without (Table p < 0.0001). The variables found to be independently associated with in hospital complications in multivariable logistic regression were: older age (aOR1.03 (95% CI: 1.01–1.05), p < 0.001); longer ICU days (aOR 1.58 (1.46–1.7), p < 0.0001) and administration of platelet units within 24 h (aOR 1.36 (1.00–1.84), p = 0.044)  developed complications. Univariate association of selected risk factors with in-hospital complications status are described in Table ) vs. 16 , 19–26,  IQR 19–3 vs. 16 [p < 0.0001. Other factors included higher ISS , p < 0.0001), development of complications , p < 0.0001) and low initial temperature , p = 0.0039) , In our retrospective cohort study, we found that trauma patients who received ≥5L of crystalloids in the first 24 h of injury had significantly higher mortality rates (26%), when compared to patients who received < 5L of crystalloids within the same period (7%). This increase in mortality represents an odds ratio of 2.55. Moreover, ≥5L fluid resuscitation in the first 24 h of injury was an independent predictor of mortality, along with older age, higher ISS, and lower admission temperature, accentuating the overall detrimental effect of injudicious fluid infusion in trauma patients. A previous study showed higher mortality related to increased crystalloid administration in the emergency department . HoweverThe importance of fluid resuscitation to maintain tissue perfusion in hemorrhagic shock has been well stablished and should not be underestimated. However, studies showed that resuscitation regimens aimed at decreasing crystalloid infusion and delivering higher ratios of blood products led to fewer complications associated with excessive fluids, less coagulopathy and ultimately increased survival –22.In keeping with those reports, our findings provide further support for a more conservative approach to fluid resuscitation.At the microvasculature and cellular levels, large volume resuscitation provokes generalized increase in interstitial fluid and cellular edema. These changes have been linked to organ dysfunction . ExcessiIn our study, the difference in fluid volume between survivors and non-survivors was more than double. Nonetheless, we speculate that higher volume differences would have had an even more pronounced effect in the parameters assessed in our study –27. SmalAs expected, our study showed that patients who developed in-hospital complications had significantly longer ICU and hospital LOS. However, volume infusion within the first 24h was not an independent predictor of the aforementioned findings. Nevertheless, infusion of ≥5L of crystalloids in the first 24h was associated with a significant increase in the duration of mechanical ventilation. Thus, it is conceivable that higher volume infusion could have a primary effect on pulmonary function interfering with weaning from mechanical ventilation. Moreover, other detrimental effects linked to high volume resuscitation also contributed to more prolonged ICU LOS. Accordingly, previous reports showed that mechanical ventilation is a known risk factor for in-hospital complications linked to significant personal and economic costs , 29, 30.The extent of physiologic derangement as reflected in laboratory variables was associated with higher crystalloid resuscitation volume. This was demonstrated by higher levels of INR, PTT, and lactate, together with lower fibrinogen and pH; previous studies corroborate these findings , 19. YouThe use of vasopressors in severe trauma patients has been described as an option to decrease fluid resuscitation volumes , 32. HowOur study has several limitations inherent in retrospective data collection. Despite a cohort of 970 patients the study population is considerably heterogeneous, even though attempt was made to match these groups as well as possible. However, in a single center study like ours the effect of group differences is considerably important in the data analysis. Another important limitation was the inability to include lactate, pH, admission blood pressure and base excess data in the multivariate logistic regression analysis because of significant missing data. Lactate and base excess are important markers of tissue perfusion, thus a reliable marker of the resuscitation efforts. Furthermore, admission blood pressure of severely injured patients help guide clinical decision making with respect to the need for ongoing volume replacement. Finally, we assessed crystalloid administration during the first 24 h after injury. It is conceivable that the effect of fluids administered during other periods of the hospital admission were not considered in the results. Nonetheless, our objective was to determine the effect of fluids received during the initial 24 h on the outcome of the patients.Large-volume fluid resuscitation (≥5L) with crystalloids in the first 24h after traumatic injury is associated with higher mortality rates and prolonged need of mechanical ventilation. In addition, age, higher ISS, and presenting temperature were independently predictive of in-hospital mortality. The current study supports the principles of judicious fluid resuscitation in trauma. These findings also highlight the need for revision of current resuscitation guidelines for fluid administration to traumatically-injured patients.


Age, obesity, and metabolic syndrome are known risk factors for gallstones; however, the combined impact of these different risk factors on gallstone formation has not yet been examined.This retrospective, cross-sectional study involved 3190 participants, including 207 participants (6.5%) with gallstones and 986 (30.9%) with metabolic syndrome. Participants were divided into four phenotypes according to metabolic syndrome and obesity status: 1378 participants were metabolically healthy and non-obese (MHNO); 826 were metabolically healthy but obese (MHO); 185 were metabolically abnormal but not obese (MANO); and 801 participants were metabolically abnormal and obese (MAO).The MAO and MANO phenotypes had more gallstones than the MHO and MHNO phenotypes, regardless of age (< 50 or ≥ 50 years old). Multivariate analyses showed that phenotype was an independent risk factor for gallstones in participants < 50 years old (odds ratio (OR) = 1.73, 95% confidence interval (CI) = 1.32–2.28). Younger participants also had a higher risk of gallstones in the MAO , MANO , and MHO  phenotypes than the MHNO phenotype.Our retrospective results demonstrate an increased association of gallstones in younger people (< 50 years old) with metabolic syndrome and obesity.The online version of this article (10.1186/s12876-019-1032-y) contains supplementary material, which is available to authorized users. Gallstones and their associated complications are common conditions worldwide. Approximately 6–20% of the population has gallstones and some studies have indicated rates of screen-detected gallstones between 0.6 and 1.39% per year in Taiwan and Europe . More thSeveral studies have shown that age, sex, body mass index (BMI), non-alcoholic fatty liver, Type 2 DM, and insulin resistance are associated with gallstones –8. Furth51 had incomplete data to define their metabolic syndrome;415 underwent repeat examination during the study period;257 refused to undergo abdominal ultrasound;491 had incomplete data for hepatitis B or C; and51 had already undergone cholecystectomy.This retrospective, cross-sectional study included patients who received voluntary physical check-ups from the Health Examination Center of Changhua Christian Hospital, Changhua City, Taiwan between 2010 and 2014. A total of 4456 participants were initially enrolled in this study; however, 774 were excluded because:In the end, 3190 participants were eligible for further analysis. All participants in the final group received blood tests , cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides, hepatitis B surface antigen, and anti-hepatitis C virus antibody), blood pressure measurement, waist circumference measurements, and abdominal sonography. The Ethics Committee of Changhua Christian Hospital approved the study protocol used in this work (CCH IRB No: 150807).2 in the study by using the classification for adult Asians proposed by WHO in 2000 [A gallstone was defined as a hyperechoic area with a typical acoustic shadow and positional change by sonography and was diagnosed by an experienced gastroenterologist who has more than 5-years of experience and was unaware of personal history or laboratory data. Cholecystectomy was defined as an invisible gallbladder can be identified by sonography or by a personal history of cholecystectomy. Metabolic syndrome was defined as fulfilling at least three of the five Adult Treatment Panel III criteria: (1) central obesity, defined as a waist circumference of ≥90 cm in Asian men and ≥ 80 cm in Asian women; (2) low HDL-C levels, defined as fasting HDL-C < 40 mg/dL in men and < 50 mg/dL in women; (3) hypertension, defined as systolic pressure ≥ 130 mmHg and/or diastolic ≥85 mmHg, or in patients with a history of hypertension receiving antihypertensive therapy; (4) high triglyceridemia, defined as fasting triglyceride levels ≥150 mg/dL; and (5) hyperglycemia, defined as fasting glucose level ≥ 100 mg/dL, or in patients with a history of diabetes receiving medications to control elevated glucose . Obesity in 2000 .Participants were further divided into four phenotypes according to metabolic syndrome and obesity profile: MHNO included participants without both metabolic syndrome and obesity; MHO included those without metabolic syndrome but with obesity; MANO included participants with metabolic syndrome but without obesity; and MAO included those who had metabolic syndrome combined with obesity.P <  0.05 (two-tailed) was considered statistically significant.Continuous variables are presented as mean ± standard deviation and were compared using an ANOVA test. Categorical data were compared using a chi-square test. Single-variable, binary logistic regression and non-parsimonious multivariable logistic regression analysis were used to analyze the significance of variables in gallstones. Statistical analyses were performed using PASW Statistics version 18 . There were 3190 participants enrolled for the final analysis, including 207 participants (6.5%) with gallstones and 986 (30.9%) with metabolic syndrome. The numbers and rates of participants in each phenotype were 1633 (51.2%) for the MHNO phenotype, 571 (17.9%) for the MHO phenotype, 292 (9.2%) for the MANO phenotype, and 694 (21.8%) for the MAO phenotype. Participants in the MAO and the MANO phenotypes were older and had higher blood pressure and more abnormal levels of blood glucose, liver function tests , lipid profiles, uric acid, and renal function (blood urea nitrogen and creatinine) compared to those in the MHNO and MHO phenotypes. Detailed results are listed in Table P = 0.837).A total of 98 (10%) participants with metabolic syndrome had gallstones but only 109 (5%) had gallstones among those without metabolic syndrome. Age, HCV infection and four phenotypes were the risk factors for gallstones after non-parsimonious multivariable logistic regression . However, in participants older than 50, only HCV infection was a significant independent factor for gallstones . Subgroup analyses were performed after multivariate regression to identify effects in each group of participants. The results are listed in Table P = 0.08), MANO  and MAO . No statistically significant differences were found between MHNO, MHO and MAO phenotypes (≥50 years old).As shown in Table Many studies have shown that several independent factors are associated with gallstone development, including age, sex, metabolic syndromes, obesity, Type 2 DM, BMI, HCV, and CKD , 19. WhiMetabolic syndrome and obesity are two of the main risk factors for gallstones and the prevalence of gallstones is higher in individuals with a greater number of abnormal metabolic factors , 9, 20. P = 0.837). Therefore, the effect of metabolic syndrome and obesity on gallstones in younger participants was similar to the effect of age on gallstones in older participants without metabolic syndrome and obesity. The HCV infection rate was lower in the younger population, with the prevalence of both metabolic syndrome and obesity in young adults increasing gradually in Taiwan. HCV may also have a close association with insulin resistance and hepatic steatosis [Many studies have reported age as a major risk factor for gallstones , 26–28. teatosis , 33. Allteatosis , 34. Wheteatosis . Our stuAlthough more than 3000 participants were included in the study, there were still several limitations. Firstly, we did not distinguish between the types of gallstones included (cholesterol or pigment stones) because we used sonographic findings. Secondly, this was a cross-sectional, retrospective study; the incident rate of gallstones in different age groups with metabolic syndrome and obesity needs to be confirmed using longitudinal studies. Thirdly, the study lacked some published data that is currently available in the literature regarding other risk factors of gallstones, such as smoking, drinking alcohol, exercise and lifestyle, pregnancy, oral contraceptive use, or family history. Fourth, the study groups were from an Asian population in Taiwan; the incidences of viral hepatitis B or C infection and definitions of obesity differ between the Eastern and Western worlds. Further studies are needed using data from different races for comparison with the current results.In conclusion, the present study has shown an increased association of gallstones in younger people with metabolic syndrome and obesity. The prevalence of gallstones in younger people with metabolic abnormality and obesity was equal to that of metabolically-healthy and non-obese elderly people. Diet and exercise intervention may be beneficial for high-risk groups to prevent gallstone formation; however, additional studies are required to confirm these findings.Additional file 1:Table S1. Baseline characteristics of participants according to participants < 50 and ≥ 50 years. (DOCX 16 kb)Additional file 2:Table S2. Risk of gallstones in multivariate analyses. (DOCX 16 kb)


Furthermore, alcoholics were less likely to complain of headache (p < 0.001) and nausea/vomiting (p = 0.005) and had lower concentration of glucose in cerebrospinal fluid (CSF) (p = 0.025). In the multiple logistic regression analysis, alcoholism was associated with lower GCS (p = 0.036), presence of seizures (p = 0.041), male gender (p = 0.042), and absence of nausea/vomiting (p = 0.040). Furthermore, alcoholism (p = 0.031), lower GCS score (p = 0.001), and higher blood urea concentration (p = 0.018) were independently associated with worse outcome measured by GOS. Compared with non-alcoholics, chronic alcohol abusers are more likely to present with seizures, altered mental status, and higher SOFA score and have an increased risk of unfavorable outcome. In multivariate analysis, seizures and low GCS were independently associated with alcoholism, while alcoholism was independently associated with worse outcome.The aim of the study was to determine the effect of chronic alcohol abuse on the course and outcome of bacterial meningitis (BM). We analyzed records of patients with BM who were hospitalized between January 2010 and December 2017 in the largest neuroinfection center in Poland. Out of 340 analyzed patients, 45 (13.2%) were alcoholics. Compared with non-alcoholics, alcoholics were more likely to present with seizures (The online version of this article (10.1007/s10096-019-03661-5) contains supplementary material, which is available to authorized users. In Poland, where the registration of bacterial meningitis (BM) cases is mandatory, the annual incidence of BM in the years 2010–2017 ranged between 1.97/100,000 and 2.5/100,000  which isWe evaluated records of adult (≥ 18 years old) patients with community-acquired BM who were admitted to the Hospital for Infectious Diseases in Warsaw from January 1, 2010, to December 31, 2017. The diagnosis of BM was based on fulfilling at least one of the following criteria: positive cerebrospinal fluid (CSF) culture, positive CSF Gram staining, and typical CSF findings (pleocytosis ≥ 100 cells/μL with ≥ 90% neutrophils and decrease of CSF glucose level < 2.2 mmol/L). Patients with CSF findings typical for BM but negative blood and CSF culture and negative microscopic CSF examination were considered to have BM of unknown etiology. Diagnosis of tuberculous meningitis was based on at least one of the following: positive culture, positive nucleic acid amplification, and positive Ehrlich-Ziehl-Neelsen staining of CSF. Glasgow Coma Scale (GCS) and Sequential Organ Failure Assessment (SOFA) scores were calculated at admission, while Glasgow Outcome Score (GOS) was assessed at the time of discharge from hospital.Initial antimicrobial treatment followed current guidelines . PatientAlcoholism was diagnosed according to the World Health Organization (WHO) criteria . QuestioU test was used to compare continuous variables, and the chi-square test was used to evaluate nominal variables. Logistic regression was used to calculate adjusted odds ratios and to determine variables independently associated with alcoholism and those independently influencing outcome reflected by GOS. Since GOS is not a nominal but continuous variable, we used the general linear model to calculate the coefficients for this analysis. Statistical analyses were performed using program R version 3.5.2 [The Mann-Whitney on 3.5.2 .The final analysis included 340 patients with bacterial meningitis . Among this group, 45 (13.2%) patients were considered alcoholics .p < 0.001) (Table p < 0.001) and nausea/vomiting . Furthermore, they scored higher on the SOFA  and lower on the GCS . Alcoholic patients were also more likely to require intensive care unit (ICU) admission (48.9% vs 37.6%), and their mortality was higher (24.4% vs 15.4%), but these differences did not reach statistical significance. The clinical outcome reflected by the GOS was significantly worse among alcoholics  and lower concentration of glucose in CSF , male gender , the presence of seizures , and the absence of nausea/vomiting . Furthermore, alcoholism, lower GCS score, and higher urea blood concentration were independently associated with worse prognosis as assessed by GOS . While high incidence of seizures among alcohol abusers presenting with BM is a well-known phenomenon, the numbers in previous reports were lower and ranged from 13 to 18% [Compared with non-alcoholics, alcohol abusers were less likely to present with fever, headache, and nausea. The absence of such typical symptoms of BM  might ca3 to 18% , 12. FurThe presence of seizures and other signs and symptoms associated with the alcohol withdrawal syndrome or even alcohol intoxication itself could also negatively affect consciousness level: in our study, alcoholic patients scored significantly lower on the GCS scale at admission compared with non-alcoholics. These findings are compatible with previous studies of van Veen et al.  and WeisWhile alcohol abuse was not an independent predictor for mortality in multivariate analysis, it was associated with worse outcome as measured by the GOS. These results are similar to those reported by Wiesfelt et al.  who alsod-dimer levels and CSF glucose levels, no differences were found in the results of routine tests between alcoholic and non-alcoholic patients. High blood d-dimer levels are common in chronic alcohol abusers and could be the effect of hemostatic system activation related to oxidative stress [In addition to alcohol, low GCS score and high blood urea levels were independently associated with worse outcome Table . The infe stress . Interese stress .Streptococcus pneumoniae was the most common causative microorganism (19.4%) in our patients


The present study aims to examine the potential in using quinoa to ameliorate the dysbiosis and colitis induced by dextran sodium sulfate (DSS). A total of 40 C57BL/6 mice were fed either an AIN-93M diet or a quinoa-based diet, separately. Colitis was induced for 10 animals/dietary group with a 5-days exposure to 2.5% DSS. The clinical symptoms were monitored every other day, and the gut microbiota was characterized by 16S rRNA gene sequencing. The results indicated that consumption of quinoa lessened clinical symptoms as indicated by the reduced disease activity index and the degree of histological damage (P < 0.05). As expected, the DSS treatment induced significant dysbiosis of gut microbiota in mice on an AIN-93M diet. However, compared to mice fed the AIN-93M diet, the consumption of quinoa alleviated the DSS-induced dysbiosis remarkably, as indicated by increased species richness and diversity, decreased abnormal expansion of phylum Proteobacteria, and decreased overgrowth of genera Escherichia/Shigella and Peptoclostridium (P < 0.05). The relative abundances of Firmicutes and Bacteroidetes were less altered in mice fed with quinoa comparing to those mice fed the AIN-93M diet. In summary, the consumption of quinoa suppressed the dysbiosis of gut microbiota and alleviated clinical symptoms induced by DSS, indicating the potential to utilize quinoa as a dietary approach to improve intestinal health.A plethora of evidence highlights that the dysbiosis of gut microbiota is a critical factor for inflammatory bowel disease (IBD). Both  IBD is directly associated with an increased risk of colorectal cancer, but there are still no effective means to treat it. In fact, long-term used of traditional immunosuppressive agents for IBD treatment, e.g. azathioprine, might lead to higher incidence of various cancers5. Thereby, it is of critical importance to develop strategies to prevent the occurrence of IBD7.Inflammatory bowel disease (IBD), categorized as either Crohn’s disease (CD) or ulcerative colitis (UC)8. IBD patients exhibit a significant reduction of biodiversity and stability of gut microbiota4. They also have a reduced abundance of phyla Firmicutes and Bacteriodetes accompanied with a considerably increased abundance of Proteobacteria and Actinobacteria10. Some specific bacterial groups, such as Enterobacteriaceae and Desulfovibrio, were found to be expanded in IBD patients12. Studies using animal models provided more direct evidence: after inoculation with microbiota from mice with colitis, the healthy recipient can develop intestinal inflammation13.Although no study has convincingly determined any specific bacterial group or strain that definitely causes IBD, it is well accepted that the dysbiosis of gut microbiota is a critical contributing factor in the etiopathogenesis of IBDChenopodium quinoa Willd (quinoa), an edible grain-like crop from the Andes region of South America, is rich in high quality protein, vitamins and minerals, and in particular possesses a wide range of various polysaccharide14. In vitro studies have suggested that quinoa has a prebiotic effect, including promoting the growth of beneficial bacteria and the production of SCFAs (Short chain fatty acids)15. Polysaccharides from quinoa have demonstrated immune-regulating activity in animal studies16. All of the evidences suggests that quinoa may have beneficial effects on intestinal health.Diet is a direct mediator for gut microbiota and thereby offers an attractive means for the prevention of IBD. In this study, utilizing the dextran sodium sulfate (DSS)-induced colitis animal model, we investigated to what extent the consumption of quinoa alleviated the dysbiosis of intestinal microbiota and reduced the symptoms of DSS-induced colitis. This was done with a consideration of developing dietary strategies for the prevention of IBD.P < 0.05).In this study, groups of mice exposed to DSS developed clinical symptoms, including body weight loss, diarrhea, and rectal bleeding. As shown in Fig. P < 0.001) and enlarged spleens . In comparing the two DSS-treated groups, the spleen weight of mice on the AIN-93M standard diet (MDSS) was significantly higher than that of mice on the quinoa diet (QDSS) (P = 0.0288). No significant difference in colonic length was observed between MDSS and QDSS groups. Histological evaluation showed that DSS treatment significantly increased colonic inflammation .As shown in Fig. ion Fig. , whereasP < 0.001; Shannon: P < 0.001). When the comparison was performed for the non-DSS control groups, the Chao1 index was lower in the QC group (P = 0.0163), and no difference for the diversity was indicated by the Shannon index (P = 0.244). However, after the DSS exposure, both of two indices were significantly higher in mice fed with quinoa when compared to the mice fed the AIN-93M control diet .As shown in Fig. The community structure of the microbiota was also analyzed using principal coordinates analysis (PCoA) with weighted and unweighted UniFrac distance matrix. As shown in Fig. Firmicutes and Bacteroidetes are the predominant phyla in non-DSS control groups (MC and QC groups). The relative abundances of Firmicutes in MC and QC mice were 49.2% and 49.7%, and the abundances of Bacteroidetes in MC and QC was 26.1% and 35.8%, respectively. After treatment with DSS, the relative abundances of Firmicutes and Bacteroidetes were significantly decreased when compared to their non-DSS counterparts. The relative abundance of Firmicutes decreased to 28.0% (MDSS) and 39.5% (QDSS), and that of Bacteroidetes decreased to 18.2% (MDSS) and 27.6% (QDSS). The magnitude of decrease was much smaller in the quinoa diet groups (QC vs. QDSS), when compared to the AIN-93M control diet groups (MC vs. MDSS), and the relative abundances of these two phyla in the QDSS mice were significantly higher than those in the MDSS mice . The phylum Proteobacteria expanded substantially and occupied dominantly in the MDSS group (41.4%), significantly higher (P < 0.001) than its non-DSS control group , whereas similar proportions of Proteobacteria were noted in QC (9.43%) and QDSS (8.96%) groups. It is specifically noteworthy that the proportions of Proteobacteria in the QC and QDSS groups were far lower than the proportions in the MC and MDSS diets group. The phylum Verrucomicrobia is significantly increased in DSS-treated groups, when compared to its non-DSS counterpart control groups; the magnitude is particularly significant for the quinoa groups (P < 0.001). The expansion of Verrucomicrobia accompanied with DSS feeding has been detected previously17 and might due to certain bacteria in Verrucomicrobia can de-polymerize DSS and thus are able to grow in a DSS-rich environment18.The microbial taxonomic composition and the details of bacterial taxonomy comparison at the phylum level were demonstrated in Fig. Bacteroidales S24-7 was significantly higher in quinoa diet groups, regardless of DSS treatment . Several other genera, including the Lachnospiraceae NK4A136 group, an uncultured bacterium from family Lachnospiraceae, Escherichia/Shigella and Peptoclostridium, were significant changed with DSS treatment in the AIN-93M diet, but the consumption of quinoa compromised their changes. The two genera belonging to family Lachnospiraceae, Lachnospiraceae_NK4A136_group and the uncultured_bacterium_f_Lachnospiraceae, significantly decreased in the MDSS diet when compared to the MC diet (P < 0.05), whereas similar high levels were maintained between the QC and QDSS groups. The other two special genera, Escherichia/Shigella and Peptoclostridium, were only detected in the MDSS group and not the QDSS group . The LEfSe Cladogram and the histogram of LDA scores are shown in Fig. P < 0.05). When comparing the two diet groups with DSS treatment, plasma level of IL-6 in QDSS was significantly reduced to 174.8 pg/ml, which was much lower than that of the MDSS group . A decrease was observed for IL-1β (P = 0.55) and a surprising increase of IFN-γ (P = 0.076) was observed in the QDSS group when compared to the MDSS group, but neither of them reached a statistically significant degree.As shown in Fig. 20. Quinoa, a well-known healthy pseudocereal, has a high content of dietary fiber, contains poly-unsaturated fatty acids, and is considered a high-quality source of protein. Furthermore, it contains an abundance of anti-inflammatory phytochemicals23 and therefore has potential protective effects against inflammation. In the present study, we evaluated the effects of quinoa on IBD using the murine colitis model induced by DSS. The results show that the consumption of quinoa significantly attenuated the clinical symptoms in the DSS-induced colitis model when compared to the condition in the mice fed the AIN-93M control diet, as indicated by reduced body weight loss, better disease activity index, less histological colon damage, and improved inflammatory status (lower plasma IL-6 level).Dietary strategies, in opposition to pharmaceutical drugs that generally have side effects, present an attractive means for the prevention of chronic inflammation-related disorders, including IBD23, it may possess the capability to modify gut microbiota. In our present study, PCoA analysis of microbial taxa from cecal content samples distinctly separated the four experimental groups, suggesting a regulatory property of quinoa on gut microbial composition. As expected, DSS treatment significantly reduced species richness and diversity indices (Chao1 and Shannon) of gut microbiota24, but the richness and diversity, as indicated by the Chao1 and Shannon indices, were significantly higher in the QDSS group than the MDSS group. Considering controls without DSS treatment, the Chao1 and Shannon indices in the QC group were not higher than those in the MC group; current results suggested that consumption of quinoa compromised the reduction of species richness and diversity induced by DSS treatment, which indicates the positive effect of quinoa on the maintenance of intestinal ecosystems.In particular, the present study aimed to examine the effects of quinoa on the symptoms of colitis and gut microbiota dysbiosis induced by DSS. As quinoa contains several prebiotic components, including polysaccharides and phenolic compoundsProteobacteria phylum induced by DSS treatment mice fed the AIN-93M diet, whereas there was no relative expansion of Proteobacteria by DSS administration in mice fed the quinoa-based diet. The unusual expansion of Proteobacteria is often considered as a “microbial signature” of dysbiosis in gut microbiota25. Therefore, the inhibitory effect on phylum Proteobacteria expansion by quinoa could be potentially beneficial to intestinal health.Our results indicate a dramatic expansion of the Escherichia/Shigella and Peptoclostridium in mice fed the AIN-93M diet, whereas neither of them were observed in mice fed quinoa. Both Escherichia/Shigella and Peptoclostridium are generally believed to be pro-inflammatory, as they contains Escherichia coli and Peptoclostridium difficile, the main pathogens associated with infectious diarrhea26. A dramatic decrease was observed for two genera belonging to family Lachnospiraceae by DSS treatment in mice fed the AIN-93M diet, whereas the change was not observed in animals fed quinoa (QC vs. QDSS). As the inoculation of Lachnospiraceae in germ-free mice can suppress the growth of Peptoclostridium difficile27, the decrease of Lachnospiraceae might be responsible for the expansion of Peptoclostridium in the MDSS group.It was also noteworthy that DSS treatment induced the bloom of the genera To our knowledge, this is the first study to analyze the effects of a quinoa-based diet on a colitis animal model. Our data demonstrated that consumption of quinoa modified the dysbiosis of intestinal microbiota, and compromised the clinical symptoms of DSS-induced colitis. The promising results from our animal study warrant further clinical studies to establish the consumption of quinoa as a dietary strategy to improve intestinal health.The quinoa seeds (white quinoa) used in this study supplied by Shandong Center of Crop Germplasm Resources , which planted in Xinzhou, Shanxi, China. Quinoa seeds were cleaned and washed several times until no foam was visible in the water; this was done in order to remove saponins from the outer skin of the seeds. The washed quinoa seeds were dried at 42 °C for at least 12 h, and then ground into quinoa flour. The resulting quinoa flour contained 15.0 ± 0.46% protein, 11.3 ± 0.35% moisture, 4.7 ± 0.41% fat, 61.8 ± 1.15% carbohydrates  and 2.0 ± 0.10% ash.ad libitum access to food and water. Mice were acclimated for one week on a standard chow diet, then randomly assigned into two experimental diets (n = 20 per diet) where they were fed either the AIN-93M purified rodent diet or a quinoa-based diet. In the quinoa-based diet, carbohydrates  were replaced with quinoa powder. Since the quinoa powder contains 15% protein and, therefore, 907 g/kg of quinoa contributes to a total of 140 g/kg protein (136 g protein from quinoa and 4 g casein), was added to match the content (140 g/kg of protein casein) in the AIN-93M diet. Detailed compositions are listed in Table Forty male C57BL/6 mice (6-wk old) were purchased from Model Animal Research Center of Nanjing University , and housed in a facility with a controlled environment  with 28. The details of each scores are listed in Supplementary Materials , spleen weight and liver weight were recorded, and the colon tissue was collected for histopathological analysis.29. In brief, scores were given based on inflammation severity (0–3), inflammation extent (0–3), and crypt damage (0–4), with the total histologic scores then calculated by adding the scores of all three parameters, resulting in a maximum potential score of 10. The detailed description of score standards are listed in Supplementary Materials . Eight randomly selected fields were viewed under a light microscope, and each view given scores according to previous descriptionsls Table .30. The paired-end sequencing with a read length of 2 × 250 bp was performed on the Illumina MiSeq platform . After the demultiplexed paired-end reads were joined (FLASH v1.2.7)31, quality filtered32, and chimerism removed33, the resulting tags were assigned to OTUs using UCLUST (version 1.2.22)34 with a 97% threshold of pairwise identity. The identified taxonomy was then aligned against the Silva reference database 35. The α-diversity analysis was performed using Mothur 36, the Chao1 index was used to characterize species richness and the Shannon diversity index was used to characterize species diversity. Two-dimensional principal coordinate analyses (PCoA) plots, including weighted and unweighted Unifrac, were used to assess the variation (β-diversity distance) between experimental groups. The raw Illumina read data for all samples were uploaded into SRA at NCBI under accession number SRP141297.Total bacterial DNA was isolated from frozen cecal contents using a QIAamp DNA Stool Mini Kit . The region V3–V4 of the 16S rRNA gene was amplified and DNA libraries were constructed according to those previously describedThe plasma samples were centrifuged at 4 °C at 3000 rpm for 10 minutes, the supernatant was collected and diluted 2-fold for further analysis. Concentrations of IL-1β, TNF-α, IL-6 and IFNγ were determined using the Proinflammatory Panel 1 (mouse) V-PLEXTM Kit and QuickPlex SQ 120  according to manufacturer’s protocols. Cytokines were expressed as ng/ml plasma.post hoc tests. The results were considered statistically significant when P < 0.05. The results are presented as the mean ± SEM. GraphPad Prism version 6.00 for Windows  was used for all calculations. As previously described37, the linear discriminant analysis (LDA) effect size  was applied to identify bacterial communities responsible for the differences of cecal microbiota compositions between different groups, using an LDA score threshold of >4.0.Comparisons of various anatomical measurements among the four groups were made by two-way analysis of variance (ANOVA), followed by Fisher’s LSD Supplementary Materials


To systematically review the evidence around the effect of ambient levels of particulate and gaseous pollutants, and the risk of hospitalisation with bronchiolitis for infants under two years of age.Systematic review of observational epidemiological studies including cohort, time series, case crossover and case control study designs.Medline, Scopus, and Web of Science searched to November 2017 with no language restrictions.2), sulphur dioxide (SO2), carbon monoxide (CO), ozone (O3)) on hospital admission for bronchiolitis.Studies investigating impact of air pollution levels on particulate pollutants (diameter <2.5 μm (PM2.5) or <10 μm (PM10) and gaseous pollutants  recommended levels.Eight studies were eligible for review. Long term exposure to PM2.5 may be associated with increased risk of hospitalisation for bronchiolitis. SOCertain particulate and gaseous pollutants may have a clinically relevant effect on hospital admissions for bronchiolitis in children below age two years old. Large cohort or time series studies are needed to examine this possible association.The protocol can be found at PROSPERO (CRD42017080643). Bronchiolitis is a common lower respiratory infection in infancy, it is caused by respiratory syncytial virus (RSV) in 80% of cases . RSV, spExposure to air pollutants in early childhood also affects pulmonary function tests, and airway inflammation . Relativ2), sulfur dioxide (SO2), carbon monoxide (CO), and ozone (O3).There is increasing awareness that air pollution contributes to respiratory morbidity  and mortWe aimed to systematically review observational studies which examined the impact of ambient levels of both particulate and gaseous pollutants on the risk of hospitalisation with a clinical or microbiological diagnosis of bronchiolitis in infants.The protocol for our review was registered on PROSPERO (CRD42017080643). Two investigators (Charlotte King and Ian Sinha) independently performed the initial screening of titles and abstracts, analysed full text reports for eligibility, extracted data, and evaluated study quality according to the steps of the PRISMA statement . DisagreWe individually searched online databases MEDLINE, SCOPUS and Web of Science for terms related to ‘bronchiolitis’, ‘air pollution’, ‘particulate matter’, ‘nitrogen dioxide’, ‘sulphur dioxide’, ‘carbon monoxide’, ‘ozone’, and ‘infants’  on the pre-specified primary outcome . Secondary outcomes were the risk of emergency department visits, unscheduled primary care visits, and critical care admission. Studies were included that evaluated exposure to air pollutants at any time period (lag) before hospitalisation occurred and were categorised as acute (less than 7 days), sub-chronic , or lifetime exposure . We specified that the primary pollutants of interest would be particulate matter and nitrogen dioxide as these are from the most common sources of pollution in urban areas  criteria  code 46.From each study, we evaluated the reported methodology with which levels of air pollutants were measured, regarding frequency of monitoring, methodology of data collection and proximity of stations to participants. Studies were considered more methodologically robust if pollutants were measured daily, using standardised techniques, and monitors were placed within ten miles of the hospitals or residences.Adjustment for meteorological confounders, socioeconomic status, age, and other clinical risk factors were examined in each study. Studies were considered to be at low risk of bias if they adjusted for at least two of these types of confounders.From eligible studies, we extracted data around sample size, odds ratio (OR) or relative risk of hospital admission for the stated air pollutant concentrations. We desired to only meta-analyse results from cohort studies, as they provide the strongest observational evidence in the absence of RCTs. Case crossover studies allow for case events to act as their own controls, with excess risk evaluated using conditional logistic regression , thus adFor acute, sub-chronic, and lifetime exposure to each pollutant, we formulated conclusions and graded evidence according to a strategy based on recommendations from the GRADE working group , such thEight studies , and for SO2 (OR 1.12[1.09–1.15]), which was also statistically significant for a lag of 0–1 days (OR 1.08[1.06–1.11]).A statistically significant association was found at a lag of 0–4 days for PM10 (OR 1.06 [1.04–1.08]), NOOf the eight included studies, five reported that one or more pollutants was associated with an increased risk of hospitalisation for bronchiolitis . Of thes2 and SO2, may also be associated with increased risk of hospitalisation. In some studies , and bronchiolitis clinical encounters (Although this systematic review only analysed the effects of air pollution to risk of hospitalisations after birth, emerging evidence suggests an association between antenatal air pollution exposure and low birthweight  which maonchitis , and alscounters . This fuThe results of the included studies were unable to be synthesised as no studies were identified that utilised a cohort design. One source of imprecision is that the diagnosis of bronchiolitis, even when made according to standardised definition, relies upon the subjective judgements by individual clinicians. Variation in the age definition between the studies may have resulted in viral wheeze being misclassified as bronchiolitis, particularly when including over the age of one year . Differe2 and SO2 exposure. There is a need for a multicentre cohort or time series to examine this possible association, and these would be strengthened by development of standardised methodological approaches. Revision of international recommendations around air quality is warranted and this should incorporate specific consideration around the impact of outdoor air pollution on infants.This review suggests an association between different air pollutants and risk of hospitalisation for bronchiolitis, particularly with particulate matter, NO10.7717/peerj.5352/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.5352/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.5352/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.5352/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.5352/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.5352/supp-6Supplemental Information 6Click here for additional data file.