Using a combination of in vitro and in silico approaches, we found that AQP7 changes from fully permeable to virtually closed at acidic pH, and that Tyr135 and His165 facing the extracellular environment are crucial residues for channel permeability. Moreover, instead of reducing the pore size, the protonation of key residues changes AQP7’s protein surface electrostatic charges, which, in turn, may decrease glycerol’s binding affinity to the pore, resulting in decreased permeability. In addition, since some pH-sensitive residues are located at the monomer-monomer interface, decreased permeability may result from cooperativity between AQP7’s monomers. Considering the importance of glycerol permeation via AQP7 in multiple pathophysiological conditions, this mechanism of hAQP7 pH-regulation may help the design of selective modulators targeting aquaglyceroporin-related disorders.The aquaglyceroporin AQP7, a family member of aquaporin membrane channels, facilitates the permeation of water and glycerol through cell membranes and is crucial for body lipid and energy homeostasis. Regulation of glycerol permeability via AQP7 is considered a promising therapeutic strategy towards fat-related metabolic complications. Here, we used a yeast  Aquaporins (AQPs) are a family of small membrane proteins expressed in almost every organism and tissue type and involved in the bidirectional transfer of water and small solutes across cell membranes in response to osmotic and hydrostatic pressure or concentration gradients. Mammalian AQPs AQP0-12) are grouped according to their primary structure and permeability. The aquaglyceroporin subfamily  facilitates permeation of glycerol and other small solutes in addition to water 2 are gro, and areStructural studies of AQPs from different species revealed a common homotetrameric assembly in the plasma membrane ,8, assumLike many other channels and transporters, AQPs can be subjected to regulation, either by transcriptional/translational mechanisms , proteinNotwithstanding the lack of knowledge in aquaglyceroporin regulation, a few studies have shown gating of AQP3 by pH, copper, and nickel ,30,31,322]Cl —a gold(III) compound reported by us as a potent inhibitor of human aquaglyceroporins, AQP3 and AQP7, in their native expression systems Cl , on hAQP3 and hAQP7, expressed in human erythrocytes and mammalian cultured cells, respectively [Pf and PGly, respectively) (Gly inhibition by Auphen resulted in an inhibitory concentration (IC50) of 12.95 ± 0.35 μmol/L (Previous studies by our group have detected the strong and selective inhibitory effect of a gold(III) coordination compound, Auphen [Au(phen)Clectively ,35,59,60ctively) G. Additi5 μmol/L H, in lin14C-glycerol uptake in the pH range of 5.5 to 8.0 and, although glycerol uptake was significantly reduced at pH 5.0, no meaningful physiological relevance was attributed to this effect [hAQP7 pH-dependency has been previously evaluated in Madin-Darby canine kidney (MDCKII) cells by measuring s effect . A recens effect . This prPf and PGly were evaluated by varying external pH from 7.4 to 5.0  corroborate the proposed channel regulation upon extracellular acidification  [To gain further insights into hAQP7’s pH regulation, we used a molecular modelling approach previously developed by our group . A homolde 1FX8)  at pH 5.de 1FX8) , from snde 1FX8) ), as it PGly  was calculated as described in the experimental section and based on reference [Dw evaluated at pH 7.0 was significantly higher than at pH 5.0.For water permeation, longer simulations (20 ns) were run at both pH values, without any pull code. The movement of water molecules was evaluated using a script developed by us and based on previous work . For thieference , simulatIn conclusion, although the MD and stopped-flow spectroscopy data may not be directly comparable since the computational studies are not performed in osmotic conditions, our in silico and in vitro data are in accordance and demonstrate that glycerol and water permeation by hAQP7 are pH-regulated.Previous research on human AQP3 using site-directed mutagenesis  demonstrTo investigate the mechanism of pH regulation, sequence alignment of hAQP3 and hAQP7 and superposition of the two homology models (the homology model of hAQP3 was prepared as described in reference ) showed Detailed analysis of the protonation sites at both pH values revealed that only four residues are protonated at pH 5.0, which are not at pH 7.0 : On the Thus, to determine the importance of His165, His140, and Tyr135, the selected amino acids were substituted by alanine residues (Ala) and, due to the proposed role of His165 on AQP3 pH gating , double a from 5.85 ± 0.01 to 6.30 ± 0.02. Interestingly, the mutants, Y135A and Y135A+H165A, rendered the protein almost inactive, reducing the maximal PGly to 10% of the observed for WT . Protonation of residues in the pore entrance (such as His165) at pH 5.0 may alter the hydrogen bond network, also affecting the residues responsible for glycerol passage. Therefore, we postulate that the contribution of His165 and Tyr135 to AQP7 pore permeability is based on a network of hydrogen bonds that dynamically readapts to changes in external acidification. In order to support this hypothesis, we prepared new homology models for all the mutants. The models were generated based on mutated sequences, refined and subsequently protonated at pH 5.0 and pH 7.0, as performed for the WT model. Each model was further analysed for pore size .Ka of water and glycerol permeation. This effect may be due to changes in the electrostatic surface of the mutants and also under different pH conditions. In fact, electric fields within protein pores have been identified as barriers to solute passage [The main difference in size was found for the double mutant, Y135A+H165A A, which  passage . Therefo passage  plugin i passage , and areOverall, the main observable difference is that the intracellular electrostatic surface of all models appears to be much more negative (shown in red colour) at pH 7.0 than at pH 5.0 B. At pH The most marked changes were observed for the H165A and Y135A single and double mutants, pointing to the crucial involvement of these two residues in pH gating and glycerol permeability. Remarkably, as observed for glycerol permeability, Y135A mutation has the strongest effect on the double mutant’s surface, also shown to have the same protonated residues as the single mutant . SimilarAnalysis of protonation at both pH values reveals that the mutations do not induce changes in protonation of the intracellular residues, Glu40 and Glu202. However, at pH 5.0, the other protonable intracellular site, His92, is deprotonated in the mutants, H165A and H140A+H165A, and partially deprotonated in the H140A mutant. Changes in protonation of this residue do not appear to affect the permeability at the lowest pH of any of the mutants, indicating that deprotonation of His92 is not a major player in the permeation mechanism. Despite the fact that none of the mutants shows protonation changes of intracellular residues at pH 7.0, it is possible to observe that the intracellular charge distribution is altered in Y135A, H165A, and in the double mutants: The electrostatic surface of the channel is more negative than WT. This indicates that changes in extracellular residues affect the overall electrostatic surface of the protein, including the intracellular surface side. Thus, changes in extracellular pH may be able to affect the intracellular charge distribution, likely by altering the H-bond network of the protein, thus, affecting the permeability.a differences appear to be due to the changes in the electrostatic protein surface, caused by the mutations, which can alter the protein’s affinity for substrates. Yet, mutating these residues does not render the protein pH-insensitive. Interestingly, beside the residues mutated in this study, most of the protonable amino acids composing the protein surface are lysines and arginines, with pKa above 10, making them very unlikely players in pH-sensitivity in a physiological range. A likely scenario is that the protein rearranges its H-bonding network according to the newly inserted residues, redistributing the electrostatic potential and changing affinity for substrates, shifting the role of pH-sensitivity to other nearby residues. The modulation of transport through electrostatic is quite typical of biological channels and has been previously observed for different porins [Moreover, the pKt porins .Aquaglyceroporins have emerging roles in energy metabolism and adipose tissue homeostasis and have been implicated in obesity and metabolic-related complications, such as metabolic syndrome (reviewed in ). AQP7 iThis study allowed disclosing the mechanism of pH regulation of human AQP7 activity at a molecular level, showing that this channel changes from a fully active to nearly inactive state when the pH drops from 7.5 to 5.0. Using in vitro and in silico approaches, we found that residues, Tyr135 and His165, facing the extracellular environment are crucial for channel permeability and we hypothesize that hAQP7’s lack of glycerol permeability may be due to changes in the overall hydrogen bond network rather than a truly open/closed pore mechanism of gating. Furthermore, due to the position of the key pH sensitive residues at the monomer-monomer interface, it is possible that a cooperativity change between the AQPs monomers within the tetramer structure affects permeability, as observed for other aquaporins ,68.+ released into the adipose micro-circulation create local acidic microenvironments, causing metabolic acidosis [According to our experimental data, the maximum of protein function is achieved at the pH range of 6.7 to 7.5. In fact, knowing that hAQP7 activity is crucial for glycerol efflux from adipose tissue to be used for gluconeogenesis in the liver, full activity of glycerol channel permeation at a mammalian physiological pH range would be expected. The observation that hAQP7 still retains 50% permeability above pH 6.00 is very interesting. Notably, during intracellular lipolysis, breakage of triglycerides into glycerol and free fatty acids (FFA) have been shown to release protons, acidifying the intracellular pH in a stoichiometric manner ,70. Howeacidosis ,70. In facidosis  and AQP1acidosis . An oppoacidosis . Thus, iacidosis , kidney acidosis , endocriacidosis ,78, capiacidosis ,80, and acidosis ,82, wheracidosis , suggestIn addition, expression of AQP7 in testis  and in hOverall, considering the importance of glycerol in multiple vital physiological processes, regulation of its permeation across hydrophobic cell membranes via AQPs may be crucial for energy homeostasis, with consequences for cell proliferation, adaptation, and survival. Thus, the mechanisms involved in the regulation of hAQP7 by acidification provide new hints for the design of new molecules targeting aquaglyceroporins and opens new perspectives for the treatment of disorders affected by glycerol unbalance.


Pelvic organ prolapse (POP) is the downward descent of vaginal walls, affecting the health of 32% to 76% female patients. Conservative interventions are considered as priority before seeking help from surgery. We plan to make the systematic review to assess the effectiveness of conservative intervention for adult women with POP.Studies will be searched in PubMed, EMBASE, and the Cochrane Library from inception to July 2017. Primary outcomes are specific POP-related symptom, severity of prolapse, Prolapse-related, and general health-related quality of life and other non-POP-specific symptoms.The data will be synthesized if possible using MD, SMD or RR. A descriptive analysis will be made if the data cannot be synthesized.The systematic review might provide solid evidence for the treatment of POP by conservative intervention. However, the relationship between anatomical change and symptoms is poor, especially in milder stages where typical symptoms may not even appear. In clinic, appropriately 6% patients have bulging feelings in vagina while up to 32% to 76% female patients accompany with loss support of vagina or uterus.,8Pelvic organ prolapse (POP), defined by anatomical change, is the descent of 1 or more of the anterior vaginal wall, posterior vaginal wall, the uterus (cervix), or the apex of the vagina . Conservative treatments tend to be considered as priority before patients seek further help from surgery, especially for those at mild-degree of POP, wishing for child, of frail body or unsuitable for surgery. In IUGA/ICS report, conservative interventions for POP are defined as non-surgical and non-pharmacological treatments, mainly including lifestyle intervention, devices and physical therapies. Lifestyle interventions mainly refer to weight loss and avoiding heavy lifting or coughing. Most frequently used devices at present are support pessaries and space filling pessaries. Physical therapies mainly include a variety of physical activity, cognitive behavioral therapy, pelvic floor muscle training, bladder training, bowel habit training, coordination training, biofeedback, electrical muscle stimulation, and others.Treatments might not be provided to POP patients until the symptoms become bothersome. Damage to the integrity of muscular, connective and nerve structures could bring threat to the normal support, which might be induced by childbirth, advancing age, and increasing body-mass index, with vaginal childbirth as the most consistent risk factor. Conservative treatments may reduce intro-abdominal pressure, build up muscle strength and prevent organ from downwards.,12Pelvic organs are mainly supported by the levator ani muscle complex and connective tissue.,13 were published in 2011 and 2013 to assess the effects of conservative treatments for POP. Conclusion was drawn that pelvic floor muscle training, as a physical therapy, was supported by some rigorous trial as effective, but evidence remained limited and no good quality evidence supported other conservative treatments. Since several other RCTs in this area have been published ever since, and innovative therapies may spring up, we plan to make this systematic review to reassess the effects of conservative interventions for adult women with POP.Two systematic reviews2The protocol of the systematic review has been registered on PROSPERO with the number of CRD42019136277 and reported under the guideline of Preferred Reporting Items for Systematic Reviews and Meta-Analyses protocols (PRISMA-P).2.12.1.1We will only consider randomized controlled trials. Quasi-RCTs, cross-over RCTs and cluster RCTs will all be excluded.2.1.2Female patients at age over 18 years will be included;Patients diagnosed as POP will be included, without limit of diagnostic criteria or prolapse segments. They may suffer from anterior vaginal wall prolapse, posterior vaginal wall prolapse, prolapse of the apical segment of the vagina or their combination.Studied involving a subset of patients at stage 0 defined by Pelvic Organ Prolapse Quantification (POP-Q) will be excluded;Studies involving only a subset of POP participants will be excluded.2.2Intervention group of the eligible trial must include 1 conservative treatment or their combinations to treat POP, which may include:Lifestyle interventions, like weight loss, avoiding heavy lifting, and avoiding coughing, etc.Devices, like support pessaries and space filling pessaries, etc.Physical therapies, like physical activity, cognitive behavioral therapy, pelvic floor muscle training, bladder training, bowel habit training, coordination training, biofeedback, electrical muscle stimulation, etc.Control group can be inactive intervention, such as standard care or a waiting list, or an active intervention, such as a different kind of conservative treatment or a combination of several different conservative treatment.RCTs with surgery or pharmacological treatment existed in any arm will be excluded. RCTs comparing an intervention with a different variant of the same intervention will be excluded.2.2.12.2.1.1Specific POP-related symptom (e.g. Pelvic Organ Prolapse Symptom Score)2.2.1.2Severity of prolapse ,Prolapse-related and general health-related quality of life,Other non-POP-specific symptoms .2.3We will conduct electronic searches in 3 databases from their inception date to July 2019, including PubMed, EMBASE, and the Cochrane Library.Search strategy will be drawn up in accordance with the Cochrane handbook guideline. Research strategy of PubMed is presented in Table 2.42.4.1The articles searched from databases will be imported into EndNote X9 software, with the help of which duplicated ones are removed. Two authors (YS and HC) will screen the titles and abstracts independently to select potentially eligible articles and then go through the full text of those articles to exclude ineligible ones according to predetermined criteria. During the process, any disagreement between the 2 authors will be discussed with ZL to make a final decision. The screening process is presented in Figure 2.4.2Two authors (RJ and JY) will extract the data separately, and a data extraction form will be applied in collection. Before actual extraction, we will put the data of 2 or 3 articles into the form to make sure the absence of obvious distinctions.The data will be extracted included: administration details of the article, methods of the trial, participants enrolled, interventions applied, controls used, primary and secondary outcomes, measurements, and results, etc. Any disagreement during the process will be discussed with ZL.2.4.3The risk of bias will be assessed by 2 independent authors (YS and XZ) in accordance with Cochrane Collaboration Risk of Bias Tool in the domains of random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias. Each trial will be evaluated as low risk, high risk, and unclear risk, based on the conduct process of the trials. Any disagreement during the process will be discussed with ZL.2.4.4If results of the same trial were multiply published in several journals, the article will be excluded. If different outcomes of the same trial were published in different journals, the data will be extracted and integrated into one.2.4.5For trials with incomplete or unclear data reported, the correspondent author of the article will be contact by phone or E-mail to seek for possible help. An intention-to-treat analysis will be performed when possible and a sensitive analysis will be made to confirm the consistence.2.4.6I2 statistic. The studies with an I2 value less than 50% will not be regarded as of heterogeneity and the data extracted will be synthesized; while those with an I2 value more than 50% will be considered as of heterogeneity, the reasons of which might be sought by subgroup analysis or from clinical and design aspects.Before synthesizing the data, the heterogeneity of the studies will be tested by 2.4.7Funnel plots will be made to detect whether there is report bias if over 10 studies are meta-analyzed under 1 outcome.2.4.8I2 value is less than 50%, and random-effects model, if I2 value is between 50% and 75%. When I2 value exceeds 75%, a descriptive analysis rather than a meta-analysis will be made, and we will try to explore the reasons behind by subgroup analysis, or from clinical and design aspects.The data will be synthesized in RevMan V.5.3 if possible. MD or SMD will be used to analysis continuous data, while RR, dichotomous data. Fixed-effects model will be applied if 2.4.9RevMan V.5.3 will be used to analyze the data extracted. Mean difference (MD) or standard MD (SMD) with 95% CI will be used to analysis continuous data, while risk ratio (RR) with 95% CI will be used to analyze dichotomous data.2.4.10Since conservative treatments are consisted of several different interventions, we plan to make subgroup analysis according to different therapies. Subgroup analysis might also need to be made to seek the cause of heterogeneity, if necessary.2.4.11We will perform sensitivity analysis to test the robustness of review process if needed. Studies with high or unclear risk of bias in allocation concealment domain will be included and excluded studies with if needed, and the results will be discussed accordingly.2.4.12Quality of the evidence will be judged according to Grading of Recommendations Assessment, Development and Evaluation working group methodology in the domains of risk of bias, consistency, directness, precision, publication bias, and additional points, based on which the assessments will be classified as high, moderate, low, or very low.3 The prolapse of vaginal segments is related to different pelvic organs, like bladder, rectum, or uterus, etc. Previous research indicated that anterior vaginal wall prolapse occurred the most frequently in clinic, followed by posterior vaginal wall and apical prolapse,,16 usually in combination. Besides, the cost of POP put large burden to the healthcare system. Several systematic reviews published before to evaluate conservative treatments for POP years ago, and results indicated that the effectiveness and safety of them needed to be supported by more trials.Though POP rarely cause mortality in clinic, the symptoms of urogenital and gastrointestinal systems can be rather bothersome to female patients and greatly reduce their quality of life.Since conservative treatment is defined as non-pharmacological and non-surgical treatment, and consist of a variety of therapies, the search strategy will be focus different names and forms of the disease, rather than the intervention itself. Ineligible trials will be excluded after research by hands.There exists limitation in this systematic review in that some articles may be missed because only articles in English will be enrolled. Since conservative treatments included a variety of specific therapies, it will be a big challenge as how to categorize them appropriately, synthesize the data and report the results.Great appreciation and respects to Simi Shao worked in National Library of China, for her patience and guidance in the development of search strategies.ZL and YS conceived the study. YS, ZL, HC and YL developed the search strategies. RJ, JY and XZ identified the key words and screened them in the three databases to simplify the search strategies. YS wrote the first draft and revised by ZL, HC and YL. All authors approved the publication of the protocol. The funder has no role in the design and conduction of the systematic review.Zhishun Liu orcid: 0000-0001-7570-8917.


Differences in game-related statistics among the continents were analyzed by an analysis of variance (ANOVA) with effect size statistics. Game-related statistics that discriminate among the continents were assessed by discriminant analysis. The ANOVA yielded significant F-values for 13 of 16 variables and large effect size differences for 10 of 16 variables. The discriminant analysis yielded three significant functions. The Asian championship was discriminated from the other continental championships by ball possessions, defensive rebounds, assists, and fouls. The African championship was discriminated from the European championship by ball possessions, successful 3-point field goals, unsuccessful free throws, and turnovers, and from the American championship by ball possessions, unsuccessful 2-point field goals, successful 3-point field goals, successful free throws, and assists. The results of this study suggest that U18 women’s basketball games are played differently in each continent.The purposes of this study were (a) to evaluate differences in basketball game-related statistics among continental championships for under-18 (U18) women, and (b) to identify game-related statistics that discriminate among the continents. The analysis was performed on all matches ( Assessing differences in performance profiles among regions of the world is one of the recent topics in the field of basketball performance analysis, and a series of papers have been published in academic journals in recent years ,2,3,4,5.Although differences in basketball performance profiles among continental championships have been studied in different categories , continental championships for junior women have yet to be studied. If there are no age and sex interactions in performance profiles, performance profiles of junior women can be inferred from those of men, women, and junior men. However, age and sex interactions in performance profiles have been demonstrated in previous researches ,7. SampaFrom a practical point of view, the knowledge about regional differences in performance profiles among continental championships for junior women would be useful when players and coaches of junior women’s national teams prepare for matches in international tournaments. In addition, this study would be valuable as part of scientific knowledge in the field of long-term development of female basketball players. Although studies on women’s basketball have been increasing ,11,12,13This study analyzed all 136 matches in the four  U18 women’s continental championships of 2016 . All datThis study did not assess inter-rater reliability of the official box score. However, the official box score has been considered as a reliable data source in basketball researches ,16. Thisp ≤ 0.05. Differences in game-related statistics among the continents were analyzed by one-way ANOVA with Bonferroni-corrected post hoc comparisons. The magnitude of effect sizes for post hoc comparisons was assessed by Cohen’s d statistic  [The statistical software R   was usedct size) . Discrimct size) ,26, and F-values for points scored, point difference, ball possessions, unsuccessful 2-point field goals, successful 3-point field goals, successful and unsuccessful free throws, defensive and offensive rebounds, assists, steals, turnovers, and fouls  to evaluate differences in basketball game-related statistics among continental championships for U18 women, and (b) to identify game-related statistics that discriminate among the continents. The ANOVA yielded significant Classification accuracy for the U18 women’s Asian championship was the highest among the four continental championships. Ball possessions, assists, and fouls greatly contributed to discriminating the U18 women’s Asian championship from the other continental championships. The number of ball possessions in the U18 women’s Asian championship was the highest among the four continental championships, whereas the numbers of assists and fouls were the lowest among the four continental championships. A high number of ball possessions indicates that the pace of the game was relatively fast, and a low number of assists indicates that most of the points were scored by individual plays. Interestingly, these performance profiles have similarly been observed in Asian championships for senior  and junid = 1.02; vs. America, d = 1.00; vs. Asia, d = 0.91) and ball possessions . The mean point difference and the number of ball possessions in the U18 women’s European championship were the lowest among the four continental championships. These results indicate that the U18 women’s European championship was characterized by a relatively slow pace and closely contested games. These characteristics have also been observed in European championships for men [Classification accuracy for the U18 women’s European championship was the second highest among the four continental championships. Point difference and ball possessions distinguished the U18 women’s European championship from the other continental championships. Large effect size differences were found in all pairwise comparisons between the U18 women’s European championship and the other continental championships for point difference . This result was in line with previous observations in American championships for senior women (43.8%) [7.5%. ThiThis study used the same method as in previous studies on continental championships for men , women , and U18This study found large effect size differences in 10 of 16 game-related statistics and identified game-related statistics that discriminate among continental championships for U18 women. The Asian championship was discriminated from the other continental championships by ball possessions, defensive rebounds, assists, and fouls. The African championship was discriminated from the European championship by ball possessions, successful 3-point field goals, unsuccessful free throws, and turnovers, and from the American championship by ball possessions, unsuccessful 2-point field goals, successful 3-point field goals, successful free throws, and assists.From a practical point of view, coaches of U18 women’s national teams can use this study to gain information on the opposing team based on the continent where the opposing team belongs. In addition, the results of this study would be valuable as part of scientific knowledge in the field of long-term development of female basketball players because studies on junior women are limited.


Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cronobacter sakazakii, Esherichia coli, Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococccus aureus, Vibrio spp. and Yersinia enterocolitica), viruses (Hepatitis A and Noroviruses) and parasites , together with some important outbreaks, are reviewed. Food safety management systems based on to classical hazard-based approach has been proved to be inefficient, and risk-based food safety approach is now suggested from leading researchers and organizations. In this context, a food safety management system should be designed in a way to estimate the risks to human health from food consumption and to identify, select and implement mitigation strategies in order to control and reduce these risks. In addition, the application of suitable food safety education programs for all involved people in the production and consumption of foods is suggested.Foodborne pathogens are causing a great number of diseases with significant effects on human health and economy. The characteristics of the most common pathogenic bacteria ( Thus, foodborne illness is generally classified into: (a) foodborne infection and (b) foodborne intoxication. In foodborne infections, since an incubation period is usually involved, the time from ingestion until symptoms occur is much longer than that of foodborne intoxications.More than 200 different food-borne diseases have been identified Salmonella spp.  and Campylobacter spp. , even though the reporting of outbreaks involving these agents has been declining over the recent years. Bacterial toxins ranked second among the causative agents in food- and waterborne outbreaks and were reported in 19.5% of the total outbreaks while viruses, which were the agents most frequently reported in 2014, accounted for 9.2% of total outbreaks in 2015 In the European Union (EU) for the year 2015, 26 member states reported a total of 4,362 food-borne outbreaks, including waterborne outbreaks. Overall, these outbreaks caused 45,874 cases of illness (209 more than 2014), 3,892 hospitalisations  and 17 deaths (10 less than 2014) Salmonella spp. in eggs was associated with the highest number of reported foodborne outbreaks and was among the top-5 food-pathogen combinations in terms of the overall number of cases of illness and hospitalisations in outbreaks. However, the number of reported outbreaks caused by Salmonella spp. and associated with the consumption of “eggs and egg products” has been decreasing in the last 5 years Salmonella spp. was the most common agent reported in private households, whereas, “bacterial toxins other than Clostridium botulinum toxins”, viruses and other causative agents were more frequently reported in public settings such as canteens, workplace catering, restaurants and pubs The implicated food vehicles were mostly of animal origin, in particular eggs and egg products and pig meat , broiler meat (9%) and cheese (8%) followed by fish and fish products (7%), milk and dairy products (5%), bovine meat (4%) and crustaceans (3%) The characteristics of the most important foodborne pathogens, the illnesses they cause, together with some of the most important outbreaks they have been implicated are studied in this review.2.Clostridium botulinum, C. perfringens, Bacillus subtilus, Bacillus cereus) Staphylococcus aureus, Clostridium botulinum). Most pathogens are mesophilic with optimal growth temperature range from 20 °C to 45 °C. However, certain foodborne pathogens (i.e. psychrotrophs), such as Listeria monocytogenes, and Yersinia enterocolitica are capable of growth under refrigerated conditions or temperatures less than 10 °C Bacteria are the most common cause of foodborne diseases and exist in a variety of shapes, types and properties. Some pathogenic bacteria are capable of spore formation and thus, highly heat-resistant  and the diarrhoeal one, causing two types of illness. The emetic syndrome is caused by emetic toxin produced by the bacteria during the growth phase in the food. The diarrhoeal syndrome is caused by diarrhoeal toxins produced during growth of the bacteria in the small intestine. The rapid onset of the emetic type is characterized by nausea and vomiting while the late onset of the diarrheal type is characterized by diarrhea and abdominal pain. Both syndromes  are a result of B. cereus endospores surviving the cooking process, after which germination and subsequent proliferation of vegetative cells occurs at some point during storage. Foods that are frequently implicated in B. cereus diarrheic food poisoning include meat products, soups, vegetables, puddings, sauces, milk and milk products B. cereus emetic food poisoning include fried and cooked rice, pasta, noodles, and pastry B. cereus is commonly present in food production environments and then spreading to all kinds of foods. They produce a range of virulence factors that may cause unpleasant disease in humans when present in food or the gastrointestinal tract and it is one of the major foodborne pathogenic bacteria, although in most cases disease is mild and of short duration Due to the formation of adhesive endospores, B. cereus is very similar to Bacillus anthracis and that some strains have plasmids resembling the toxin plasmids of Bacillus anthracis. 310 genomes have been completed up to now according to the data retrieved from NCBI, 2017. The median total length of the genome is 5.6626 Mb Genetic and genomic analyses have revealed that B. cereus related food poisoning is not a notifiable disease in most countries; therefore, incidence data is limited. It is recognized that there may be significant under reporting of B. cereus illness due to the generally mild, short duration and self-limiting symptoms, however, fatal incidences have been reported B. cereus caused 0.7% of foodborne illness among the 31 major pathogens in the US B. cereus outbreaks B. cereus outbreaks were associated with meat or poultry dishes. Meat or poultry dishes were cooked and served immediately (50%), roasted (33%), or part of liquid or semisolid mixtures (17%) In a study for B. cereusB. cereus was isolated from basmati rice and fecal specimens. Poor food handling and storage was most probably the cause of the outbreak In an outbreak at a birthday party in Bari, Italy, the characteristics were consistent with available reports on foodborne outbreaks caused by B. cereusB. cereus was reported as a major causative agent of foodborne illness in the Netherlands in 2006 (causing 5.4% of the foodborne outbreaks) and in Norway in 2000 (causing 32% of foodborne outbreaks) B. cereus as the causative agent At a college sport day in Thailand, 470 individuals were ill with vomiting, nausea, and abdominal pain; approximately half of the individuals reported diarrhea 2.2.Campylobacter spp. are members of the family Campylobacteriaceae and Campylobacterjejuni is one of the most common causes of diarrheal illness. C. jejuni is responsible for approximately 850,000 illnesses, 8,500 hospitalizations, and 76 deaths in the US each year C. jejuni can colonize the intestines of both mammals and birds, and transmission to humans occurs via contaminated food products. This organism can invade the epithelial layer by first attaching to epithelial cells, then penetrating through them. Diarrhea results from damage to the epithelial cells. Systemic infections can also occur causing more severe illnesses Campylobacter spp. are small (0.2–0.9 µm wide and 0.2–5.0 µm long), spiral formed, Gram-negative bacteria with 18 species, six sub-species and two biovars Campylobacter genomes are relatively unstable; several mechanisms that may lead to this genetic instability have been proposed, including bacteriophage activity, DNA recombination and transformation 2 and approximately 5% O2. The species pathogenic for man also have a rather narrow temperature range for growth with a maximum temperature of ∼46 °C and a minimum of 30 °C. These are classified as thermophilic campylobacters In 2015, Campylobacter continued to be the most commonly reported gastrointestinal bacterial pathogen in humans in the EU and has been so since 2005 Campylobacter spp. are part of the normal intestinal flora of a wide variety of healthy domestic and wild animals, including cattle, sheep, goats, pigs, chickens, ducks, geese, wild birds, dogs, cats, rodents, and marine mammals, and are often found associated with bodies of water such as water troughs and streams. Most cases of campylobacteriosis are associated with eating raw or undercooked poultry meat, unpasteurized milk, contaminated water, or from cross-contamination of other foods by these items. All animals used for food can be campylobacter-positive as can many companion species (domestic pets). Samples from the natural environment, such as groundwater, will also frequently contain these pathogens When stressed, campylobacters enter a “viable but non-culturable state”, characterized by uptake of amino acids and maintenance of an intact outer membrane but inability to grow on selective media; such organisms, however, can be transmitted to animals Campylobacter spp. outbreaks associated with consumption of poultry liver pâtè have been described, especially in the UK In June 2012, 44 persons who attended a wedding reception in Sweden became ill Campylobacter spp. was associated with the consumption of raw milk C. jejuni was isolated in 50 of 88 raw milk samples in New Zealand after a gastrointestinal illness among children in two different camp sites C. jejuni enteritis involving more than 500 participants in a jogging rally in Switzerland, with an attack rate of over 75%. An outbreak of C. jejuni enteritis followed the consumption of unpasteurized milk at an attack rate of around 50%; there were cases in all age groups, with the highest number in the 1 to 10 year old group C. coli was isolated from a 9-year-old British boy with persistent diarrhea, whose family had consumed raw goat's milk from a local farm. C. jejuni and E. coli were found in the feces of goats from the farm, and C. jejuni was identified in samples of bulk milk A serious outbreak of C. jejuni infection, were identified in 235 eligible respondents C. jejuni diversity in epidemiologically related human and food isolates recovered during outbreaks linked to poultry liver In October 2013, public health authorities in Australia were notified of a suspected outbreak of gastroenteritis in students and guests following a catered function at a university residential college; a total of 56 cases of gastroenteritis, including seven laboratory-confirmed cases of 2.3.Clostridium spp. are spore-forming bacteria, members of the family Bacillaceae and includes obligately anaerobic or aerotolerant, sporeforming rods that do not form spores in the presence of air and, at least in early stages of growth, are usually Gram-positive. In most species, vegetative cells appear as straight or curved rods, varying from short coccoid rods to long filamentous forms with rounded, tapered, or blunt ends, that occur singly, in pairs, or in various chain lengths Cl. botulinum are motile by means of peritrichous flagella and produce botulinum neurotoxins, the most lethal poison known. There are seven types of botulinum neurotoxin, A through G, based on the antigenic specificity of the toxin produced by each strain Cl. botulinum spores.Mollicutes (Eubacteria) have evolved regressively  from gram-positive clostridia-like ancestors with a low GC content in DNA. Several species of clostridia  are known opportunistic toxin-producing pathogens in animals and humans Cl. botulinum that produce type A toxin. This toxin finds its use in various applications requiring neuroparalitic intervention, including cosmetology (Botox®). 177 genomes have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 3.898 Mb From the evolutionary perspective, clostridia are considered to be the most ancient bacteria. It is believed that present day Cl. botulinum is present in soils, freshwater, marine sediments, and the intestinal tracts of animals. Food sources commonly sampled include primarily honey, which should not be fed to infants less than 1 year of age, as well as fish, meats, vegetables, and infant foods. A variety of foods, such as canned corn, peppers, green beans, soups, beets, asparagus, mushrooms, ripe olives, spinach, tuna fish, chicken and chicken livers, liver pate, luncheon meats, ham, sausage, stuffed eggplant, lobster, and smoked and salted fish have been associated with botulinum toxin Traditionally, foodborne botulism has been associated with underprocessed and abused sausages or home canned foods; however, in recent years botulism has been acquired through the consumption of contaminated foods such as potato salad, sauteed onions, garlic sauce, cheese, yogurt, bean paste, and olives.Cl. botulinum neurotoxin has not been determined, but from a human health and food safety standpoint, there should be no tolerance either for the neurotoxin itself or for conditions allowing growth of the organism in foods Symptoms of botulinum neurotoxin ingestion appear 12–36 h after consumption of contaminated food and initially may include nausea and vomiting . HoweverBotulinum neurotoxin is synthesized during cellular growth and is subsequently released during cell lysis, where proteolytic cleavage activates the molecule Cl. botulinum Group I (proteolytic Cl. botulinum) and Cl. botulinum Group II (non-proteolytic Cl. botulinum). Two different approaches were used to study genomics in these bacteria; comparative whole genome microarrays and direct comparison of complete genome DNA sequences Recent developments in whole genome sequencing have made a substantial contribution to understanding the genomes, neurotoxins and biology of Cl. botulinum Group I. For example, a large outbreak in Thailand in 2006 (209 cases) was associated with consumption of inadequately home-canned bamboo shoots The failure to effectively apply the botulinum cook (121 °C for 3 min) to canned or bottled foods has led to many outbreaks of foodborne botulism associated with 2.4.Clostridium perfringens, previously known as Clostridium welchii, belongs to the family Bacillaceae and is an important cause of foodborne disease. They are nonmotile, encapsulated rod-shaped cells that produce protein toxins and form spores resistant to various environmental stresses such as radiation, desiccation, and heat wa, of 0.93, a sodium chloride concentration less than 5–8% depending on the strain, and a pH of 5.0–9.0, although 6.0–7.2 is preferred Cl. perfringens are the most prevalent Clostridium species found in human clinical specimens, excluding faeces, and has been implicated in simple wound infections to myonecrosis, clostridial cellulitis, intra-abdominal sepsis, gangrenous cholecystitis, postabortion infection, intravascular hemolysis, bacteremia, pneumonia, thoracic and subdural empyema, and brain abscesses Cl. perfringens are estimated to be the second most common bacterial causes of foodborne illness in the US, causing one million illnesses each year Cl. perfringens outbreaks to the US CDC through the Foodborne Disease Outbreak Surveillance System. From 1998 to 2010, 289 confirmed outbreaks of Cl. perfringens illness were reported with 15,208 illnesses, 83 hospitalizations, and eight deaths Cl. perfringens occur regularly, are often large, and can cause substantial morbidity yet are preventable if contamination of raw meat and poultry products is prevented at the farm or slaughterhouse or, after contamination, if these products are properly handled and prepared, particularly in restaurants and catering facilities Foodborne illness almost always is a result of temperature abuse, and in many instances, the food vehicle has been improperly cooked meat or meat product that has been left to cook and/or cool too slowly or has undergone insufficient reheating, allowing surviving spores to germinate leading to vegetative cell proliferation. After ingestion and an incubation period of 7–30 h, symptoms typically include cramping and abdominal pain, although nausea and vomiting may also ensue, persisting for 24–48 h Cl. perfringens have been identified (A through E), and all produce an alpha-toxin (phospholipase) that plays a role in myonecrosis Cl. perfringens are a result of type A infection after the ingestion of highly contaminated foods with greater than 106–107 viable vegetative cells, which undergo sporulation in the small intestine and produce enterotoxin Five toxin-producing types of Cl. perfringens occurred in a care home and fifteen residents reported illness. The likely cause was consumption of mince and vegetable pie and/or gravy Cl. perfringens enterotoxin gene and four samples were indistinguishable by fluorescent amplified fragment length polymorphism, indicating a likely common source An outbreak of 2.5.Cronobacter consists of a diverse group of Gram-negative bacilli and comprises seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimentiThe genus Cr. sakazakii, formerly Enterobacter sakazakii, is associated with infant septicemia, meningitis, and necrotizing enterocolitis. Originally isolated from powdered formula, it has also been shown to compartmentalize cerebral ventricles and cause brain abcesses in neonates. This species produces a yellow pigment when grown at 30 °C, but this fades at 37 °C Among these, Cronobacter spp. have been detected in this wide assortment of foods, only contaminated powdered infant formula has been linked epidemiologically with infant infections and outbreaks caused by Cr. sakazakiiInfections in elderly and immunocompromised adults have also been reported 2.6.Escherichia coli is a Gram-negative, non-spore forming rod. It may or may not be mobile; some rods are flagellated and some are not E. coli comprise a large and diverse group of bacteria. Most strains of E. coli are harmless; other strains have acquired characteristics, such as the production of toxins, which make them pathogenic to humans E. coli (pathovars or pathotypes) cause much morbidity and mortality worldwide; many of these pathotypes are a major public health concern as they have low infectious doses and are transmitted through ubiquitous mediums, including food and water E. coli occurs when food or water that is contaminated with feces of infected humans or animals is consumed. Contamination of animal products often occurs during the slaughter and processing of animals E. coli can survive for long periods in the environment and can proliferate in vegetables and other foods.E. coli have been categorized into six groups according to the pathogenic mechanism: (1) Enteropathogenic E. coli (EPEC); (2) Enterohemorrhagic E. coli ; (3) Enterotoxigenic E. coli (ETEC); (4) Enteroaggregative E. coli (EAggEC); (5) Enteroinvasive E. coli (EIEC); and (6) Attaching and Effacing E. coli (A/EEC) Pathogenic E. coli O157:H7 outbreaks. Food producers must report the presence of E. coli O157:H7 to health authorities STEC infection can cause episodes of mild to severe diarrhea, and 5–10% of infections develop into Hemolytic Uremic Syndrome (HUS)—a severe complication marked by profuse bleeding that can lead to kidney failure and death. STEC strain O157:H7 is estimated to cause 63,000 illnesses, 2,100 hospitalizations, and 20 deaths each year E. coli, one that expressed O-antigen 157 and H-antigen 7, that had not previously been recognized as a pathogen E. coli that produce toxins which are similar to Shiga toxin of Shigella dysenteriae and distinct from previously described E. coli heat-stable and heat-labile toxins. As data were accumulating on the role of E. coli O157:H7 as a pathogen, parallel work in Canada was uncovering high rates of infection with this and other Shiga toxin-producing E. coli in patients with the HUS E. coli O157:H7 is the cause of 85–95% of cases of hemolytic uremic syndrome in North America, and that non-O157 Shiga toxin-producing E. coli are responsible for another 5–15% In 1982, an investigation by the CDC of two outbreaks of severe bloody diarrhea, associated with the same fast-food restaurant chain, led to the identification of a strain of E. coli O104:H4 caused the second largest and the deadliest outbreak of E. coli-associated disease ever recorded. Between May 21 and July 22, 2011, more than 4,000 people became ill in 16 countries, and 50 individuals died E. coli and the capacity to produce Shiga toxin In 2015, 5,901 confirmed cases of STEC infections were reported in the EU E. coli O157:H7 and at least 4 deaths The proportion of non-typable STEC strains continued to increase in 2015 E. coli O157:H7 In 2006, investigators linked at least 183 illnesses and one death to the consumption of fresh spinach contaminated with Interestingly, for the outbreak in Germany, investigators initially identified fresh produce—including leafy greens, tomatoes, and cucumbers as likely sources Salmonella and STEC O157 outbreaks Flour and flour-based mixes have been suspected or implicated as the source of other foodborne 2.7.Listeria monocytogenes is one of the leading causes of death from food-borne pathogens especially in pregnant women, newborns, the elderly, and immuno-compromised individuals L. monocytogenes. Although it is a relatively rare disease with a high mortality rate (20–30 %) that makes it one of the deadliest food-borne threats Listeria multiplies in cold environments such as refrigerators Listeria bacteria get into a food-processing factory, they can live there for years, sometimes contaminating food products Listeriosis is a serious infection usually caused by eating food contaminated with In the EU for the year 2015, 28 member states reported 2,206 confirmed human cases of listeriosis L. monocytogenes causes on average 1,591 episodes of domestically acquired food-borne illnesses, 1,455 hospitalizations, and 255 deaths annually in the US L. monocytogenes in food processing plants for years or even decades is an important factor in the transmission of this foodborne pathogen and the root cause of a number of human listeriosis outbreaks. L. monocytogenes persistence in other food-associated environments  may also contribute to food contamination and transmission of the pathogen to humans It is estimated that L. monocytogenes persistence at various stages of the food chain contributes to contamination of finished products, continued efforts to quantitatively integrate data on L. monocytogenes persistence  will be needed to advance our understanding of persistence of this pathogen and its economic and public health impacts Although the available data clearly indicate that L. monocytogenes; investigators should perform single-interviewer open-ended interviews when a food is not readily identified Whole apples have not been previously implicated in outbreaks of foodborne bacterial illness. A nationwide listeriosis outbreak associated with caramel apples was investigated L. monocytogenes is killed by pasteurization and cooking; however, in some Ready-To-Eat (RTE) foods contamination may occur after factory cooking but before packaging. RTE foods pose higher risk for listeriosis as they are ingested without any further processing, such as cooking, that would kill L. monocytogenes. Many of these foods use refrigeration, among other methods, to restrict bacterial growth during their shelf-life. While these standard practices work well for most bacteria, they are not adequate for Listeria control as the organism is capable of growth at refrigeration temperature and is often tolerant to freezing temperature, high salt and low pH Listeria spp. contamination had been linked to whole cantaloupe and one of the few times it had been linked to fresh produce One of the largest and deadliest multi-state outbreaks of listeriosis in the US occurred in late summer of 2011. The incident marked the first time that 2.8.Enterobactericiae have pathogenic characteristics and are one of the most common causes of enteric infections (food poisoning) worldwide. They were named after the scientist Dr. Daniel Salmon who isolated the first organism, Salmonella choleraesuis, from the intestine of a pig Salmonella is divided into two species that can cause illness in humans: S. enterica and S. bongoriSalmonella is further subdivided into serotypes, based on the Kaufmann-White typing scheme first published in 1934, which differentiates Salmonella strains by their surface and flagellar antigenic properties. Salmonella spp. are commonly referred to by their serotype names. For example, Salmonella enterica subsp. enterica is further divided into numerous serotypes, including S. Enteritidis and S. Typhimurium Salmonella enterica are responsible for more serious diseases such as Typhoid fever. The presence of several pathogenicity islands (PAIs) that encode various virulence factors allows Salmonella spp. to colonize and infect host organisms. There are two important PAIs, Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) that encode two different type III secretion systems for the delivery of effector molecules into the host cell that result in internalization of the bacteria which then leads to systemic spread. 5323 Salmonella enterica genomes have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 4.783 Mb This group of Salmonella spp. are the leading bacterial causes of food-borne illness in the US Salmonella each year, with an average of 19,000 hospitalizations and 380 deaths Salmonella spp. live in the intestines of most livestock and many wild animals. Salmonella spp. infection usually occurs when a person eats food contaminated with the feces of animals or humans carrying the bacteria. Salmonella outbreaks are commonly associated with eggs, meat, and poultry, but these bacteria can also contaminate other foods such as fruits and vegetables. More recently, the CDC has reported a total of 258 persons infected with the outbreak strain of Salmonella Bareilly (247 persons) or Salmonella Nchanga (11 persons) from 24 states and the District of Columbia Salmonella serovars in 2014 were S. Enteritidis and S. Typhimurium, representing 45.7% and 15.8%, respectively, of all reported serovars in confirmed human cases. Cases of Salmonella Infantis, the fourth most common serovar continued to decrease in 2015. Cases of Salmonella Stanley still remained, as in the last 2 years, at a higher level than before the large outbreak reported in 2011–2012 In EU for the year 2015, a total of 94,625 confirmed salmonellosis cases  were reported by 28 member states, resulting in an EU notification rate of 21.2 cases per 100,000 population. This represented a 1.9% increase in the EU notification rate compared with 2014. There was a statistically significant decreasing trend of salmonellosis in the 8-year period between 2008 and 2015 Salmonella. This “single batch” of ice cream was consumed by individuals in 15 states, where it sickened an estimated 225,000 individuals Salmonella spp. contamination of peanuts and peanut products led to one of the largest product recalls in US history. More than 714 people in 46 states were sickened in this outbreak and 9 individuals died In 1994, 138,000 gallons of ice cream were contaminated by Salmonella Newport, which infected 78 people in 13 states Pathogens may be passively internalized during produce processing, and this occurred in 1999, when mangoes imported to the US from Brazil were treated to kill possible Mediterranean fruit fly by dipping them in hot water, after which they were chilled in a cold-water bath Salmonella enterica serotype Chester; S. Chester was more likely to have eaten in a restaurant and visited the coast of Morocco S. Chester have been reported: in Australia, associated with sea turtle meat in 1998 and with tap water in 2005; in the US, associated with frozen meals (cheesy chicken and rice) in 2010 and in Canada, associated with headcheese in 2010 In EU between 2014 and 2015, a total of 162 cases, mostly from France, followed by Belgium, the Netherlands, Spain, Denmark and Sweden were reported, including 86 (53%) women 2.9.Shigella is a member of the family Enterobacteriaceae and possesses four serogroups that have been traditionally treated as species: serogroup A as Shigella dysenteriae, serogroup B as Shigella flexneri,serogroup C as Shigellaboydii, and, serogroup D as Shigella sonnei. Whereas serogroups A, B, and C consist of 38 serotypes, serogroup D possesses only one Shigella are non-motile, non-spore-forming, facultative anaerobic Gram-negative rods. They can grow at temperatures ranging from 6 to 48 °C, but prefer 37 °C, and S.sonnei appears to be able to tolerate lower temperatures better than the other serogroups. Optimum growth occurs between pH 6.0 and 8.0, although growth has been reported between pH 4.8 and 9.3 The genus Shigella spp. are closely related to E. coli in their DNA homology and share some biochemical characteristics as well as reactivity to some of the same antibodies, but despite these similarities, their differentiation should be considered clinically significant based, at least in part, on differences in symptoms expressed by infected individuals Shigella spp. are found most frequently in environments of compromised sanitation and poor hygiene, and although the primary route of transmission is by person-to-person contact, shigellosis can occur after the ingestion of focally contaminated water or food Shigella spp. have not been associated with one specific type of food; foods associated with outbreaks of shigellosis have included milk, salads, chicken, shellfish, and other fresh produce served at a wide range of establishments including restaurants, homes, schools, sorority houses, commercial airlines, cruise ships and military mess halls sonnei being the most prevalent and S. flexneri being the second most common in developed countries S. flexneri and S. dysenteriae type 1 are the most common serogroups, with S.dysenteviae type 1 having been involved in a lengthy epidemic in southern Africa and major epidemics in other parts of Africa, in Asia and in Central America Shigella serogroups can cause gastrointestinal infections after an incubation period of 12–50 h, after which time individuals experience watery diarrhea in conjunction with fever, fatigue, malaise, and abdominal cramps  of 65 football players and staff developed shigellosis that was associated with consumption of cold sandwiches; the sandwiches were prepared at the airline flight kitchen S. sonnei infection who attended a food fair during July 31-August 3 S. sonnei infection who had eaten at a specific kiosk at the fair or at the restaurant that had supplied the kiosk Outbreaks associated with fresh produce have emerged as an important public health concern. On 10 August 1998, the Ontario Ministry of Health was notified of a family of three persons with 2.10.Staphylococcus aureus are nonmotile, gram-positive cocci that appear singly or in pairs, tetrads, short chains, or characteristic “grapelike” clusters. Staphylococci are facultative anaerobes that, with the exception of Staphylococcus saccharolyticus and Staph. aureus subsp. anaerobius, grow more rapidly under aerobic conditions Staphylococcus spp. are widespread throughout nature and can be found on the skin and skin glands of mammals and birds, in addition to the mouth, blood, mammary glands, and intestinal, genitourinary, and upper respiratory tracts of infected hosts Staph. aureus can survive for long periods of time in a dry state, and have been isolated from air, dust, sewage, and water, making it one of the most resistant non-spore-forming pathogens Staph. aureus containing foods include ground beef, pork sausage, ground turkey, salmon steaks, oysters, shrimp, cream pies, milk, and delicatessen salads Staph. aureus grow, depending on the strain, at temperatures ranging from 7 to 47.8 °C and produce enterotoxins between 10 and 46 °C but prefer an optimum temperature between 40 and 45 °C. The bacterium grows between pH 4.5 and 9.3, with an optimum between 7.0 and 7.5, and is very tolerant to high levels of salt (>10% sodium chloride); enterotoxin production requires a minimum aw of 0.86, whereas growth has been demonstrated at an αw of 0.83 Staph. aureus typically causes infections involving the skin, such as boils, cellulitis, impetigo, and postoperative wound infections, but can also be associated with more serious infections like bacteremia, pneumonia, osteomyelitis, cerebritis, meningitis, and abscesses of muscle, urogenital tract, central nervous system, and various abdominal organs Staph. aureus infection Staph. aureus, and contamination of food can occur through direct contact, indirectly by skin fragments, or through respiratory tract droplets, with most staphylococcal food poisoning cases being traced to food contamination during preparation because of inadequate refrigeration, inadequate cooking or heating, or poor personal hygiene. After ingestion of the enterotoxin and an incubation period of less than 6 and up to 10 h, symptoms may include vomiting, nausea, abdominal cramps, headache, dizziness, chills, perspiration, general weakness, muscular cramping and/or prostration, and diarrhea that may or may not contain blood Staph. aureus in food may be considered a public health hazard because of its ability to produce enterotoxin and the risk of subsequent food poisoning. Although there are nine identified staphylococcal enterotoxins, designated as A, B, C1, C2, C3, D, E, F, and G, types A and D are responsible for the majority of the outbreaks The presence of 2.11.Vibrio, belonging to the family Vibrionaceae, contains more than 35 species, of which nearly half have been described in the last 20 years and more than one-third are pathogenic to humans Vibrio species, including Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, require sodium for optimum growth. They are found primarily in brackish or marine environments located in tropical or temperate areas, because their incidence decreases significantly as water temperature falls below 20 °C V. cholerae are also motile by means of a single polar-sheathed flagellum; these curved rods thrive in their environmental reservoir as part of the microflora found in estuaries. In addition to its primary environmental source, V. cholerae has been isolated from areas not associated with a marine or brackish water supply, including freshwater lakes and rivers and from birds and herbivores Vibrio cholerae O1 is composed of the classic biogroup that has been isolated during previous pandemics and El Tor, which is the predominant biogroup of the current pandemic The genus V. cholerae is between 30 and 37 °C, although growth can occur between 10 and 43 °C. Vibrio cholerae grow at pH 5.0–9.6 but prefer a pH of 7.6. They grow at a aw of at least 0.97 but prefer 0.984. Optimum growth occurs in an environment with a sodium chloride concentration of 0.5%, although V. cholerae growth can occur at concentrations of 0.1–4.0% The optimum temperature for growth of V. cholerae typically gain entrance into the human body through ingestion of a contaminated food, such as mollusks (raw oysters) or crustaceans eaten raw, undercooked, or even contaminated after cooking, or exposure of an open wound to a contaminated water source. Conditions resulting from V. cholerae O1 infection range from asymptomatic to the most severe form known as “cholera gravis” and in part depend on which biogroup is involved, because 75% of the El Tor biogroup and 60%) of the classic biogroup lead to asymptomatic infections After an incubation period of several hours to 5 days, depending on inoculum size and the amount of food ingested, typical symptoms include muscle cramping caused as a result of severe dehydration (fluid loss up to 500–1000 ml/h) resulting from vomiting, increased peristalsis followed by loose stools progressing to watery stools, and mucus-flecked diarrhea that is characteristic of cholera V. cholerae O139 Bengal is clinically identical to the symptoms exhibited by V. cholerae O1-infected individuals. Other V. cholerae serogroups, in addition to V. cholerae O1 and V. cholerae O139 Bengal, are known as non O1, non agglutinating vibrios or noncholera vibrios and are not known to cause epidemic disease. However, noncholera vibrios are known to cause self-limiting gastroenteritis and also may cause wound infections, bacteremia, and septicemia when associated with a preexisting liver condition V. cholerae is approximately 1011, but with the ingestion of food, the infectious dose is reduced to about 106 depending on the buffering capacity of the food The disease caused by Vibrio parahaemolyticus include crabs, prawns, scallops, seaweed, oysters, and clams V. parahaemolyticus grow at temperatures between 5 and 44 °C, with an optimum temperature and pH for growth between 30 and 37 °C and 7.6 and 8.6, respectively; the organism will grow in an environment at pH 4.8–11.0, in sodium chloride concentrations of 0.5–10.0%, and in environments with a minimum aw of 0.94; however, it prefers a concentration of sodium chloride in the range of 2 to 4% and a αw of 0.981 Food sources implicated as vehicles of transmission for V. parahaemolyticus is the Vibrio species most frequently isolated from clinical samples obtained in the US V. parahaemolyticus induced gastroenteritis include nausea, vomiting, headache, abdominal cramps, slight fever, chills, and watery diarrhea that is occasionally bloody V. parahaemolyticus possess four hemolytic components, including a thermostable direct hemolysin (TDH), a thermolabile direct hemolysin, phospholipase A, and lysophospholipase V. parahaemolyticus are invasive and can penetrate the lamina propria and enter circulation, as they have been found in the heart, spleen, pancreas, and liver Although symptoms are usually self-limiting, lasting only 2–3 days, severe cases may result in dysentery, primary septicemia, or cholera-like illness with the possibility of death V. parahaemolyticus has been the most common cause of the bacterial foodborne outbreaks and among the leading causes of bacterial foodborne disease outbreak in many Asian countries, including Japan and India V. parahaemolyticus gastroenteritis outbreaks in 12 provinces were investigated from China National Foodborne Diseases Surveillance Network. 322 gastroenteritis outbreaks due to V. parahaemolyticus were reported, resulting in 9,041 illnesses and 3,948 hospitalizations V. parahaemolyticus gastroenteritis outbreaks, food workers and consumers should receive training on avoiding cross contamination of ready-to-eat foods with uncooked seafoods, particularly in warm weather months During the past two decades in China, V. parahaemolyticus infection has been considered the leading cause of bacterial illnesses mainly associated with seafood consumption in Guangdong province in China V. parahaemolyticus were reported China National Foodborne Diseases Surveillance Network, resulting in 933 illnesses and 117 hospitalizations without death V. parahaemolyticus was isolated from food or based on epidemiologic evidence; aquatic products , meat and meat products , plant-based foods , mixed foods  were the most commonly implicated foods. Outbreaks most frequently occurred in restaurants (50.7%), private residents (21.1%), and cafeteria (12.7%) V. parahaemolyticus outbreaks caused by cross contamination, improper cooking and improper storage in high-temperature seasons, regulations for seafood safety from the production stage to the consumption stage should be strengthened V. parahaemolyticus illness outbreaks in the US Consumption of raw shellfish, primarily oysters, was linked to several multistate 2.12.Yersinia belongs to the family Enterobacteriaceae and includes 10 established species, although only 3 are considered pathogenic to either humans or animals. Yersinia pestis is the causative agent of plague, Yersinia pseudotuberculosis is primarily an animal pathogen but may infect humans after the ingestion of contaminated food or water, and Yersinia enterocolitica has surfaced as a cause of foodborne gastroenteritis in humans Yersinia spp. are Gram-negative or gram-variable, non-spore-forming rods that grow under both aerobic and anaerobic conditions but are considered facultative anaerobes. With the exception of Y. pestis, all Yersinia spp. possess peritrichous flagella and are motile at 22–30 °C but not at 37 °C The genus Y. enterocolitica was the most common species reported to be isolated from human cases Twenty-six member states reported 7,202 confirmed cases of yersiniosis in 2015, making it the third most commonly reported zoonosis in the EU Y. enterocolitica are widely distributed throughout the environment and have been isolated from raw milk, sewage-contaminated water, soil, seafood, humans, and many warm-blooded animals such as poultry and, most importantly, pigs Y. enterocolitica may pose a health hazard in contaminated refrigerated foods, although under refrigeration temperatures the pathogen is usually outgrown by other competing psychrotrophs Y. enterocolitica grow at temperatures between 0 and 45 °C but prefer an optimum temperature between 25 and 30 °C Y. enterocolitica can grow in the presence of sodium chloride at concentrations as high as 5% Y. enterocolitica are enteropathogenic, and the specific serotypes of Y. enterocolitica involved in hunian yersiniosis are prevalent primarily in swine. Ingestion of contaminated water or food. more specifically raw or undercooked pork, is a source of foodborne infection in humans, resulting in symptoms appearing after an incubation period of a few days to a week. Intestinal yersiniosis may persist for 1–2 weeks in adults and as long as 4 weeks in children and may include symptoms such as watery, sometimes bloody, stools or bloody diarrhea in conjunction with fever, vomiting, and abdominal pain Y. enterocolitica toxin is heat stable, resists enzymatic degradation, remains stable during prolonged storage, and is of similar pH stability as the thermostable enterotoxin produced by ETEC Not all serotypes of Y. enterocolitica infections was detected in southwestern Pennsylvania, US Y. enterocolitica serogroup O:8 was isolated from 24 fecal specimens of 21 patients and 3 kitchen staff in an outbreak in Japan; fresh vegetable salad was confirmed as the incrimination food of this outbreak In July 2011, a cluster of 3.Viruses are particulate in nature and multiply only in other living cells. Thus, they are incapable of survival for long periods outside the host. More than 100 types of enteric viruses have been shown to cause foodborne illness; the most common foodborne virus pathogens are Hepatitis A and Noroviruses. These viruses are frequently transmitted via food; bivalve molluscs, such as clams, cockles, mussels, and oysters, are especially prone to transmit viruses. The waters in which they grow are increasingly subject to human fecal contamination, sometimes from sewage discharges and sometimes from infected shellfish harvesters. The shellfish collect viruses in the course of their filter feeding activity. Human viruses do not infect these species, but they are harbored for days or weeks in the shellfish digestive tract and are apparently more difficult to remove than bacteria during processes intended to cleanse the shellfish (e.g. depuration) 3.1.HepatovirusHepatitis A virus particles are environmentally hardy organisms that can be transmitted by contaminated food, water, environmental surfaces  and through direct or indirect person-to-person contact Escherichia coli. The presence of these bacteria, however, has been shown to be a poor predictor of the presence of human enteric viruses The first recorded outbreak of shellfish-associated viral disease resulted from storing clean oysters in a fecally contaminated harbor while awaiting sale In 2003, a series of Hepatitis A outbreaks resulted in 1,000 cases of illness across multiple states and 3 deaths. The outbreaks were linked to green onions imported from four farms in Mexico where hepatitis A is endemic and the FDA subsequently banned imports from these farms The multinational Hepatitis A outbreaks occurring in Europe in 2013 and 2014 with over 1,400 cases linked to fresh and frozen strawberries and berry mix evidenced the usefulness of virus sequencing to link sporadic cases reported in different EU countries in outbreaks The numbers of reported foodborne illnesses are fewer than actually occur because the CDC's passive data collection system records only illnesses occurring as outbreaks, rather than those occurring sporadically. Hepatitis A, which is notoriously under reported in the US 3.2.Norovirus cause the majority of acute viral gastroenteritis cases worldwide, including an estimated 5.4 million episodes of foodborne illnesses in the US annually Noroviruses are nonenveloped viruses with a diameter of 30–35 nm and a single-stranded RNA genome of approximately 7.5 kb. The viruses are very diverse and are classified into six genogroups of which only three cause infection in humans; within these genogroups, 30 genotypes have been described to date via contaminated water) or during preparation Fecal-oral spread is the primary mode of transmission. The virus's abilities to withstand a wide range of temperatures (from freezing to 60 °C) and to persist on environmental surfaces and food items contribute to rapid dissemination, particularly via secondary spread (via food handlers or to family members) Cruise ships provide ideal conditions for the introduction and the rapid, global spread of Norovirus infection. Thousands of passengers from different geographic areas are transported in close quarters to multiple destinations around the world. Passengers and crew often disembark at multiple ports throughout the cruise where they can sample the local foods and culture. Cruise ships account for 10% of all reported outbreaks of Norovirus in the US An outbreak of Norovirus gastroenteritis that affected as many as 24 players and staff members from 13 National Basketball Association teams were affected with gastroenteritis symptoms was reported In another study, 286 fecal specimens from 88 oyster-associated gastroenteritis outbreaks were examined for the presence of 10 human enteric viruses using antigenic or genetic detection methods in order to determine the prevalence of these infections Norovirus outbreaks occur frequently in EU and it can be difficult to establish whether apparently independent outbreaks have the same origin Foodborne viruses cause considerable morbidity and mortality. Controlling them still means relying on good personal and food hygiene, good agricultural practice, post-harvest controls and effective management of human sewage to prevent onward transmission Risk assessment for transmission of emerging viruses through the food chain should include consideration of all means by which food could pose a hazard, that is not just consumption. New technologies have demonstrated the widespread nature of viral contamination in the food chain, but this does not necessarily correlate with the risk of disease. Finally, understanding people's knowledge and behaviour is just as important as understanding virus characteristics and epidemiology when assessing risks of foodborne transmission 4.cyst. Parasites are organisms that derive nourishment and protection from other living organisms known as hosts. They may be transmitted from animals to humans, from humans to humans, or from humans to animals. Several parasites have emerged as significant causes of foodborne and waterborne illness. These organisms live and reproduce within the tissues and organs of infected human and animal hosts, and are often excreted in faeces. The most common foodborne parasites are Cyclospora cayetanensis, Toxoplasma gondii and Trichinella spiralis.Parasites are one-celled microorganisms without a rigid cell wall, but with an organized nucleus. They are larger than bacteria. Like viruses, they do not multiple in foods, only in hosts. The transmissible form of these organisms is termed a In 2015, 156 confirmed trichinellosis and 41 cases of congenital toxoplasmosis were reported in the EU. The EU notification was 0.03 cases per 100,000 population, and decreased by 57.1% compared with 2014 when the highest notification rate was reported since 2010 The significant burden in low- and middle-income countries where cycles of parasitic infection are highly specific to food sources all over the world has been emphasized 4.1.Cyclosporra cayetanensis are protozoan parasites, belonging to the family Eimeriidae, that inhabit the small intestine, where they spend the intermediary life cycle stages in the cytoplasm of enterocytes and subsequently produce oocysts containing two sporocysts encapsulating four sporozoites C. cayetanensis have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 44.2991 Mb C. cayetanensis is capable of causing prolonged illness (6 weeks or longer) in both immunocompromised and immunocompetent individuals, with characteristic symptoms including nonbloody diarrhea, nausea, vomiting, anorexia, bloating, abdominal cramping, malaise, fever, and fatigue C. cayetanensis was identified as the etiologic agent in several outbreaks in the US and Canada involving raspberries, baby lettuce, and basil. Currently in the US, C. cayetanensis is estimated to cause about 15,000 cases of foodborne illness annually Between 1996 and 1998 C. cayetanensisIn 1996, 1,465 persons in 20 states, the District of Columbia, and two Canadian provinces became ill after consuming fresh raspberries that were imported from Guatemala and infected with the parasite 4.2.Toxoplasma gondii is a protozoan parasite member of the phylum Apicomplexa, and an obligate intracellular pathogen that is the causal agent of toxoplasmosis in humans. T. gondii uses cats as its primary reservoir and any other warm-blooded animal as an intermediate host T. gondii have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 64.1936 Mb T. gondii can be acquired in several ways, including the ingestion of contaminated food or water containing the oocyst, contaminated blood transfusion or organ transplantation, transplacental transmission, or accidental tachyzoite inoculation. T. gondii infections typically result from the ingestion of cysts in raw or undercooked meat, with fresh pork and beef appearing to be the primary sources In humans, T. gondii is one of the world's most common parasites. Although cats are the only known host in which the parasite can complete its life cycle, this parasite can use almost all warm-blooded vertebrates, including humans, as hosts. T. gondii infections are estimated to cause approximately 87,000 illnesses, 4,400 hospitalizations, and 330 deaths each year in the US, making it the second leading cause of foodborne mortality in the US and the third leading cause of food-borne hospitalizations T. gondii is highly amenable to experimental manipulation in the laboratory, and serves as a model system for genetic exploration of parasite biology and host-parasite interactions; this organism has been successfully used in transformation studies with genes from the closely related apicomplexan relative Plasmodium falciparumOf particular concern are women of childbearing age who have not acquired immunity against this parasite since it can be transmitted via placenta to the fetus . The consequences of congenital toxoplasmosis range from mild to severe to fatal and include: mental retardation, seizures, blindness and death 4.3.Trichinella spirulis is a parasitic roundworm belonging to the Phylum Nematoda, responsible for most human trichinosis infections. Besides humans, T. spiralis can infect most carnivorous mammals. 2 genomes of T. spiralis have been completed up to now according to the data retrieved from NCBI. The median total length of the genome is 56.7757 Mb The adult worms are 1.4–1.8 mm in size and are found embedded in the epithelium of the host's small intestine, where females and males mate T. spiralis is estimated to cause about 52 cases of foodborne illness annually, with a case fatality rate of 0.003 Symptoms, after an incubation period of 3–14 days, include nonspecific gastroenteritis, nausea, vomiting, headaches, fever, visual deficiencies, difficulty breathing, chills, night sweating, eosinophilia, myalgia, and circumorbital edema 5.E. coli O104 in Germany was estimated to cost more than $USD 3.5 billion in medical costs and a further $USD 304 million was paid by the European Commission for crop losses due to not selling the fresh produce The WHO Foodborne Disease Burden Epidemiology Reference Group provided in 2015 an estimate of global foodborne disease incidence, mortality, and disease burden in terms of DALYs Continued surveillance for foodborne disease outbreaks is important to reveal trends in the foods, regions and pathogens associated. In this field, genotype and subtype information from food contaminant strains is required to trace the transmission source, and to characterize and compare strains. The use of WGS as a tool for subtyping foodborne pathogen isolates has considerable potential for improving the detection of foodborne disease outbreaks, rapidly From the studies reviewed, the foods implicated in foodborne outbreaks are: fish and seafood Risk-based food safety approach is significantly different, compared to the classical hazard-based approach, leading to a major shift in thinking about the ways that science and policy-making in food safety should interplay Foodborne diseases are a global issue, and a unified and joint approach by all countries and the relevant international organizations is a prerequisite for the identification and control of all emerging foodborne problems that threaten human health and international trade


The present study is undertaken to investigate the fibrinogen levels in type 2 diabetes mellitus (T2DM) and its relation to peripheral artery disease (PAD) based on a more accurate and applied noninvasive measurements of duplex ultrasonography.  We performed a cross-sectional study including 1096 T2DM patients . The odds ratios (ORs) and 95% confidence intervals (CIs) were presented to show the association between PAD and fibrinogen in the subjects divided by fibrinogen levels quarterly. Furthermore, the univariate and multiple logistic analyses were performed to explore the correlation between PAD and fibrinogen levels, individual components in the cross-sectional study. P < 0.001). Multiple logistic analyses revealed that higher fibrinogen quartiles were positively related with the development of PAD in the adjusted model. After adjusting for known confounding parameters, the ORs for PAD were 1.993 , 2.469 , and 2.942  for Q2, Q3, and Q4, respectively .  Finally, 887 (80.9%) T2DM patients meet the diagnostic criteria of PAD and these patients had considerably higher serum fibrinogen concentration than non-PAD group ( Our results suggest that serum fibrinogen concentration can be considered as an independent risk factor for PAD in T2DM patients. Type 2 diabetes mellitus (T2DM) enhances the risk of lower extremity peripheral artery disease (PAD) and hemostatic and inflammatory disturbances with serious consequences on morbidity and mortality . PAD is Fibrinogen, as one of hemostatic and inflammatory markers, is present in diabetic patients and has generated considerable attention. Previous reports declared that circulating fibrinogen is increased in T2DM –11. MoreBased on the relationship of the fibrinogen and PAD which is controversial and the insufficiency of the ABI, more indicators to evaluate whether PAD exists in diabetes are essential. High-resolution duplex ultrasonography provides both anatomic location and functional information about the lower-extremity arterial system and has good sensitivity and specificity as compared with invasive angiography –21. The Between June 2008 and July to August 2013, Chinese patients with type 2 diabetes aged 18 years or above who got treatment in the endocrinology department and in line with inclusion criteria at the First Affiliated Hospital of Wenzhou Medical University were selected. Subjects were doctor-diagnosed as T2DM according to 1999 World Health Organization criteria . We perfOf all subjects, the clinical research coordinators collected physical assessment data, including age, sex, height, weight, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), waist circumference, and hip circumference. At every visit, height, weight, waist circumference, and hip circumference were measured by a team of trained nurses in the morning twice during the health examination. While weight was measured without heavy clothing and recorded to the nearest 100 g, height was measured without shoes and recorded to the nearest mm with a stadiometer. Waist circumference and hip circumference were measured to the nearest 1 mm using a stadiometer and a metal anthropometric tape, respectively. BMI was calculated as dividing the weight by the height squared. The waist/hip ratio was calculated as waist circumference divided by hip circumference. Blood pressure was measured at the brachial artery of the person in a seated position after they had had an at least fifteen-minute rest with an automatic instrument .Blood samples were collected and analyzed from antecubital vein sampling. Among them, laboratory parameters including fibrinogen, high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), total cholesterol (TC), triglyceride (TG), and HbA1c were subsequently analyzed by an automated analyzer .In this study, the diagnosis of PAD depended on the duplex ultrasonography examination in reference to Guidelines for the Assessment and Management of Patients with Peripheral Arterial Disease from the American Association in 2005 . Accordit-test was used for comparisons of the baseline characteristic in PAD subjects and non-PAD subjects, while 1-way analysis of variance (ANOVA) or the Kruskal-Wallis test for continuous variables and χ2 test for categorical variables were used for comparisons in different groups. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to present the relative risks for each category of fibrinogen level with the prevalence of PAD. Multivariable logistic regression analysis was performed to determine whether the association persists after adjusting confounding variables. A two-sided P < 0.05 was considered as statistically significant.Data analyses were performed with SPSS software (SPSS version 23.0 for Windows) and MedCalc software . In order to assess the correlation of fibrinogen concentrations and the influence of PAD, the study population was divided into quarters according to the fibrinogen concentration . Continuous variables are presented using mean with standard deviation (SD) according to the distribution of data, and categorical parameters were presented as number (%) for incidence rates. Student's Baseline clinical and biochemical measurements of 1096 subjects  are summarized in P < 0.001), waist circumference , fibrinogen , and HDL-C . Finally, fibrinogen was found to be the independent risk factor for the prevalence of PAD in T2DM subjects.All potential variables were initially evaluated in a univariate model to assess the functional relation with the prevalence of PAD. Parameters associated with PAD in the univariate analysis were male, age, smoking, alcohol use, diabetes duration, stroke hypertension, weight, height, BMI, waist circumference, hip circumference, waist/hip ratio, fibrinogen, TC, TG, HDL-C, LDL-C, and HbA1c as shown in P < 0.001), 2.738 , and 3.639 , respectively. In the adjustment for age and sex (model B), the ORs for PAD were 2.112 , 2.765 , and 3.651  for Q2, Q3, and Q4, respectively. After adjusting the full confounding parameters , the ORs for PAD were 1.993 , 2.469 , and 2.942  for Q2, Q3, and Q4, respectively , computed tomography angiography (CTA), and angiography. To our knowledge, the diagnosis of PAD was defined by the values of ABI ≤ 0.9 in almost all previous studies. The ABI test is a noninvasive method and is simple to perform . HoweverBased on these evidences, we performed a cross-sectional study to further assess whether the circulating levels of fibrinogen are associated with subjects with PAD measured by duplex ultrasonography in T2DM patients. The findings of our study reconfirm the positive association between serum fibrinogen levels and PAD. The strengths of this study include the assessment of PAD by a more accurate measurement-duplex ultrasonography and the exhaustive evaluation of biochemical and clinical parameters of atherosclerotic risk. Limitations of this study should also be acknowledged. First, the cross-sectional design limits the ability to establish a cause-effect relationship. Second, a selection bias could have been introduced because of the retrospective nature of the study. In order to minimize this issue, we maximized the sample size included in the study.In summary, our observation demonstrated that fibrinogen was positively correlated with the prevalence of PAD in T2DM patients, which should pay more attention to double lower extremity ultrasonic examination in diabetes patients with high fibrinogen level.