Salmonella spp., Campylobacter spp., and Yersinia enterocolitica. All specimens were tested with a routine clinical diagnosis algorithm and with five real-time PCR assays. A total of 98 specimens (24.5%) were positive for enteropathogens. We found 24 samples positive for Salmonella enterica, 71 positive for Campylobacter spp., and 4 positive for Yersinia enterocolitica. All evaluated methods exhibited a good performance in identifying Salmonella and Yersinia enterocolitica, being the highest positive percent agreement (PPA) value of 95.8% and 100%, respectively. The clinical algorithm showed the highest PPA value identifying Salmonella, due to the enrichment in selenite broth. However, the evaluated methods showed notable differences in the identification of Campylobacter species, obtaining a wide range of PPA values: 59.2%–100%. The clinical algorithm showed the lowest PPA value since it was only able to detect Campylobacter jejuni and Campylobacter coli species. This study revealed the importance of implementing the real-time PCR technique in a clinical algorithm: it improved the accuracy of the diagnosis and provided results in a shorter time compared to routine clinical methods.This study aims to validate the current diagnostic method for the clinical detection of gastroenteritis. We analyzed 400 stool samples to detect three of the most common enteropathogens:  We included three bacteria in this study: Salmonella spp., Campylobacter spp., and Yersinia enterocolitica, which are among the most common enteropathogens that cause gastrointestinal infections. Thus, their rapid, accurate identification is crucial for infection control, and in selected cases, for determining the most suitable therapy. According to the Centers for Disease Control and Prevention, during 2016, the Foodborne Active Surveillance Network identified 24,029 cases of foodborne infections, which led to 5,512 hospitalizations (22.9%) and 98 deaths (0.4%). Some of these cases were detected by culture, but others were detected with culture-independent diagnostic tests (CIDTs). Current CIDTs mainly comprise two types: antigen-based and nucleic acid-based assays. In 2016, one study showed that many infections were identified with a CIDT and without a culture confirmation; most frequently, Campylobacter (32%) and Yersinia (32%); but also Shiga toxin-producing E.coli (STEC) (24%), Shigella (23%), Vibrio (13%), and Salmonella (8%)2. That study revealed the consequences of the current change in diagnostic trends. The implementation of CIDTs in clinical laboratories has led to more exhaustive diagnoses. Most of these tests are ordered by clinicians, because CIDTs are easier to perform and they return results faster than traditional culture techniques. Moreover, samples are subjected to deep screening when multiplex panels are used; consequently, the number of reported infections has increased3. Routine detection of enteropathogens in clinical laboratories typically consists of a complex algorithm, based on selective culture-, biochemistry-, and immunology-based tests. Most studies have shown that culture-based methods lacked sensitivity, particularly in identifying Campylobacter, and most researchers agree that nucleic acid-based CIDTs have great potential. Consequently, CIDTs ) are increasingly implemented in clinical laboratories8, due to their high sensitivity and their ability to screen a considerable number of enteropathogens simultaneously. This approach avoids complex algorithms and reduces the hands-on time. However, the use of stool antigen-based CIDTs remains controversial, because they have displayed highly variable sensitivities and specificities, depending on the study10.Acute gastroenteritis is a leading cause of morbidity and mortality worldwideThis study aimed to compare five molecular CIDTs, based on qPCR technology, to culture-dependent techniques to evaluate their performance and determine the most suitable alternative for routine clinical diagnoses of enteropathogens.Stool samples were submitted from October to December 2015 to the Hospital Clinico Universitario Lozano Blesa . This hospital had 803 beds and attended a population of 286,774 inhabitants, with 29,506 admissions annually. It included an outpatient care facility with 2,315,197 visits annually and an emergency department with 127,694 visits annually. The samples were randomly chosen from specimens received with a request for routine detection of enteropathogens . This study was approved by the Clinical Research Ethics Committee of Aragon . This evaluation does not require informed consent as it was performed with medical waste. After routine analysis, the leftover of DNA, which is considered medical waste, was analyzed with molecular methods. These non-hospital routine diagnosis methods implied no risk or burden for any individuals and the patient did not have to provide any additional sample. The study focused on the presence/absence of bacterial DNA, without attending to residual human DNA. For each patient, the following data were collected: gender, age, hospitalization status, and stool sample consistency. The non-clinicians researchers never had access to personal identifiable information or clinical record. All recorded data were robustly pseudonymized at source, so no subject-identifiable data were generated.Several comparisons were performed simultaneously. All specimens were tested with five qPCR assays. In addition, all specimens were analyzed with the routine clinical algorithm. Discrepant samples among the qPCR assays were resolved with additional qPCR tests. Discrepancies between the qPCR assays and the routine clinical algorithm were resolved with conventional PCR and sequencing.When an incongruence could not be resolved, the information derived from the other tests was used to resolve it. The growth in culture was considered a positive result, regardless of other results.Samples that were identified as positive in culture media.Discrepant samples, with positive results supported by resolution tests. We consider as resolution tests those that help us to resolve the incongruences. In this study they were the additional qPCR test and the conventional PCR and sequencing.Discrepant samples, without successful resolution tests, but a positive result was supported by at least three qPCR assays.Consequently, taking into account the above mentioned factors, we designated the following as “cases”:Once samples arrived at the Microbiology laboratory, they were routinely plated onto various selective and differential media, including selenite broth, MacConkey (MCK) agar, Hektoen enteric (HEK) agar, cefsulodin-irgasan-novobiocin (CIN) agar, xylose lysine deoxycholate (XLD) agar, and charcoal cefoperazone deoxycholate agar (CCDA). These cultures were grown to isolate and identify the enteric pathogens. All culture media were obtained from Oxoid .Stool samples were inoculated into enrichment selenite broth and incubated at 37 °C for 24 h prior to culturing in XLD agar. All media were incubated at 37 °C for 24 h, except for CCDA plates, which were incubated at 42 °C for 48–72 h under microaerophilic conditions, with a GasPak EZ Campy system .Salmonella is expected to grow on MCK, HEK and XLD agar, Campylobacter on CCDA plates and Yersinia enterocolitica on MCK and CIN agar.11 and they were further analyzed with an automated system  to determine the sensitivity to antibiotics. Results from the MicroScan® panels were read on the MicroScan Walk Away 96 plus platform .Colonies recognized as presumptive pathogens were confirmed with matrix-assisted laser desorption/ionization-time of flight mass spectrometry Campylobacter were determined using MALDI-TOF11.The species of Salmonella was determined following the Kauffmann-White scheme with the Difco Salmonella O Antiserum Group Kit .The serotype of Yersinia enterocolitica was determined with the Yersinia enterocolitica O:3 Antiserum Kit .The serotype of Nucleic acids were extracted from fresh stool samples with the VIASURE RNA-DNA Extraction Kit . Solid (0.1 g) or liquid (200 µl) stool was added to 200 µl of phosphate-buffered saline, and extraction was performed according to the manufacturer’s instructions. Samples were eluted in 100 µl and stored at −20 °C until use in the PCR detection assay.Salmonella Real-Time PCR Detection Kit, VIASURE Campylobacter Real-Time PCR Detection Kit, VIASURE Yersinia enterocolitica Real-Time PCR Detection Kit, VIASURE Salmonella, Campylobacter & Y.enterocolitica Real-Time PCR Detection Kit , and RIDA®GENE Bacterial Stool Panel .Five different Real-Time PCR assays were evaluated in this study: VIASURE invA gene , 16S rRNA gene (Campylobacter), and ail gene (Yersinia enterocolitica), whereas RIDA®GENE Bacterial Stool Panel allows the simultaneous detection of ttr gene , 16S rRNA gene (Campylobacter), and ail gene (Yersinia enterocolitica). All Real-Time PCR assays were performed following the manufacturer’s protocol. Positive and negative controls were included in each run. All runs were performed on the AriaMx thermocycler .The targets of the three monoplex and one multiplex VIASURE assays are the Campylobacter spp kit  was used for resolving Campylobacter discrepant samples. This Real-Time PCR assay was performed according to the manufacturer’s protocol.The Mericon Salmonella (hilA gene) and Campylobacter (16S rRNA gene) were described previously13; we used 1 µM of each primer. We modified Salmonella primers by deleting the first nucleotides; the following forward (5′-ATTTGCGCCATGCTGAGGTAG-3′) and reverse (5′- CCGCCGGCGAGATTGTG-3′) primers were used.Conventional PCR was performed with the VIASURE ESSENTIALS DNA Master Mix kit , according to the manufacturer’s instructions. The kit contained all necessary PCR reagents lyophilized in the wells, except for the primers, which were customizable, depending on the target. The primers used for detecting PCR products were separated on a 1.0% agarose gel by electrophoresis.http://www.ncbi.nlm.nih.gov/BLAST/).The same conventional PCR primers were used for sequencing. Sequencing was performed by the General Research Support Service at the Health Research Institute of Aragon (IIS Aragon) and the University of Zaragoza. Sequencing was performed with the capillary sequencer, 3500 XL . Sequences were analyzed with Chromas software, and they were identified with the basic local alignment search tool  and negative percent agreement (NPA), instead of sensitivity and specificity. This approach conforms to FDA recommendationsq) value determines the initial copy number of the target. Samples with low target concentrations display high Cq values. A Cq value greater than 40 was considered negative.The quantification cycle , except for the 95% confidence intervals (CI), which were calculated with the modified Wald method, with GraphPad QuickCalcs. The Chi-square test was performed to determine correlations between enteropathogen infections and patient groups. A A total of 400 stool samples, acquired from 385 patients, were included in this study. Samples were grouped by stool consistency, and they were also classified according to the patient’s age group, gender, and hospitalization status. These data are shown in Table Campylobacter and Yersinia enterocolitica. A more detailed analysis is given in Table Among 400 stool samples, 98 24.5%) met the case definition. Of these, 69 were found positive in culture media, 25 were discrepant samples that were resolved, and 5 were discrepant samples that were found positive at least in three qPCR assays. Although there were 98 cases, there were 99 positive results, because one sample corresponded to a co-infection by 4.5% met Salmonella enterica ; 71 cases positive for Campylobacter ; and 4 cases positive for Yersinia enterocolitica O:3. The information of those Campylobacter species that were determined by 16 S rRNA sequencing is collected in Table We found 24 cases positive for Salmonella positive samples with all methods. The routine clinical algorithm values were, as follows: PPA = 95.8%, CI = 78.1–99.9 and NPA = 100%, CI = 98.8–100. The VIASURE and R-Biopharm assays provided the same level of results. All gave a PPA = 87.5%, CI = 68.2–96.5 and a NPA = 100%, CI = 98.8–100.We could detect most Campylobacter were found. All of these were detected with the VIASURE monoplex assay ; the next most accurate assays, in descending order were: the VIASURE multiplex ; the R-Biopharm , and finally, the routine clinical algorithm .Nine different species of Yersinia enterocolitica O:3 positive samples could be detected with all methods studied .The four No false positive results were found in this evaluation; consequently, all NPA values were 100%. Moreover, no inhibition occurred in any qPCR reaction; therefore, it was not necessary to re-test any sample.q value in three categories: Cq <25 (high copy samples), Cq = 25–35 (medium copy samples) and Cq >35 . They were also classified into two groups: positive by culture and negative by culture. The minimum and maximum Cq values were calculated for each group. These data are shown in Table Yersinia enterocolitica samples that were negative by culture as none of them were positive by qPCR assays.Samples were grouped according to the CP = 0.199), hospitalization status (P = 0.556), or stool sample consistency (P = 0.073). Hence, we can only report trends. When analyzed according to age, most patients affected seem to be the youngest; infection rates were 32.2% in pre-school children; 30.6% in infants; 23.1% in school-age children and adolescents; and 20.9% in adults. When analyzed according to hospitalization status, infection rates were higher among patients that required hospitalization (28.0%), compared to those attended in outpatient clinics (25.1%) and in the emergency department (18.2%). Finally, when analyzed according to sample consistency, infections were found most frequently in stools with mucous (43.8%), liquid (38.2%), and crumbly (35.7%) consistencies and in stools with traces of blood (28.6%).We also evaluated whether some factors  might be positively associated with infections by these three enteropathogens. We performed a Chi-square test, but we did not find any statistically significant link between enteropathogen infections and the age group .This study aimed to validate the current routine clinical diagnosis method for identifying the three main bacterial enteropathogens: 4, ProGastro SSCS assay7, Allplex GI-Bacteria assay8 or FilmArray GI Panel15. These previous studies emphasized the importance of not considering the culture technique as the reference method, due to its lack of sensitivity. Consequently, we created a reference method based on different techniques. According to this reference, samples were classified as “cases” or “non-cases” .In recent years, a large number of studies have been conducted to validate novel gastrointestinal molecular panels as BD Max enteric bacterial panelSalmonella spp. The highest PPA value was obtained with the routine clinical algorithm. This could be explained by the fact that stool samples were inoculated into a selenite enrichment broth, as reported in previous studies16. This broth contributed to bacterial replication, which enhanced the culture method. The culture method was able to detect samples that showed Cq values extremely near the detection limit (Cq > 35).The maximum Cq values obtained in Salmonella identification were 39.7, 35.8, and 39.6 with VIASURE monoplex, VIASURE multiplex, and R-Biopharm assays, respectively  for identifying ly Table . This obCampylobacter, the qPCR assays exhibited notable differences in their abilities to detect many Campylobacter species. The highlight was the high capacities of the VIASURE monoplex and multiplex assays  in diagnosing the majority of Campylobacter species that are considered pathogenic in humans19.Relative to Campylobacter species (other than C.jejuni and C.coli) rather than in the sensitivity of the test, since most of the samples were in high concentration (Cq < 25). The R-Biopharm assay could not detect any positive sample for C. upsaliensis, C. hyointestinalis, C. helveticus, or C. rectus (Table The R-Biopharm test displayed less diagnostic capacity (PPA = 81.7%). It seems to be due to a failure in the recognition of less common ion Cq < 5. The R-q > 35) ; 3/13 showed a Cq = 25–35  and 7/13 samples showed a Cq < 25  (Tables Only 3/13 samples that R-Biopharm missed were near the detection limit (CC. jejuni and C. coli species (PPA = 59.2%)  could inhibit the growth of several species, including C. hyointestinalis, C. upsaliensis, and C. fetus. Moreover, not all Campylobacter species are thermophilic; consequently, the incubation at 42 °C was not suitable for all species. Additionally, some species, like C. concisus, C. rectus, C. curvus, C. gracilis, and C. showae require a special atmosphere enriched with hydrogen.The routine clinical algorithm was only capable of detecting PPA = 59.% .Regarding 10 stated that the problem with stool antigen-based CIDTs was that they exhibited highly variable sensitivities and specificities. That problem has not occurred with molecular-based CIDTs, which, to date, have shown excellent sensitivity and specificity compared to cultures. Our findings correlate with those previous results; therefore, molecular CIDTs could be considered a reliable diagnostic tool, although more studies are needed. Additionally, some molecular CIDTs, like VIASURE assays, are very easy to use. All the components are provided lyophilized inside the wells, which implies that the user is not required to mix any reagents. Also, the lyophilized format facilitates the storage and shipping conditions as lyophilized reagents are stable at room temperature.An important aspect of this study was that no false positive result was found in any qPCR assay. This high specificity (100%) could represent a key difference between molecular-based and antigen-based CIDTs. CouturierWe did not encounter inhibition in any of the qPCR assays, although we tested a vast range of stool consistencies. This finding suggested that the qPCR assays used in this study are robust tests with a stable matrix formulation, which neutralized PCR inhibitor effects.The main limitation of this study was that the number of positive samples depended on the prevalence of each pathogen in the study area. Consequently, we recommend that a multicenter study should be performed, and that the sample collection time should be extended.Campylobacter diagnosis. Additionally, they exhibited good performance in Salmonella and Yersinia enterocolitica identification too, and they substantially reduced the turnaround time (from days to hours). The ease of using molecular methods and the time they save will allow laboratories to increase the number of samples processed. Moreover, saving time is critical for avoiding the spread of infections and for providing effective treatment plans.We conclude that the three types of qPCR assays  tested could improve the routine clinical diagnosis algorithm, because they had a noticeable impact on the


This study addressed stress experiences and emotion regulation in allergic and non-allergic adults. Allergics scored higher on emotion suppression, had higher pre-stress concentrations of plasma oxytocin and exhibited a stronger salivary cortisol response to stress than healthy people. The research outcomes indicate that allergic individuals cope less efficiently with acute stress but may benefit from adaptive emotion regulation strategies such as reappraisal.Numerous studies have demonstrated that acute psychological stress, induced by the Trier Social Stress Test (TSST) paradigm, affects salivary cortisol secretion and self-reported stress measures including anxiety. Allergy has been related to altered cortisol responsiveness and increased stress vulnerability. Here, we investigated acute stress responses and emotion regulation strategies in cohorts of allergic and healthy individuals. Groups of allergics and healthy individuals were subjected to the TSST and experienced levels of stress and anxiety, as well as emotion regulation strategies, were assessed. Cortisol and oxytocin concentrations were measured in saliva or plasma. The present findings confirm earlier results of altered stress responsiveness in allergic individuals. Acute stress by the TSST evoked higher physiological arousal in allergics by means of salivary cortisol secretion. Allergics also scored higher on emotion suppression. However, individuals who were more likely to use reappraisal recovered more efficiently from the cortisol increase. No such effect for reappraisal was found in the healthy population. No differences in self-reported anxiety and stress emerged between the groups. Plasma oxytocin levels prior to the TSST were significantly higher in allergics. Our data corroborate earlier findings on altered stress susceptibility in allergics. Moreover, we identified differences in emotion regulation and oxytocin secretion which should be further explored. Accounting for the emerging global prevalence of allergy, more in-depth research into the experience of stress, coping strategies and stress-related molecules in allergic people is warranted. Stress responsiveness has been identified as a crucial factor associated with the onset and progression of diseases including allergy , who reported typical seasonal or perennial clinical symptoms as well as doctor-diagnosed allergies to grass pollen, birch pollen and/or house dust mites and (b) a healthy control group (n = 21), characterized by the absence of clinical allergy symptoms and without doctor-diagnosed allergies. None of the allergic patients had symptoms other than mild-to-moderate allergic rhinitis at the time recruited, with some reporting mild oral allergy syndrome to apple due to IgE crossreactivity with birch pollen. Prestudy screening of volunteers via skin prick testing (SPT) and determination of IgE levels are reported elsewhere  participated in the study. The study protocol was approved by the ethics committee of the Medical University of Vienna (EK Nr 1030/2015). The study was carried out in AllergyCare®, Allergy Diagnosis and Study Center Vienna.Study volunteers were recruited through advertisement at the University of Veterinary Medicine Vienna, the Medical University Vienna and the University of Vienna and selected via an online screening questionnaire, resulting in 40 age and gender matched study participants  who met the inclusion criteria. As demonstrated in 2.2.State-Trait-Anxiety-Inventory  at four time points during the experiment (see study protocol below).State anxiety was assessed before and after the TSST using the German state subscale of the s Gross, . Finally2.3.Commercial sampling devices  without any saliva-stimulating additives were used to obtain saliva. After signing the informed consent, study participants were instructed how to collect their saliva via putting the cotton roll from the sampling tube into their cheek pouch, letting it saturate with saliva for approximately 60–80 seconds and finally replacing it in the device container. Sampling tubes were immediately stored in a freezer at −20 °C in the laboratory facility at the research site.2.4.®). The samples were centrifuged for 10 min at 3000×g at room temperature and plasma samples were collected in Eppendorf vials and immediately stored in a freezer at −80 °C in the laboratory facility until further analyses were done.Blood samples were drawn from the right arm before the stress test and from the left arm after the test and stored in EDTA containing vials  of clear saliva were used for the analysis of cortisol. Analyses were carried out at the University of Veterinary Medicine in Vienna using a highly sensitive enzyme immunoassay kit. Samples were assayed in duplicates and cortisol concentrations were assessed in a double-antibody biotin-linked enzyme immunoassay  were determined using enzyme-linked immunoassay (ELISA) according to the manufacturer's recommendations . Concentrations that varied by more than 5% were subject to repeat testing or dismissed if sample volumes were too low before a coefficient of variation <5% was achieved.Prior to analysis, samples were thawed on ice and centrifuged at room temperature at 1000×2.8.p-values supplied by the lmer package  for cortisol levels in response to the TSST. Power analysis using G*Power  to analyze differences in cortisol reactivity, and cortisol recovery. All statistical analyses were conducted using R version 3.5.0  with changes in cortisol levels, we therefore did not include sex in our models.Two models were fit. Our first model included change trajectory slopes and their cross-level interaction with allergy status . Our second model additionally included changes in oxytocin concentration (from −10 min pre-TSST to immediately after the TSST), trait anxiety and ERQ suppression/reappraisal with both change trajectory slopes and allergy status. ERQ subscales, changes in oxytocin concentration, and trait anxiety variables were standardized to have means of 0 and standard deviations of 1. A preliminary unconditional model revealed that sex was not significantly associated (Model 1:Level 1Level 2Model 2:Level 1Level 23.suppression strategies (t(37)=2.761, p=.009, d = 0.89) with allergic participants reporting to use suppression of emotions more frequently  than their nonallergic counterparts . Regarding the emotion regulation strategy reappraisal, however, there were no significant group differences (t(37)= −1.015, p = .316).3.1b = 0.182, SE = 0.062, p=.004) and the cortisol response can be characterized by a quadratic change trajectory . Our first model with allergy status indicated a significant interaction of allergy*time , indicating a steeper rise in cortisol levels for allergics. The interaction between allergy and time2 (i.e. curvature) was significant as well  with allergics displaying a less steep recovery slope than nonallergics.Cortisol levels for the allergy and nonallergy groups are presented in b = 0.075, SE = 0.033, p=.026). Simple slope analysis revealed that this association between the acceleration of cortisol levels over time (i.e. steeper recovery slope) and reappraisal was found in allergics  but not in non-allergics . The remaining interactions tested in model did not reach significance. Model summaries are depicted in In model 2, reappraisal showed a significant interaction with allergy, and quadratic change  than in nonallergics =2.335, p=.077, d= −0.651). Post-TSST oxytocin values of allergics  and nonallergics  did not differ significantly (t(34)=1.117, p=.272). In addition, differences in oxytocin concentrations (Delta oxytocin) were calculated at the time points B1 minus B2. Delta oxytocin exhibited a trend to significant differences suggesting that the stress experience led to decreases in oxytocin levels in allergic participants, while in healthy controls, concentrations of the neuropeptide hormone increased (t(34)=1.853, p=.073, d = 0.62).Oxytocin was assessed as an additional physiological parameter before and after the TSST via blood sampling and as depicted in 3.3F=15.498, p<.001, par. η2=0.295), but no significant interaction of VAS ratings*allergic status was found =1.860, p=.140, par. η2=0.048). State anxiety measures before and after stress exposure only revealed higher STAI ratings after the TSST procedure =5.177, p=.029, par. η2=0.123), but no interaction effect of STAI x allergic status groups =0.203, p=.655, par. η2=0.005). See The VAS stress ratings over 4 time points  showed a significant main effect over time (4.According to the World Allergy Organization (WAO), recent evidence about the increasing prevalence of allergic diseases worldwide warrants extensive research into the subject . The discrepancy between self-reported stress levels and physiological arousal may be interpreted as an effect of interfering emotion regulation strategies (Gross & Levenson, Our data support earlier results by Lam et al.  who founHowever, allergic individuals recovered more efficiently from the stress-induced cortisol rise if they used reappraisal. This association of appraisal on the HPA stress cascade is in line with results from Jamieson et al. , who repA limitation of our study is that in the context of the pilot nature of the study, according to the relatively small sample size, we were not able to control for seasonality and chronicity of allergy. Controlling for these factors would be worthwhile in light of results by Buske-Kirschbaum, Ebrecht, and Hellhammer  who demo


Recent data suggest that the risk of young-onset colorectal cancer (yCRC), in adults less than 50 years of age, is increasing. To confirm findings and identify contemporary trends worldwide, we conducted a systematic review of studies examining population-level trends in yCRC epidemiology.We searched MEDLINE (1946–2018), EMBASE (1974–2018), CINAHL (1982–2018), and Cochrane Database of Systematic Reviews (2005–2018) for studies that used an epidemiologic design, assessed trends in yCRC incidence or prevalence, and published in English. Extracted information included country, age cut-off for yCRC, and reported trends in incidence or prevalence . We pooled similarly reported trend estimates using random effects models.p < 0.0001) that is largely driven by findings from North America and Australia. Also contributing to these trends is the increasing risk of rectal cancer as among 14 studies assessing cancer site, nine showed an increased risk of rectal cancer in adults less than 50 years with APCi up to + 4.03 (p < 0.001).Our search yielded 8695 articles and after applying our inclusion criteria, we identified 40 studies from 12 countries across five continents. One study assessed yCRC prevalence trends reporting an APCp of + 2.6 and + 1.8 among 20–39 and 40–49 year olds, respectively. 39 studies assessed trends in yCRC incidence but with substantial variability in reporting. Meta-analysis of the most commonly reported trend estimate yielded a pooled overall APCi of + 1.33 (95% CI, 0.97 to 1.68; Our systematic review highlights increasing yCRC risk in North America and Australia driven by rising rectal cancers in younger adults over the past two decades. Colorectal cancer (CRC) is a heterogeneous disease of the colon and rectum predominantly arising from adenomatous polyps or adenomas . The IntWhile CRC has long been considered a disease of older adults , recent An information scientist searched Medline 1946-), Embase (1974-), and Cochrane Database of Systematic Reviews (2005-) on the Ovid platform, and CINAHL (1982-) and PsycINFO (1880-) on Ebscohost. Database searches were conducted on January 17, 2018, and then updated on December 3, 2018. To ensure comprehensive capture of both articles that may assess CRC with sub-group reporting allowing extrapolation of yCRC and those that specifically assessed yCRC, we combined two separate but complementary searches. First, we used a broad search strategy with concepts of “colorectal cancer”, “prevalence”, and “incidence” to identify articles on CRC across all ages from which data could be extracted for individuals with yCRC. Second, we used a specific strategy where we additionally incorporated concepts of “young age” and “early” to identify articles that specifically examined yCRC. We used both database dependent subject headings  and keywords  original study using epidemiologic design; 2) published in a peer-reviewed journal as a full-length article or letter; 3) patient population with CRC or yCRC; 4) published in English; and 5) assessed trends in the incidence and/or prevalence yCRC, using regression methods  and reported corresponding estimates . While yCRC has been largely defined in individuals under 50 years , this maWe extracted information on country, data source, sample size, sex distribution, age cut-off for yCRC, and cancer site . The primary outcome was measures of trends in the incidence (e.g. APCi) and prevalence (e.g. APCp) of yCRC. As we noted substantial variability in the reporting of trends during data extraction, we contacted authors to request specific estimates  to facilitate pooling. Where available from the included studies, we also extracted reported incidence rates. As some of the studies meeting inclusion criteria additionally reported on outcomes such as yCRC mortality and/or survival, we considered these as secondary outcomes and extracted relevant information. Two researchers (KS and MDV) independently screened titles and abstracts, reviewed manuscripts, and extracted data, resolving any discrepancies by consensus.We assessed the quality of included studies with a checklist adapted for this systematic review based on the Joanna Briggs Institute Prevalence Critical Appraisal Tool, developed to address the lack of critical appraisal tools for systematic reviews of studies reporting prevalence , and thep-values. When a p-value was reported only as <X , we based our computation of the standard error on the conservative assumption that p = X. The intercept-only fixed effects solution represents the synthesized common APCi within the group of studies. We obtained pooled estimates of APCi and corresponding 95% confidence intervals (CI) and p-values across included studies. We pooled overall APCi and sex-specific APCi, given that studies varied in reporting. We also pooled APCi according to continent. Primarily analysis considered all studies that reported APCi. We also conducted various sensitivity analyses to account for potential overlap between included studies from the United States of America (USA) that used Surveillance, Epidemiology, and End Results Program (SEER) data . For each group of studies pooled, we computed the Cochran’s Q-test and the I-squared statistic to measure the presence of, and degree of heterogeneity [To synthesize findings on trends in yCRC epidemiology across included studies, we pooled the most commonly reported estimate, in particular, the APCi. We applied methods described by Sheu et al. for meta-analysis using linear mixed method  and fit The combined search strategies yielded 8695 (6612 with the broad search strategy and 2083 with the specific search strategy) titles  among 20–39 year-olds and + 1.8 (p < 0.01) among 40–49 year-olds, suggesting an increasing burden over the study period of 2002 to 2008 [A 2012 Canadian study by Ellison et al. evaluating trends in the prevalence of various cancers reported APCps of yCRC of + 2.6  alone , 40, 46. alone , With respect to our primary outcome of trends in yCRC incidence, included studies varied across reported trend measures – for example APCi in 22 studies , 49, 50,p < 0.0001). When we meta-analyzed studies that reported sex-specific APCi, we obtained a pooled APCi of + 1.02  for women and + 0.99  for men. Further columns in Table Table p < 0.001) for localized, + 3.05 (p < 0.001) for regional, and + 2.66 (p < 0.001) for distant disease and estimated that incidence rates of rectal cancers for patients under 50 years are expected to increase up to 124.2% by 2030 [The majority of studies in our systematic review are from North America with 25 studies altogether , 48, 51  by 2030 , and two by 2030 , 48 that by 2030 , 19, 24. by 2030 , 48, 51. by 2030 , 48, Whi by 2030 , 19, 51, by 2030 .We also identified two studies from Canada , 20, 26.p < 0.0001). Various pooling scenarios that attempted to account for potential overlap in data from included USA studies did not result in marked changes, though somewhat higher pooled APCi’s .We included four studies from Oceania –37. Threp < 0.03) for 20 to 29 year-olds, + 4.6 (p = 0.1) for 30 to 39 year-olds, and + 0.83 (p = 0.45) for 40 to 49 year-olds [p = 0.72) for European studies.We included three studies from Europe –40. In ap < 0.05) and men  [p < 0.05) for yCRC [p = 0.49) for studies from Asia.We identified six studies from Asia –46. The  < 0.05) . In cont < 0.05) . Three s < 0.05) , 43, 46.for yCRC ; while ZOne included study from Africa used the Cancer Registry of Setif, Algeria from 1986 to 2010 and reported no change in yCRC incidence with APCi’s of CRC among patients 15 to 44 years of − 2.1  for women and − 0.8  for men .p < 0.001) and, compared to patients with aCRC, those with yCRC had a 33% lower risk of disease-related death  [Among included studies, seven reported additional information on survival , 28, 35 to 0.74) . Howeverto 0.74) .p < 0.0001), that is largely driven by increasing incidence in the USA, Australia, and Canada with reported overall APCi’s up to + 7.9  [p < 0.001) [We identified 40 studies spanning 12 countries across five continents evaluating temporal trends in the incidence and prevalence of yCRC. Altogether, we found an increasing incidence of yCRC with a worldwide pooled APCi of + 1.33   and nearto 15.1) , 50 studTo our knowledge, this is the first systematic review assessing the changing epidemiology of yCRC. While narrative reviews of yCRC have been published , 52, 53,n = 31) have been published since 2010 with seven published in the past year (2018) alone. Synthesizing these published, peer-reviewed evidence including quality assessment and where feasible, pooling of commonly reported estimates brings areas for attention based on key findings. One of our key findings is the contribution of findings from the USA, Australia, and Canada to the worldwide increased risk of yCRC. Quite timely, our systematic review also builds on Liu et al.’s recently published work taking data from the IARC and using joinpoint regression to calculate APCi’s to show the increasing incidence of yCRC in 11 out of 12 countries [The increasing incidence of yCRC across a number of jurisdictions seen in our systematic review may indeed signal a recent paradigm shift in CRC. Of note, the majority of included studies  [Along with key findings, it is also important to discuss knowledge gaps that we identified from our systematic review. Notably, only one included study evaluated trends in the prevalence of yCRC . It is i to 1.4) .1) younger age ; 2) African-American ethnicity ; and 3) insurance status  [Aside from identifying gaps, our systematic review also has implications for informing future research needs for better understanding of yCRC. Indeed, key findings of an increasing risk of yCRC highlights the need for future research examining reasons for this. As well, findings from our systematic review suggesting that the increased risk in yCRC may be driven by increased risk of rectal cancer among younger adults underscores the need for research on etiologic reasons for the differences in the risk between rectal and colon cancers. Among included studies in the systematic review, only one evaluated population-level determinants of yCRC . Specifi to 1.8) , which m to 1.8) . NoteworAlong with providing further confirmatory evidence on the increasing risk of yCRC, findings of our systematic review also lend to the question of the appropriate age for screening for CRC. In 2018, the American Cancer Society lowered the recommended age for average-risk adults to initiate screening from 50 to 45 years , 57. RecStrengths and limitations of our systematic review deserve discussion. All database searches were conducted by an experienced information scientist. Combining two separate but complementary searches was a unique feature of our study that maximized our capture of eligible studies. However, the inclusion of relevant studies may have been limited by publication bias as in any other systematic review. We did not consider abstracts given the importance of being able to assess the quality of the included studies. In addition, although we considered articles from many countries, inclusion of only those published in English is a limitation. Also, a potential limitation is that we did not consider reports or data from national cancer registries unless they have been published as peer-reviewed studies and reported trend estimates of yCRC incidence and/or prevalence, in line with our inclusion criteria. The majority of included studies from the USA using potentially overlapping SEER data may also be considered a limitation. However, drawing more nuanced information on yCRC such as sex- and ethnicity-specific trends is only possible with consideration of all included studies; we did not attempt to exclude studies based on potential overlapping data but rather, conducted various sensitivity analyses, which showed no appreciable differences in pooled APCi.Overall, by synthesizing findings from peer-reviewed, epidemiologic studies, our systematic review provides empirical evidence that confirms the increased incidence of yCRC, particularly in North America and Australia. Continued efforts for awareness and education to address this increasing risk are warranted along with research to explain this risk so as to identify areas for prevention and intervention.Additional file 1: Figure S1 Forest plot for studies reporting overall incidence rates for yCRC. Figure S2 Forest plot for studies reporting incidence rates for yCRC among. Figure S3 Forest plot for studies reporting incidence rates for yCRC among menAdditional file 2: Table S1. Database Search StrategyAdditional file 3: Table S2. Quality appraisal checklistAdditional file 4: Table S3. Reported incidence rates  for yCRC overall and according to sex among included studies


Background: Fifteen years after a nationwide voluntary iodine prophylaxis program was introduced, the aims of the present study were: (a) to obtain an up-to-date assessment of dietary iodine intake in the Veneto region, Italy; and (b) to assess dietary and socioeconomic factors that might influence iodine status. Methods: Urinary iodine concentration (UIC) was obtained in 747 school students . Results: The median UIC was 111 μg/L, with 56% of samples ≥ 100 μg/L, but 26% were < 50 μg/L, more frequently females. Iodized salt was used by 82% of the students. The median UIC was higher among users of iodized salt than among non-users, 117.0 ug/L versus 90 ug/L (p = 0.01). The median UIC was higher in regular consumers of cow’s milk than in occasional consumers, 132.0 μg/L versus 96.0 μg/L (p < 0.01). A regular intake of milk and/or the use of iodized salt sufficed to reach an adequate median UIC, although satisfying only with the combined use. A trend towards higher UIC values emerged in regular consumers of cheese and yogurt. Conclusion: Iodine status has improved (median UIC 111.0 μg/L), but it is still not adequate as 26% had a UIC < 50 μg/L in the resident population of the Veneto region. A more widespread use of iodized salt but also milk and milk product consumption may have been one of the key factors in achieving this partial improvement. Iodine deficiency (ID) affects two billion people around the globe and is the world’s greatest single cause of preventable brain damage . A low iWhen the physiological requirements of iodine are not met in a given population, developmental and functional abnormalities occur, including thyroid function anomalies and, when ID is severe, endemic goiter and cretinism . An areaAs Italy has historically been characterized by endemic goiter , a natioIn addition to the national voluntary iodine prophylaxis program, since 2013, the Italian Ministry for Education, University, and Research (MIUR) has been running a nationwide education program on the importance of iodine for human health and the benefits of using iodized salt use in all secondary and higher-level schools, with the aim of improving iodine nutrition. The educational program on iodine prophylaxis in the Veneto region consists of several steps. Physicians from our Endocrinology Unit provide training for the medical staff at the Food Hygiene and Nutrition Services (SIAN) with the aid of several computer-based materials provided by the MIUR illustrating the main concepts behind iodine prophylaxis and the importance of a responsible use of salt. Then the SIAN staff train the schoolteachers at the designated schools, and the teachers in turn conduct educational sessions with their students, using targeted oral presentations, based on a multistep program already used in a previous survey .Prior to the introduction of 2005 voluntary iodine prophylaxis program, iodine status assessments conducted between 1994 and on SAC living in north-east Italy demonstrated a non-negligible prevalence of thyroid goiter , and ioIn 2012 , seven yThe influence of socioeconomic and educational factors on voluntary iodine prophylaxis program is presented. In many regions of the world, a low education level proved a predictor of reduced efficacy of such programs ,14. In EPreliminary data from the Italian National Observatory for Monitoring Iodine Prophylaxis (OSNAMI) for the years 2015–2019, on a sample of 2523 SAC (11–13 years old) residing in 7 Italian regions , indicated that the UIC was now sufficient (118 ug/L), and iodized salt was used in 75% of Italian school canteens. The Veneto region was not among the regions included in this preliminary analysis .a) to conduct an up-to-date assessment of iodine status in our Veneto region; and (b) to examine dietary and socioeconomic factors that might influence it.The aims of the present study were: , were selected. These schools represent the 1.7% of middle schools in Veneto region (10/578). This study was conducted in accordance with the Declaration of Helsinki. For all participants, verbal informed consent was obtained from the parent/guardian on behalf of the minor, witnessed, and formally recorded. The investigation was conducted between September 2018 and September 2019 on 11- to 16-year-old SAC in grades 6 to 8. To survey the students’ awareness about iodine prophylaxis and iodine status, we enrolled students living in 10 different cities located in all 7 provinces of the Veneto region. Pupils attending middle schools adhering to the iodine prophylaxis monitoring program were asked to participate to the investigation.Pupils were requested to complete a questionnaire, with the help of their parents and to collect a non-fasting early-morning spot urine sample. The inclusion criteria were an obtained informed consent, a fully completed questionnaire and urine samples properly collected. The questionnaire comprised two sections: one recorded personal socioeconomic information , any personal history of thyroid diseases, and how much students knew about iodine prophylaxis; the other addressed their dietary habits, and particularly their consumption of the following products: cow’s milk, dairy products, yogurt, beef, chicken/turkey, eggs, fish, canned food, cold cuts, soft drinks, chips, soy products, and sodium-rich foods .These types of food were chosen to obtain a measure of the amount of iodine deriving from different iodine sources: from naturally-rich in iodine food (fish); from the policy of cattle food iodine enrichment and breeding’s diet in general . Since law n.55/2005 encourages the use of iodized salt in food preparation and conservation, also the contribution deriving from industrial food processing was considered, including prepackaged food , processed food (cold cuts), and sodium-rich foods (pizza and chips).Nationality was dichotomized in Italian or foreign.Parental education level was recorded as the highest qualification obtained . The total number of years of education completed was also recorded, with the data considered for each parent separately and also as both parents summed.The knowledge about iodine prophylaxis campaign was assessed by asking if the pupils already knew about the program and about the existence of a legislation regulating that and from what information source .As regards iodized salt use, pupils’ parents were asked if he/she used iodized salt (yes/no) and for how many years. Cow’s milk intake was scored as: 1 = no consumption; 2 = occasional consumption; 3 = 1 cup a day (at least 200 mL/d); and 4 = more than 1 cup a day. Cow’s and soy milk consumption was also dichotomized as: 1 = regular (daily or more than once a day) or 0 = occasionally (less than once a day). For other foods, intake was scored as: 1 = never; 2 = at least once a week; or 3 = more than once a week.A 20 mL sample of early-morning urine in non-fasting conditions was collected from all students. Samples were divided into aliquots and refrigerated at –20 °C until assay. UIC was expressed as μg/L and measured in duplicate using the colorimetric ceric ion arsenious acid method in a second-analyzer Technicon Auto-Analyzer  . Betweenp value of < 0.05 was considered statistically significant. The Kolmogorov–Smirnov test was used to check the normal distribution of the UIC. As the values were not distributed normally, they are reported here as medians and interquartile range (IQR). The Mann–Whitney U test was consequently used to analyze the differences in UIC in relation to different variables, when dichotomized. The Kruskal–Wallis test was used to study possible changes in median UIC when more than two possible scores of a given variable were taken into account. The χ2 test was used to explore the relationship between different iodine settings  and dietary and socioeconomic factors . Univariate analysis (by logistic regression) was used to explore the relative influence of each independent variable on the UIC. A p < 0.01)  . This po < 0.01) .In p = 0.01) (p = 0.15). The percentage of students with UIC values ≥ 100 μg/L was also higher among the families using iodized salt for more than a decade (61% as opposed to 49% in the remainder of the sample), although this difference did not reach statistical significance (p = 0.07).Eighty-two percent (610/747) of the students interviewed used iodized salt. Iodized salt was used by 336/411 (81.8%) of males and by 279/336 (83.0%) of females. The median UIC was higher among students using iodized salt than among non-users, being 117.0 μg/L (interquartile range: 54.0–192.0 μg/L) in the former and 90.0 ug/L, (interquartile range: 36.6–174.6 μg/L) in the latter ( = 0.01) . The stup < 0.01. The median UIC was higher in regular consumers of cow’s milk than in occasional users, being 132.0 μg/L (IQR: 72.0–198.9 μg/L) in the former and 96.0 μg/L (IQR: 36.0–177.3 μg/L) in the latter (p < 0.01) (Daily cow’s milk consumption was reported by 47% (349/474) of the students, with a higher percentage among males  compared to females ,  < 0.01) . The per < 0.01) .p < 0.01  for users of iodized salt who never or only occasionally drank cow’s milk. This level indicates an adequate iodine intake, though it is significantly lower than in iodized salt users who also drank cow’s milk regularly, whose median UIC was 141.0 μg/L (IQR: 79.5–201.3 μg/L), p < 0.01 . p = 0.02)  among the occasional cow’s milk consumers, and 110.4 μg/L (IQR: 39.3-189.6 μg/L) among the regular cow’s milk consumers, ( = 0.02) . In short, regular milk consumption and iodized salt use both sufficed alone to reach an adequate median UIC. p = 0.04 ) did not have higher median UIC than those who ate it less often ), however, although the former showed a trend towards higher values . On the p = 0.04 .Our analysis identified much the same picture for other milk products (yogurt): among the students not using iodized salt the median UIC was 116.4 μg/L (IQR: 56.7–183.9 μg/L) for those who ate yogurt more than once a week (213/747 (28.6%)) and 66.0 μg/L (IQR: 36.0–132.9 μg/L) for those who did not (205/747(27.4%)) , but thep < 0.01). The influence of milk on UIC was confirmed by logistic regression model, taking into consideration milk frequency (at least once a day or more versus less than once a day or never), iodized salt use and cheese consumption frequency (more than once a week versus never). Milk frequency has a higher impact on UIC adequacy than iodized salt , p = 0.04. When students were asked about their main source of nutritional information on iodine, 55% mentioned the mass media , and only 45% cited the school education program, although it had been running in Italy since 2013. Less than 1% of the sample mentioned their physician or some other institution .In 42.5% of cases (317/747), the students knew there was legislation to deal with iodine deficiency . Among these students, those who were aware of the significance of iodine prophylaxis used iodized salt more than those who were not, i.e., 85.6% of the former (271/317) versus 79.5% of the latter (342/430), No association was found between the use of iodized salt or median UIC and the educational level of student’s parents (which ranged from elementary school to a university degree). Nor was there any association between the use of iodized salt or median UIC and student’s nationality .A partially adequate iodine status (median UIC of 111 μg/L) has been reached in the population living in the Veneto region since the start of an iodine prophylaxis campaign in 2005 and systematic monitoring of iodine intake in SAC. However, following WHO recommendations, no more than 20% of UIC should be <50 μg/L to define a population iodine-sufficient. Since 26% of our population still has a UIC that falls into a moderate iodine deficiency (<50 μL), much more work should be made in Veneto region to reach iodine adequacy. A more widespread use of iodized salt, if compared to the past consumption in our region [The amount of iodine added to salt established by Italian law is 30 mg per kilo (tolerance range: 24–42 mg/Kg) and was calculated estimating an average consumption of salt of 5 g per day (following WHO recommendations) and the median amount of iodine provided by food in a varied diet . Data onIn 2012, it was necessary to combine the use of iodized salt with daily cow’s milk consumption to ensure an adequate median UIC in our region . AccordiWe also confirmed the role of cow’s milk suggested in our previous reports ,21, whicThe percentage of pupils with a UIC < 50 μL is higher in females than in males (30.3% versus 23.6%), with a superimposable iodized salt consumption in males and females, but a less frequent use of milk in the latter. Adolescent females might be induced to reduce their milk and dairy product consumption due to a misconception that they are fattening ,25. UIC Iodized salt use resulted as quite unchanged when its use was analyzed based on sex and age range, while milk use frequency percentages were much more affected by sex. Eleven to twelve-years-old and 15–16-years-old females resulted as iodine insufficient, although the small sample sizes limited our ability to further analyze this data.Whatever their reasons, females were confirmed as being poor milk consumers in this latest survey also, when we recorded a median UIC in female SAC of 103 μg/L, barely enough to be considered an adequate iodine supply. This situation warrants attention because these girls could be future mothers and, to prevent iodine deficiency and its consequences, they should be informed that they need a higher iodine intake—not only in pregnancy and breastfeeding, but also prior to conception. Their iodine intake should be raised to 150 μg/day, according to recent ATA guidelines .It is worth noting how milk products are also able to influence UIC: regular consumption of cheese or yogurt (more than once a week) proved effective in achieving an adequate median UIC even in students not using iodized salt. The present study is the first in Italy to find a significant association between cheese consumption and UIC. Unfortunately, our survey did not collect information on the type of cheese the SAC ate. In a previous report by our group we assessed the iodine content of milk commercially available in Veneto region. Based on that data, the median and mean iodine values of fresh and long-life commonly commercially available cow milk samples were 262 and 264 μg/L, respectively . UnfortuThe SAC’s awareness of legislation concerning iodine deficiency documented in this report has not changed when compared to the previous survey, undergone in 2012 (42.5% versus 45%) . In our Our study has many limitations. We included in the survey many middle schools selected by our local hygiene service, that should be representative of all Veneto region. However, since not schools are included, a possible bias may come from the representativeness of our series, mostly in the 15–16- and 11–12-year old age range. Moreover, the number of pupils included in the study was lower than expected, since not all students complied with the inclusion criteria.Finally, based on our data, adolescent females should be the main target of the next iodine prophylaxis campaigns.


To examine the association of dietary behaviors, lifestyle, and biochemical factors with metabolic phenotypes of obesity among obese Iranian children and adolescents.This cross-sectional study was conducted within the framework of the fifth phase of CASPIAN study. Of 3840 students aged 7–18 years of 30 Iranian provinces, 408 subjects were diagnosed as obese; they were divided into metabolically healthy obese (MHO) and metabolically unhealthy obese (MUO) groups. Biochemical factors, anthropometric measures, dietary, and lifestyle habits were compared between groups.Of the 408 obese subjects, 68 (16.7%) were the MUO; the remaining 340 (84.3%) fall in the MHO group. The MUO group had significantly higher systolic and diastolic BPs, FBS, TG, ALT, anthropometric measures, and lower HDL levels than MHO groups . The frequency of high birth weight (> 4000 gr) was significantly higher in the MUO group than the MHO group . A higher percentage of individuals with breastfeeding duration ≥ 6 month was found in the MUO group (95.5% (95% CI 86.1–98.6%)) compared to MHO group (85.7% (95% CI 80.4–89.7%)) . Among dietary and lifestyle-related behaviors, only the frequency of salty snack consumption and eating food according to the parents’ request was significantly higher in the MUO group than the MHO group .Dietary habits and lifestyle factors may determine the obesity phenotypes in children and adolescents. One of the most preventable causes of mortality is obesity. This is while about 100 million obese children were reported in 195 countries in 2015 worldwide, with studies for instance from the US reporting its prevalence among teenagers to be tripled from 4.6 to 20.6% in the past four decades [Obesity is linked with a long list of diseases, including metabolic disorders, cardiovascular disease, non-insulin-dependent diabetes, sleep apnea, osteoarthritis, and asthma, many of which are believed to persist from childhood into adulthood . As a reDespite all this, current recommendations for the management of childhood obesity has shown limited efficacy. Many believe this is due to the fact that the essence of obesity lies in the adipose tissue and therefore body mass index is an indirect estimation of the amount of fat in the body and poor health . These pObese individuals have heterogeneous phenotypes, each associated with various health conditions . MetabolAs expected, there is a long list of factors that contribute to these phenotypes. This list includes but is not limited to positive family history of obesity, anthropometric profile, environmental factors such as dietary intake behaviors and lifestyle habits, and certain biochemical markers , 9. A beRecent data shows a surge in the obesity rates in the developing countries, including the Middle Eastern region, mainly due to an increase in the dietary energy surpluses secondary to the dietary changes from traditional diets to fast foods and westernized foods , 3. IranThis study was conducted within the fifth phase of a national school-based survey entitled “Childhood and Adolescence Surveillance and Prevention of Adult Non-communicable Disease” (CASPIAN-V study). CASPIAN-V was conducted on 14,400 primary and secondary school-aged students in urban and rural areas of 30 Iranian provinces in 2015. The detailed protocol of this survey has been published previously . From thThe demographic questionnaire for pediatrics, including information about birth weight, duration of breastfeeding, eating behaviors, screen time, physical activity, and sleep duration, was completed. For evaluating eating behaviors, information about breakfast consumption , eating speed , consumption at least 3 meals/day (yes/no), consumption of sugar-sweetened beverages, junk foods , fruits, and vegetables was filled out by all the students. Information concerning eating speed was self-reported according to the question “How fast is your eating speed compared to others?” in the questionnaire. The response was based on three possible semi-quantitative categories: “slow”, “medium”, and “fast”.Screen time (ST) was defined as the average hours per day spent watching television or using a personal computer . The phy2). Waist circumference (WC) was measured at the midpoint between the lower margin of the rib cage and top of the iliac crest with a nonelastic tape to the nearest 0.1 cm [The anthropometric measurements were performed by trained healthcare staff. Height was measured without shoes with 0.5 cm accuracy. Weight was measured with minimal clothes and no shoes, using a digital scale  placed on flat ground to the nearest 0.1 kg. Body mass index (BMI) was calculated as weight (kg) divided by square of height , total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C) and triglycerides (TG) were measured by enzymatic methods using Hitachi auto-analyzer  .We obtained written informed consent from all parents and participants older than 16 years, and oral informed consent from those who were under 16 years. The study protocol was approved by the Research and Ethics Council of the Isfahan University of Medical Sciences (project number: 194049).All variables were checked for normality and presented as mean and 95% confidence interval (CI) for quantitative variables. Qualitative variables were presented as a percentage and 95% CI. Quantitative and Qualitative variables were compared between the MHO and MUO groups using independent samples t-test and Chi square test respectively. All analysis was adjusted using the Bonferroni post hoc test to control multiple testing problems. Multivariate logistic regression model was run for variables which were significant in univariate model, adjusting for age, sex and living area. Data analysis was done using STATA Statistical Software version 11.0 . P-values < 0.05 were considered as statistically significant.Among 408 children and adolescents with obesity, defined as age and sex-specific BMI more than 95th percentile, a metabolically unhealthy and obese phenotype (MUO) was detected in 16.7% (n = 68).Table Besides, as compared to MHO group, the MUO group had significantly higher ALT levels (mean (95% CI) 11.58 (8.78–14.39) vs. 8.41 (7.18–9.64), p-value < 0.001), BMI (28.11 (24.81–31.42) vs. 26.10 (25.52–26.67), p-value = 0.03), and z-BMI (2.03 (1.33–2.74) vs. 1.61 (1.48–1.73), p-value < 0.001).Table The MUO group had a higher percentage of individuals with breastfeeding duration ≥ 6 month (% (95% CI) 95.52% (86.15–98.65%)) than the MHO group (85.76% (80.48–89.79%)) .In addition, daily salty snack consumption was significantly more frequent among individuals in the MUO group (14.71% (6.693–29.3)) compared to the MHO group (4.451% (2.504–7.79%)) . The frequency of eating food according to the parents’ request was higher in the MUO group in comparison with the MHO group . Results of multivariate logistic regression model showed significant association between metabolic phenotypes of obesity and salty snack consumption , breastfeeding duration , and when eating food , after adjusting sex, age and living area.Other dietary behaviors , eating speed, and lifestyle behaviors  in the MUO group were not statistically significant compared to those in MHO group .Our study shows a significant difference in the profile of obesity-related biochemical markers and some lifestyle habits between the MUO and MHO adolescents. Among dietary behaviors, only daily consumption of salty snack was more frequent in the MUO group.MHO group consisted 83% of our obese population. This is while many studies have suggested that about 10–40% of the obese adult population do not experience any metabolic complications [Demographic factors are considered as an important predictor of obesity phenotypes, with some studies reporting a higher incidence of MHO among girls and younger adolescents , 10. SimRecent advancement in medicine has shifted the focus towards precision medicine, which refers to the “tailoring of medical treatment to the individual characteristics of each patient” and the “ability to classify individuals into subpopulations that differ in their susceptibility to a particular disease, in the biology or prognosis of those diseases they may develop, or in their response to a specific treatment” . This apAs mentioned earlier, the current approach to obesity, especially childhood obesity, has not been successful and behavioral phenotyping can thus help provide a more personalized solution to this problem. Behavioral phenotyping refers to distinct personal behaviors that result from the interaction of genes and environment . They caThe dietary pattern  determine the cardiometabolic health status in adolescents, especially boys . SkippinThere is no consensus about the association of certain lifestyle habits with the metabolic phenotypes of obesity. Several studies have shown a higher incidence of obesity among children and adolescents with high screen time (TV and PC) , 8. ThisPoor sleeping quality  and low sleep duration are reported to be associated with MUO, mainly among boys , 9. Our Several biochemical markers are among other determinants of obesity phenotypes, with many studies reporting lower levels of fasting triglycerides and fasting glucose but elevated levels of high-density lipoprotein (HDL) among MHOs compared with MUOs . On the Obese children are reported to have higher blood pressure values compared with their normal weighted counterparts, which is believed to make them susceptible to many diseases, mainly cardiometabolic disorders . SimilarApart from the adolescent behavior and habits, their weight at birth and the duration of breastfeeding are also reported to affect the obesity phenotypes. This is inconsistent with previous studies that had considered lower birth weight and longer breastfeeding duration as protective factors against obesity , 18, 19.The present study is one of the largest cohorts on obese adolescents from different parts of the country examining various determinants of metabolic phenotypes. As a result, its finding could be generalized to the Iranian adolescents. Among various factors linked with obesity and its phenotypes, the present study has assessed the influence of most important risk factors of obesity, such as dietary factors, lifestyle habits, and certain biochemical factors (lipid and liver function). As for the anthropometric profile, however, we only used BMI as a measurement of obesity, while this value cannot distinguish between fat and lean tissue . This isBehavioral phenotyping through defining obesity phenotypes helps identify the variation in children’s dietary behaviors and lifestyle factors, determine those at risk and adopt the personalized intervention based on the individual’s predisposition to prevent obesity later in the child’s life. However, additional research is needed to improve risk stratification for these phenotypes. The results of such studies could in near future result in targeted screening along with cost-effective detection and personalized treatment of children at higher risk for cardiometabolic disorders.


In patients with moderate/severe FMR, MA diameters correlated with PISA radius, EROA, and regurgitant volume (Rvol), as also did the MA area . MAFAC correlated inversely with EROA and Rvol , with both active and total LA emptying fractions and with LV ejection fraction as well. In a stepwise multivariate regression model, decreased MAFAC and increased LA volume independently predicted patients with severe FMR.  Patients with FMR vs. controls presented increased MA size and sphericity during the entire systole, whereas MA fractional area change (MAFAC) and MA displacement were decreased (15 ± 5 vs. 28 ± 5%; and 5 ± 3 vs. 10 ± 2 mm,  Patients with DCM and FMR have MA geometry remodeling and contractile dysfunction, correlated with the severity of FMR. MA contractile dysfunction correlated with both LA and left LV pumps dysfunctions and predicted patients with severe FMR. Our results provide new insights that might help with better selection of patients for MV transcatheter procedures. Functional mitral regurgitation (FMR) is a common complication in patients with ischemic or nonischemic dilated cardiomyopathy (DCM), increasing mortality, and the risk of rehospitalization . In patiRecent studies suggested an association between the anteroposterior diameter of the mitral annulus (MA) and FMR recurrence after MV interventional treatment . HoweverAccordingly, we designed our study (i) to characterize MA geometry and dynamics changes, as the remodeling of the MA, by using 3DTTE in patients with DCM and FMR, in comparison with healthy controls; (ii) to assess the relationship between MA remodeling and severity of FMR; and (iii) to assess the relationship between MA dysfunction and severity of LV and LA dilation and dysfunction.We conducted a prospective study, in which we enrolled 56 patients with mild-to-severe FMR due to ischemic and nonischemic DCM and compared them with 52 control subjects, similar in age, gender, and BSA. All subjects were in sinus rhythm and had complete 3DTTE datasets for MV, LA, and LV. Patients with more than mild aortic, pulmonic, or tricuspid valve disease, significant MV calcifications, or poor-quality image of the 3DE datasets were excluded. Control subjects were healthy volunteers free of any cardiovascular history or risk factors. Heart rate, blood pressure, height, and weight were measured in all subjects immediately before the echocardiographic examination. Body surface area (BSA) was calculated . EtiologEchocardiography was performed in accordance with a standardized acquisition protocol, with a Vivid E9 system , equipped with M5S and 4V probes. Standard transthoracic echocardiography was performed to assess LV size and function and etiology and severity of FMR in patients with DCM. Then, to obtain high temporal resolution of the datasets, separate 3D full-volume multibeat acquisitions of the MV, LV, and LA were obtained by combining six consecutive ECG-triggered subvolumes, during breath-hold and avoidance of patient or probe movement. Care was taken to encompass the MV in the full volume throughout the cardiac cycle and the entire LV and LA in the dedicated dataset. By using multislice display, the absence of stitching artifacts in the acquired dataset was carefully checked.The severity of FMR was graded as trivial/mild, moderate, and severe, using two-dimensional (2D) echocardiography, according to the current guidelines . QualitaLV 3DTTE datasets were stored digitally in raw-data format for offline analysis , as previously described and validated against cardiac magnetic resonance (CMR) , 15 Fig.LA 3DTTE datasets were converted to DICOM format and analyzed using dedicated software package designed for volumetric analysis of the LA and recently validated against CMR  . At the static analysis) and then during the cardiac systole  , as previously described . Brieflynalysis) . MA meast-test. Pearson's correlation was used to analyze the relationships between MR severity parameters and MA function and between MA function and LA and LV parameters. Multiple linear backward regression tests were used to identify the parameters that independently correlated with severe MR. All analyses were performed using SPSS version 20.0  and MedCalc version 10.0.1.0 . Differences among variables were considered significant at p < 0.05. Our group has previously reported the reproducibility for the echocardiographic methods, in unselected patients with MV disease [Normal distribution of variables was checked by Kolmogorov–Smirnov test. Continuous variables were summarized as mean ± SD, and categorical variables were reported as percentage. Variables were compared between groups using unpaired  disease .2 and RVol 26 ± 19 ml, available in 33 patients (59%). Temporal resolution of the 3D datasets for MA analysis was 32 ± 7 volume/sec in patients with FMR and 35 ± 6 volume/sec in control subjects (p=0.87). The number of systolic frames in each dataset ranged from 9 to 17, depending on heart rate or acquisition settings. Static analysis of the MA was feasible in all subjects. Dynamic analysis of MA was feasible in 54 patients with FMR (96%) and in 48 control subjects (92%).The general characteristics of the study population are summarized in Mitral annulus static analysis is shown in Comparisons between LV, LA, and MA parameters in patients with trivial/mild, moderate, and severe FMR are shown in r = 0.56 and r = 0.43; with EROA: r = 0.66 and r = 0.45; and with RVol: r = 0.53 and r = 0.39, all p < 0.05), as did also MA area . MV tenting volume correlated with PISArad  and with Rvol , while MV tenting correlated only with PISArad . MA fractional area change correlated inversely with EROA and Rvol .In patients with moderate and severe FMR, MA diameters (AP and ALPM) correlated with all quantitative parameters of FMR severity (with PISArad: p=0.03), AP diameter fractional change (p=0.02), and MA fractional area change (p=0.003), respectively.In a multiple backward linear regression model using indexed LVEDV; LAVmax; MA area; LVEF; active and total LAEFs; AP diameter fractional change; and MA fractional area change, independent predictors for severe MR were the LAVmax . MA area fractional change correlated with both active and total LAEFs, as well as with LVEF, while MA AP diameter fractional change showed similar behavior; MA displacement correlated only with LVEF  increased MA size and sphericity, during the entire cardiac systole, was associated with a concomitant remodeling of the MV leaflets; (ii) MA was flatter, with increased MV tenting height, area, and volume during the cardiac systole; (iii) maximum MA contraction was not only decreased in amplitude, but also delayed in time; (iv) MA size and contractile dysfunction correlated with FMR severity; and (v) MA contractile dysfunction correlated with both LA and LV dysfunctions.MA remodeling and dysfunction have been reported as mechanisms of both ischemic and nonischemic FMR , 20, 21.Patients with mild-to-severe FMR showed MA dilation throughout the entire systole, as previously reported , 21. HowMA was flatter in patients with FMR than in control subjects. Similar with the data reported by Topilsky et al. , our patBeside MA enlargement and remodeling, patients with FMR also showed altered relation of the MA with the surrounding structures, presenting wider Ao-AP angle than control subjects. This has to be taken into account for annuloplasty techniques, as the angle Ao-AP proved to predict the occurrence of systolic anterior movement of the anterior mitral leaflet after MV annuloplasty . MoreoveLV dyssynchrony has been reported as a mechanism of FMR, while cardiac resynchronization therapy (CRT) can reduce early systolic FMR in selected patients –31, but MA area fractional change was increasingly impaired in patients with FMR. We showed that the “contractile dysfunction” of the MA was associated with both LA and LV dysfunctions. In patients with DCM, Kwan et al.  reportedOtsuji et al.  showed tIn our study, we did not assess the complex relationship between papillary muscle position, orientation, and FMR severity; however, this has been extensively assessed in previous studies –40. The Patients with dilated cardiomyopathy and functional mitral regurgitation have mitral annulus geometry remodeling and contractile dysfunction, which correlated with the severity of mitral regurgitation. Mitral annular contractile dysfunction correlated with both left atrial and left ventricular pump dysfunctions. Thus, these results enhance the association of atrial remodeling and dysfunction with the severity of functional mitral regurgitation severity, mediated through the mitral annular dysfunction. Our results may ensure further studies to use parameters of the mitral annular size and geometry, in order to help us to better select patients for transcatheter procedures, targeting the mitral annulus or the mitral valve.


Pueraria lobata is a traditional Chinese herb which has been used medically since ancient times. In this study, we sequenced the complete chloroplast genome of P. lobata based on next-generation sequencing and used the data to assess genomic resources. The chloroplast genome of P. lobata is 153,411 bp in length consisting of large and small single-copy regions of length 84,142 and 17,989 bp, separated by two IR regions of 25,640 bp. The overall GC content was 35.4%. De novo assembly and annotation showed the presence of 112 unique genes with 78 protein-coding genes, 30 tRNA genes, and four rRNA genes. A maximum-likelihood phylogenomic analysis showed that Pueraria was sister to Pediomelum + Glycine. Pueraria lobata (Willd.) Ohwi is a traditional Chinese herb which has been used medically since ancient times. However, only a few genomic resources have been explored. Chloroplast genomes are valuable sources of genetic markers for phylogenetic analyses, genetic diversity evaluation, and plant molecular identification , China Academy of Chinese Medical Sciences with accession number 431028LY1034. Total genomic DNA was extracted and purified following the method of Li et al.  in RAxML 8.0 using the concatenated coding sequences of 77 chloroplast coding genes for a group of 23 species (Stamatakis Pueraria was sister to Pediomelum+Glycine (Pueraria.The chloroplast genome of +Glycine . The who


An 80-year-old woman with a positive RT-PCR (reverse transcription polymerase chain reaction) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on nasopharyngeal swab was admitted to our COVID-19 department during the pandemic with fever, dyspnea, and need for oxygen therapy; two large hematomas were present bilaterally in the axillary zones. Her past medical history included Crohn's disease diagnosed 30 years ago after perforation of the ascending colon, hypertension, bilateral hip replacement, appendectomy, and tonsillectomy; no history of bleeding complications was reported during or after surgical interventions or in the postpartum, and no history of menorrhagia or easy-bruising. One month and a half before admission, she underwent right knee replacement surgery with no complications, no blood units, or plasma transfusions were required. At that time complete blood count and screening clotting test were within normal range.3; hemoglobin 8 g/dL; mean corpuscular volume 89 fL; platelets 251,000/mm3; PTr 1.16 (n.v. 0.80–1.21); PTTr 1.49 (n.v. 0.80–1.18); D-dimer 2,161 μg/L (n.v. < 790); fibrinogen 455 mg/dL (n.v. 180–400); C-reactive protein 78.4 mg/L (n.v. < 5.0). A chest computed tomography scan outlined a picture of bilateral interstitial pneumonia with ground-glass areas and thickening of interstitium, consistent with a viral infection; moreover, the presence of hyperdense hematomas in the axillary zones was recorded (thickness 20–30 mm), no active bleeding was detected. Antiretroviral therapy with darunavir and ritonavir was started; hydroxychloroquine was not administered for prolonged QTc on electrocardiogram. Enoxaparin 4000 IU/day was introduced for prophylaxis and continued until hospital discharge.Investigations on admission were as follows: white blood cell 11,380/mmThe isolated prolonged PTTr (silica) was confirmed in day +5 and +7 from admission , PTTr  was prolonged too, so a PTT MIX test was required: the PTTr MIX at time 0 was normal but PTTr MIX at 2 hours at 37°C was above the normal range, consistent with the presence of an inhibitor ; the fXInhibitors against clotting factors generally occur in patients with severe congenital deficiencies who underwent periods of replacement therapy. Acquired factor inhibitors in the absence of congenital deficiency are rare events, among them the production of autoantibodies inhibiting fVIII is the most frequent acquired bleeding disorder. On the contrary, inhibitors against fXI have been reported only anecdotally in literature mostly associated to autoimmune disorders, that is, systemic lupus erythematosus,intrinsic pathwayof coagulation waterfall the fXIIa activates the zymogen fXI which in turn activates fIX and so, through a series of enzymatic reactions, it conducts to thrombin formation. It is nowadays well documented that fXII has only a marginal role in the hemostasis in vivo, contributing more to inflammation and pathologic coagulation; in fact, fXI seems to be activated in a stronger way directly by thrombin, thus supporting and amplifying thrombin formation in loop.extrinsic pathway).In the classical chart of theBut fXI is not only a component of the coagulation pathway, it also participates to the process of contact activation by mean of the bradykinin-generating kallikrein–kinin system (KKS)  contributing in this way to several host-defenses and to the initial response to infection: in fact, the inflammatory response to viral infections is in part mediated by contact activation and it has already been demonstrated how hantavirus and herpes simplex virus 1 can lead to the activation of KKS pathway.milieu(Crohn's disease), as evolutionary advantaged in this condition having the possibility to counterbalance the state of hypercoagulability and inflammation vascular thrombosis, still to demonstrate


Upper respiratory tract infections (URTIs) are common and burdensome infectious illness. Several trials have reported that probiotics can prevent URTIs in adults.  To evaluate the efficacy and safety of probiotics in the prevention of URTIs in adults.  PubMed, Web of Science, Embase, and Cochrane Library were searched for reports published from database inception to May 14, 2020. Randomized controlled trials (RCTs) comparing probiotics with placebo for the prevention of URTIs in adults were included. P < 0.0001; I2 = 26%), the episode rate of URTIs , and the mean duration of one episode of URTI . The adverse events of probiotics were mainly mild gastrointestinal symptoms. There were no significant differences in occurrence rate of adverse effects between probiotics intervention and placebo group .  Six RCTs with 1551 participants were included. Compared with the placebo group, the probiotics intervention group significantly reduced the incidence of URTI episodes (RR: 0.77; 95% CI: 0.68 to 0.87;  Low-quality evidence provides support that probiotics have potential efficacy for preventing URTI episodes in adults. More trials are required to confirm this conclusion. Upper respiratory tract infections (URTIs) are the most common diseases, including the rhinitis, sinusitis, tonsillitis, pharyngitis, laryngitis, and common cold. Adults suffer from cold two to three times each year, and the global incidence of URTIs was estimated to be 17.2 billion in 2015 . SymptomClostridium difficile infection [Probiotics are live microorganisms that promote health benefit when consumed in adequate amounts . Studiesnfection . The effnfection , 14. Hownfection , 16. ThuParticipants with the age from 18 to 65 years, RCTs comparing probiotics with placebo for the prevention of URTIs, were included. The probiotics included various strains, forms, and dosages. Studies were excluded for the following reasons: (1) they included other probiotics in placebo; (2) participants were vaccinated or took potential immune-enhancing dietary supplements during the trial process; (3) participants had congenital or acquired immune dysfunction; (4) participants had chronic allergies; and (5) participants took part in regular high-intensity physical exercise.The primary outcomes were the incidence of URTI episodes and the number of episodes of URTIs. Secondary outcomes included the mean duration of one episode of URTI and adverse events.∗ OR sinusit∗ OR tonsillit∗ OR laryngit∗ OR pharyngit∗ OR “respiratory tract infection” OR “respiratory tract infections” OR “upper respiratory infection” OR “upper respiratory infections” OR “common cold” OR “common colds”) AND (probiot∗ OR prebiot∗ OR bifidobacterium∗ OR enterococ∗ OR Lactobacil∗ OR Lactococ∗ OR streptococ∗ OR saccharomyc∗) AND . Duplicate articles were eliminated. We screened potentially eligible trials by titles and abstracts of articles obtained from the broad search, and then, full texts of these screened trials were assessed for eligibility according to the inclusion and exclusion criteria.A systematic search of PubMed, Web of Science, Embase, and Cochrane Library was performed for studies from database inception to 14 May 2020. Language was limited to English. The following search string was applied: .Five studies reported the incidence of URTI episodes in the probiotic intervention group and placebo group , 20–23. P=0.0002; I2 = 99%; P < 0.00001). We conducted sensitivity analyses by excluding each study individually. We found that this was not significantly different with the original analyses.Five studies reported the number of episodes of URTIs or the episode rate of URTIs , 20–22. P = 0.01; I2 = 80%; P = 0.03).Only two studies , 15 coulP=0.96; I2 = 99%; P < 0.00001), which was not significantly changed by using sensitivity analyses.Five studies reported adverse events, including nausea, vomiting, flatulence, abdominal pain, diarrhea, and bloating. Most of adverse events were mild , 21, 23.It remains as an unsolved public health problem of finding effective prevention strategies for URTIs . RecentlWe excluded participants who did regular high-intensity physical exercise because previous studies suggested that high-intensity physical exercise may affect immunity . We alsoγ in blood and sIgA in the gut. In addition, natural killer (NK) cells play an important role in the prevention of URTIs. Probiotics could inhibit the reduction of NK cell activity and increase the level of salivary cortisol [The potential mechanism of probiotics in the prevention of URTIs is probably related to the modulation of the immune system. The study has shown that Lactobacillus casei Zhang can activate T-cells and B-cells and improve the levels of anti-inflammatory cytokines IL-4 and IL-10 . Zhang ecortisol . Besidescortisol , 29.This meta-analysis has some limitations. First, high heterogeneity was observed in our meta-analysis. The study population, probiotics strains, forms, and dosages varied among the included studies, which might be the reason for substantial heterogeneity. For example, we observed that the episode rate of URTIs in the B. bifidum R0071 group was lower than that in the L. helveticus R0052 group . In addiIn summary, probiotic supplementation may be an effective strategy to prevent episodes of URTI in adults. However, the quality of the evidence is low because of publication bias and substantial heterogeneity. More high-quality RCTs are required to confirm this conclusion and to assess which species of probiotics, forms, and dosages is the most efficacious in preventing URTI episodes in adults.


The data on the association between the microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) during pregnancy and risk of preeclampsia (PE) is limited.We, therefore, conducted a prospective nested case control study during Sep 2017 to Dec 2018 to examine the association between plasma TMAO measured during pregnancy and the risk of PE. Total of 17 patients diagnosed with early onset PE (EOPE), 49 with late onset PE (LOPE) and 198 healthy controls were enrolled. Blood samples were collected at 15–23 weeks gestation and time at delivery. The Logistic regression model was used to assess the odds ratio (OR) and 95% confidence interval (CI) for TMAO and risk of PE, EOPE, LOPE, mild PE, and severe PE.3 (standard deviation (SD) =45.91) and 175.01 μg/m3 (SD = 160.97), respectively. No significant spearman correlation was found between the TMAO in those two periods (p > 0.05). T2 TMAO was not significantly associated with risk of PE or risk of any PE subtypes (p > 0.05). However, TD TMAO was significant associated with risk of PE, EOPE and severe PE , 1.62, and 1.41) per 50 μg/m3 increment, respectively).We found that the mean TMAO levels of overall subjects in the second trimester (T2) and at the time of delivery (TD) were 90.39 μg/mOur study found that plasma TMAO level would alter over the course of pregnancy. The major role of TMAO in PE development might be in the accelerating process not in the initiation. Preeclampsia (PE) complicates 2–5% of pregnancies and is a major cause of maternal, fetal, and neonatal morbidity and mortality . Recent Microbes that live in the gut could produce a vast number of metabolites, many of which are dependent on the diet of the host  and absoPE shared similar mechanisms with ASCVD whereby both of them involved oxidative stress and endothelial dysfunction –15. TheyWe enrolled subjects from an ongoing prospective cohort which aimed at maternal depression screening and associated risk factors. The study was performed at the Third Xiangya Hospital of Central South University (TXYHCSU) from September 2017 until December 2020. The enrollment criteria for the cohort were singleton pregnancy, ≥20 years old, with no history of mental disease. All qualified pregnant women at 12–14 weeks gestation were recruited when they attended first prenatal visit and would be followed till 6 weeks after delivery. In the present nested case-control study, all patients in the cohort diagnosed with PE and have delivered before the end of 2018, with no history of chronic diabetes mellitus, renal disease, or other chronic cardiovascular disease were recruited in case group. We matched each PE case with 3 controls who were normotensive singleton pregnant women, without chronic diseases, with similar age (within ±3 years), and blood samples were collected. Finally, 17 patients diagnosed with early onset PE (EOPE), 49 with late onset PE (LOPE) and 198 healthy controls were enrolled.The study protocol were reviewed and approved by the Ethical and Confidentiality Committee of Central South University. Written informed consent was obtained from all participants. A face-to-face interview was conducted at baseline using a structured questionnaire to collect demographic, environmental and medical information. Enrolled subjects were followed at the following 5 time points: 15–23 weeks gestation , 24–32 weeks gestation, time at delivery (TD), 2–4 weeks and 6 weeks postpartum. At T2 and TD, 5 ml fasting blood samples were collected in tubes with heparin as anticoagulant and centrifuged at 1620 g at 4 °C for 5 min. Then, the plasma was detached and stored at − 80 °C for subsequent analysis of TMAO. Information on maternal complications was abstracted from medical charts. PE was diagnosed as de novo hypertension  after 20 weeks gestation accompanied with proteinuria (≥ 1+ on dipstick). PE was further classified as mild PE , severe PE , EOPE (diagnosed before 34 weeks gestation), or LOPE (diagnosed at or after 34 weeks gestation).We measured TMAO concentrations in Hunan Key Laboratory for Bioanalysis of Complex Matrix Samples using ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) as described by Awwad HM et al.  and the p < 0.05). Median and range were used to describe the distribution of TMAO among overall subjects. Individual change of TMAO was TD TMAO minus the T2 TMAO. The spearman correlation between the change of plasma TMAO and TMAO in T2 or TD was further estimated. Unconditional logistic regression model was used to calculate odds ratios (OR) and 95% confidence intervals (CI) for the associations between TMAO and risk of PE, EOPE, LOPE, mild PE, and severe PE, by different times. The exposure of TMAO was evaluated in two ways: 1) categorized by quartiles; 2) treated as a continuous variable, and results were presented for the change in health outcome per 50 μg/m3. Potential confounding variables included in multivariate models were year of education , active and/or passive smoking during pregnancy (yes or no), pre-pregnancy body mass index , and fetal gender . Statistical significance was all assessed at the two-tailed 0.05 level. All analyses were performed using SAS, version 9.2 .The distribution of maternal characteristics was compared between PE and control groups by Chi-squared tests. Prior to describe the distribution of TMAO concentration, the normality assumption of residuals was tested using Shapiro-Wilk test. However, the significant departures from normality were observed (p = 0.040). There were no significant differences in the distribution of pre-pregnancy BMI, exposure to smoking during pregnancy and fetal gender between preeclampsia and controls  (Table p-trend = 0.018); for EOPE 14.86 ; and for severe PE were 14.37 .With adjustment for education, smoking during pregnancy, pre-pregnancy BMI, and fetal gender, T2 TMAO was not significantly associated with risk of PE or risk of any PE subtypes (Our nested case-control study found that the maternal plasma TMAO would alter over the course of pregnancy. Maternal plasma TMAO in second trimester was not associated with PE. However, the plasma TMAO level among PE, EOPE and severe PE patients was significantly increased at the time of delivery.Circulating TMAO is formed via a two-step metaorganismal pathway , 8. SpecPlasma TMAO levels have been observed associated with risk of ASCVD –15, kidnLOPE and EOPE are two distinct phenotypes of PE with different mechanisms . Our stuStrengths and limitations should be considered when interpreting the study findings. The nested case control study with prospective design allowed us observing the TMAO variation during pregnancy in the same individual. Information on maternal complications was obtained from medical records, which minimized the potential misclassification of the outcomes. Furthermore, we have data on maternal education, smoking during pregnancy, pre-pregnancy BMI, and fetal gender which allowed adjusting for several important potential confounding factors. However, the limitation of our study should be considered when interpreting the study findings. In present study, we have only accessed the plasma TMAO in second trimester and at the time of delivery. The distribution and alteration of TMAO in other period, such as, preconception, the first trimester and postpartum need further investigation. The TMAO level was associated the various factors including diet pattern, gut microbes, liver function and FMOs genotype , 8, 22, Maternal plasma TMAO would alter over the course of pregnancy. Maternal plasma TMAO in second trimester was not associated with PE. However, the plasma TMAO level among PE patients was significantly increased at the time of delivery. The major role of TMAO in PE development was in the accelerating process not in the initiation.Additional file 1. Methods for Plasma TMAO concentration assayAdditional file 2 Table S1. Comparison on maternal plasma TAMO concentration between preeclampsia and control groups