Background: Nonalcoholic fatty liver disease is considered to be the hepatic component of metabolic syndrome (MetS). However, the association between changes in MetS status and the risk of liver cirrhosis (LC) has not been investigated to date. This study assessed the association between changes in MetS and subsequent nonviral LC development. Methods: Data were obtained from the Korean National Health Insurance Service. Individuals who participated in health screenings from both 2009 to 2010 and 2011 to 2012 were included. The primary outcome was LC development according to the static and dynamic MetS status. Subjects were stratified into four groups according to the change in MetS status observed from the two-year interval screening (2009–2011). Cox regression analysis was used to examine the hazard ratios of LC. Results: During a median of 7.3 years of follow-up, 24,923 incident LC cases developed among 5,975,308 individuals. After adjusting for age, sex, smoking, alcohol, regular exercise, and body mass index, the adjusted hazard ratios  for LC development were 1.39 (1.33–1.44) for the MetS-Developed group, 1.32 (1.26–1.37) for the MetS-Recovered group, and 1.51 (1.45–1.56) for the MetS-Sustained group, relative to the MetS-Free group. Stratified analyses according to age, sex, smoking, alcohol intake, exercise, diabetes mellitus, hypertension, dyslipidemia, and chronic kidney disease showed similar results. Conclusions: Both static and dynamic MetS status are independent risk factors for LC development. The risk of LC was the highest in people with sustained MetS and was lower in the MetS-Recovered group than in the MetS-Sustained group. These results suggest that improving a person’s MetS status may be helpful in preventing LC.  Liver cirrhosis (LC) is the end-stage of various chronic liver diseases and is characterized by the pathological features of necro-inflammation and fibrosis with regenerative nodules . It is aMetabolic syndrome (MetS) is a cluster of metabolically related risk factors for cardiovascular disease . Since MThe Korean government has a single mandatory health insurance system that covers nearly 97% of South Koreans. The Korean National Health Insurance Service (NHIS) manages all administrative processes and reimburses medical providers and pharmacies based on their claims data. The Korean NHIS is a mandatory social insurance that covers virtually all Koreans except for Medicaid beneficiaries in the lowest income bracket (who make up approximately 3% of the population). It conducts biennial health examinations for all Korean employees of any age and dependent subscribers who are older than 40 years also receive the examination every 2 years. It consists of anthropometric measurements, laboratory tests , and questionnaires on lifestyle behaviors . Therefore, the Korean NHIS database includes health information from all Korean people based on eligibility, medical utilization, and results from health examinations. This database has been widely used for various epidemiologic studies ,20.The present study was approved by the institutional review board of the Seoul National University Hospital . Anonymized and de-identified information was used for analyses; therefore, the collection of informed consent was not required. The Korean NHIS database is open to use by all researchers whose study protocols are approved by the official review committee.n = 309,775) and those with pre-existing viral hepatitis , codes B15–B19), LC (defined by ICD-10 codes K703 and K746), or cancer diagnoses . As patients who develop LC immediately after a health examination may present an unclear temporal relationship with MetS status as identified at the health examination, we allotted a one-year lag time and further excluded subjects diagnosed with LC or hepatitis within one year after their second health examination. Therefore, 5,975,308 subjects were included in the final study population. Among the 7,212,102 subjects (age ≥ 20 years) who participated in health screenings both from 2009 to 2010 and 2011 to 2012, respectively, we excluded individuals with missing data  was calculated as weight in kilograms divided by height in meters squared. WC was measured at the midpoint between the lower margin of the ribs at the mid-axillary plane and the top of the iliac crest. Hypertension was defined as any of the following: a systolic blood pressure of at least 140 mmHg, a diastolic blood pressure of at least 90 mmHg, or treatment with an antihypertensive medication that was linked to the hypertension ICD-10 codes (I10–I13 and I15) and resulted in at least one claim in a year. Diabetes mellitus (DM) was defined as a blood glucose level of at least 126 mg/dL or a history of a hypoglycemic medication prescription that was linked to a diabetes ICD-10 code (E11–E14) and resulted in at least one claim in a year. Dyslipidemia was defined as a total cholesterol level of at least 240 mg/dL or a history of a lipid-lowering medication that was associated with the ICD-10 code E78. Chronic kidney disease (CKD) was defined as an estimated glomerular filtration rate of less than 60 mL/min/1.73 m² of the body surface area. Blood specimens including a liver function test such as aspartate transaminase (AST) and alanine transaminase (ALT) were obtained from each participant after an overnight fast lasting at least eight hours.p-values of less than 0.05 were considered to be statistically significant.The comparison of baseline characteristics according to the change in MetS was conducted using independent t-tests for continuous variables and the chi-squared test for categorical variables. The incidence rates of liver cirrhosis were assessed as incident cases divided by 1000 person-years. Cox proportional hazards regression was performed to estimate the risk of LC for each of the four MetS change groups . Multivariable analyses were adjusted for age, sex, smoking history, alcohol consumption, physical activity, and BMI. Stratified analyses were performed according to age (<65 vs. ≥65 years old), sex , smoking, alcohol (average drinking < 30 g/day vs. heavy drinking ≥ 30 g/day), and regular exercise . The statistical analyses were performed using SAS version 9.4 , and A total of 5,975,308 subjects were enrolled in this study . In the The baseline characteristics among the four groups are shown in The median follow-up duration of the study population was 7.3 years. The incidence rate of LC for the normal group was 0.53 cases per 1000 person-years and 1.02 cases in the MetS group . The risConsidering individual components, those who presented high values of WC , fasting glucose , and blood pressure , and low HDL  were at a significantly increased risk of LC development relative to those in the sustained normal group. Those who showed sustained high values of WC , fasting glucose , blood pressure , and TG  also demonstrated a greater risk of LC relative to those in the sustained normal group. Finally, those who showed normalization of WC , fasting glucose , and blood pressure  were at an elevated risk for LC relative to those in the sustained normal group, but the risk was lower than that in the sustained high group .We performed stratified analyses according to age (<65 vs. ≥65 years), sex , smoking, alcohol intake (<30 vs. ≥30 g/day), regular exercise, and the presence of DM, hypertension, dyslipidemia, and CKD. Stratified analyses also showed generally similar associations between the change in MetS and the risk of LC development . HoweverIn this nationwide, population-based cohort study, we found that MetS status was an independent risk factor for LC development. We demonstrated that a baseline MetS status increased the LC risk by 36%, while a sustained MetS status further increased the risk of LC up to 51%. In addition, those who developed MetS showed a significantly greater risk of developing LC as compared to those who maintained a MetS-free status, suggesting the existence of a causal association between MetS and the development of LC. Among the components of MetS, central obesity, impaired fasting glucose, high blood pressure, and low HDL were associated with an increased risk of LC.The most common primary etiologies for cirrhosis are known to be chronic hepatitis B, alcoholic liver disease, chronic hepatitis C, and NAFLD . In receAlthough the mechanism regarding the association between MetS and the increase in LC has not been fully elucidated, glucose intolerance is thought to be at the center of the mechanism. Several studies have suggested an association between insulin resistance and hepatic fibrosis. Chronic hyperinsulinemia, induced by insulin resistance, directly activates stellate cells ,29, MoreIn this study, when the risk of LC was analyzed by changes in individual MetS components, the development of central obesity, impaired fasting glucose, low HDL, and high blood pressure led to a significantly increased risk of LC development, with impaired fasting glucose among them correlated with the largest increase in LC risk. This shows that glucose intolerance plays a key role in the influence of MetS on LC. By individual components, a raised TG level, low total cholesterol, a low low-density lipoprotein level, raised glucose, diabetes, and a low HDL level were also associated with an increased risk of cirrhosis in a Swedish cohort study . ConsistTo the best of our knowledge, this is the first study to assess the risk of LC according to MetS state changes. Recovery from MetS was associated with a reduced risk of developing LC as compared with sustained MetS, suggesting that improving MetS status may have a preventive effect against LC development. On the other hand, the risk of LC was higher in the MetS-Recovery group than in the MetS-Free group, although both groups were equally free of MetS during the follow-up health examination period, indicating that a history of MetS, as well as current MetS status, influences LC development.The association between sustained or developed MetS and LC risk was more prominent in men, those aged younger than 65 years, and those who were heavy drinkers and/or current smokers, suggesting a greater focus is necessary on these high-risk individuals. As the MetS status can be dynamically changed , a comprThere were several limitations to this study. First, as is true with all epidemiological studies, it is difficult to ascertain causality. Second, although we used the Korean NHIS database, which includes the entire Korean population, and a large number of study participants underwent national health examinations (66.0% in 2009) , there mIn conclusion, the dynamic status of MetS is closely associated with the risk of nonviral LC, and the risk was greater when MetS was sustained. Recovered MetS lowers the risk of developing LC as compared with sustained MetS, and the history of MetS, as well as the current status of MetS, influences the risk of LC. Since dynamic changes in MetS affect the risk of LC, a comprehensive evaluation of the dynamic status of MetS is required and careful attention should be paid to the high-risk group.


Introduction: The benefit of cytoreductive nephrectomy (CN) for metastatic kidney cancer has been challenged recently. The study aimed to evaluate the prognostic roles of surgical resection of primary tumor site for metastatic kidney cancer under a real-world setting.Methods: The Surveillance, Epidemiology, and End Results (SEER) database (2010–2015) and the overall survival (OS) and cancer-specific survival (CSS) were evaluated using the Cox proportional hazards regression model. One-to-one matching using the propensity score was used to estimate and compare the survival rates.Results: The SEER data contain records of 8,932 patients from 2010 to 2015. The data showed that 61.7% of the patients underwent CN while 38.2% did not receive any surgery. The median survival month for a patient without surgery was 4 months and for a patient with surgery was 19 months. The multivariate analysis showed that surgical resection of the primary tumor site was an independent favorable predictor for both OS and CSS  in the original and the matching cohort.Conclusions: In the era of target therapy, CN might still be a vital method to treat metastatic kidney cancer. Kidney cancer remains one of the most commonly diagnosed cancers worldwide. The estimated new cases ranked sixth in men and 10th in women, and ~15% are metastatic at diagnosis .The standard of care for metastatic kidney cancer has been under debate over the decades. Cytoreductive nephrectomy (CN) is used to resect the primary tumor in place and single or oligo-metastatic disease. In the era of cytokines (before 2004), the study showed that combining CN with immunotherapy improved overall survival (OS) rate compared with immunotherapy alone, which favored CN as a standard of care for patient with metastatic disease for a long time , 4. HoweSeveral meta-analysis and retrospective studies supported CN combined with target therapy instead of target therapy alone , 8. HoweThus, this study aimed to investigate the controversial issue again to check whether CN brought more survival benefits compared with the conservative management of M1 kidney cancer, based on the Surveillance, Epidemiology, and End Results (SEER) database, one of the largest population-based cancer databases. This study is still significant as this topic needscontract-sponsor id="cn001" more discussion in a large real-world situation. Furthermore, we performed a 1:1 propensity score matching (PSM) analysis, which is a powerful method to minimize the selection bias, to create a 1:1 matched cohort with well-balanced baseline characteristics.Patients diagnosed with metastatic (M1 stage) kidney parenchyma carcinoma from 2010 to 2015 were identified from the SEER database due to the unavailability of metastasis information before 2010. Kidney parenchyma carcinoma with distant metastasis as the first primary malignancy was the inclusion criterion followed in the study. Information of the distant metastatic organs included the bones, brain, liver, and lungs. As the study aimed to investigate the impact of cytoreductive surgery on patients with kidney cancer, patients without sufficient surgery information and those who only underwent local treatment procedures  were excluded. The current study was granted an exemption from the Ethics Review Board in the institution because the SEER program collects data from population-based cancer registries with anonymous information. It is a publicly available database with no human participants involved. Hence, no ethical approval was needed. A detailed description of patient selection is shown in Clinical information was collected, such as demographics , tumor characteristics . Follow-up information was collected, such as survival months, survival status, and cancer-specific survival (CSS) status. The major end point was overall mortality and cancer-specific mortality. The duration of OS and CSS was defined as the time from kidney carcinoma diagnosis to the date of all-cause and cancer-specific death, respectively.The propensity scores were estimated using the logistic regression model that had been established from the factors which potentially affected a decision of treatment modalities: no surgery vs. cytoreductive surgery group. Those factors were age, gender, pathologic T stage, N stage, laterality, bone metastasis, brain metastasis, liver and lung metastasis, and histology. With the estimated propensity score, a one-to-one matched cohort was constituted using the nearest-neighbor methods, within a 0.000001 caliper size.t-test was used for continuous parametric variables. Survival curves were estimated using the Kaplan–Meier method. The log-rank test was used to assess significant differences between OS and CSS. The multivariate Cox proportional hazards regression analysis was employed to evaluate the prognostic factors in the original and matched group, and hazard ratios (HRs) along with 95% confidence interval (95% CI) were calculated. Only variables of significance in univariate analysis were put into the multivariate analysis. The differences were considered to be significant if p < 0.05. The statistical analysis was performed using IBM SPSS version 19.0 .A descriptive study of all the variables was conducted. The patients were divided into a group with no surgery and a group with resection of primary tumor site. Pearson's chi-square test was used to compare the categorical variables between two groups, and the A total of 8,932 patients with metastatic kidney parenchyma carcinoma at the time of diagnosis from 2010 to 2015 were included. The detailed patient characteristics are displayed in Using the propensity score, the matched cohort composed of 582 patients was constructed The main metastatic sites of kidney cancer were the bones, brain, liver, and lungs, which composed of 7,891 (88.3%) of all patients. The information of the four main distant metastatic organs is summarized in Those patients with different metastatic sites were extracted separately, and OS was compared between the surgery and no surgery groups. The Kaplan–Meier analysis indicated that patients receiving surgery had OS benefits among those patients with bone metastasis status , brain mP < 0.001). The Kaplan–Meier analysis indicated that patients receiving surgery had OS benefits in both the original and the matched cohort   and the matched cohort . In addition, multivariate analysis showed that older age, late T or N stage, presence of distant metastases, and non-clear cell renal cell carcinoma were all unfavorable factors associated with OS in the original cohort . In the matched cohort, tumor on the left side, T4 stage, brain metastasis, and non-clear cell renal cell carcinoma were linked with poorer survival.The 1-year OS rate was 68.9% for the surgery group and 38.2% for the no surgery group in the matched cohort (< 0.001) . As showP < 0.001). Similarly, patients receiving surgery had better CSS in both the original and the matched cohort  (P < 0.001). The trend was also observed in the matched cohort . Besides, elderly patients, late T or N stage, larger tumor size present of distant metastases, and non-clear cell renal cell carcinoma had significantly decreased CSS in the original cohort in the multivariate results . In the matched cohort, only tumor on the left side, T4 stage, brain metastasis, and non-clear cell renal cell carcinoma were associated with poorer CSS  The 1-year OS rate was 72.6% for the surgery group and 45.2% for the no surgery group in the matched cohort (< 0.001) . The patIn the current study, we first demonstrate the survival advantage of CN over no surgery for the treatment of M1 kidney cancer using data from the SEER database since 2010. To overcome the selection bias in order to decide the treatment modality (CN vs. no surgery) from retrospective data, a propensity matching score was employed, which is commonly used in large observational studies.There are various reasons for the gradual decline of CN. Although nephrectomy is a fairly safe procedure, CN in metastatic kidney cancer still remains a challenge, which is often accompanied with aggressive tumor feature, larger dimension, and severe adhesion to surrounding tissue, making cytoreductive surgery more difficult to perform and increases the risk of complications . Anotherde novo metastases, or alleviating malignant symptoms  nor the matched cohorts (27 vs. 7 months). In CARMENA study, the median OS was 18.4 months for sunitinib alone group vs. 13.9 months in the group starting with nephrectomy followed by sunitinib. The survival discordance could be explained by the difference between a real-world and a well-designed study. Besides, there are still patient selection bias and unbalance between the groups in the CARMENA study. Only intermediate and poor-risk patients were included in a higher proportion of advanced patients, which is the group already known to be least likely to benefit from CN. The points mentioned above might influence the interpretation of the CARMENA results . In the To our knowledge, there are two other similar studies utilizing the SEER database, all showed that patients with mRCC undergoing CN have improved survival rate in the era of target therapy. Conti et al. enrolling a total of 20,104 patients from 1993 to 2004, showing that median OS with CN was 19 months and outweighed 4 months without CN . Abern eNevertheless, we realized that the current study was limited by the retrospective and non-randomized design. First, although the patients were from a large national database, the systematic treatment information was lacking. There is a group of patients in both the surgical and non-surgical groups who were not eligible for systemic therapy. This could produce bias between the two groups and prevent further interpreting the impact on CN. Second, other physiological indicators related to prognoses, such as lactate dehydrogenase (LDH), serum hemoglobin, albumin, and calcium, were also unavailable in our dataset and might differ between the surgical and no surgical groups . Third, In a word, the recent study again highlights the importance of surgery for patients with M1 kidney cancer. The optimal choice is to find the most suitable candidate for upfront CN, such as those with good physical condition and limited metastatic tumor burden, to avoid CN or to defer in poor risk patients .Despite newly-emerging pieces of evidence slashing the importance of CN in M1 kidney cancer, we showed that in the real-world setting, patients undergoing cytoreductive surgery continue to have improved OS compared to those who do not. The optimal sequencing of surgery and systemic therapy warrants further study.https://seer.cancer.gov/about/overview.html).Publicly available datasets were analyzed in this study. The datasets generated for this study are available in the SEER database (XY and SL contributed to the conception of the study. RW performed the data curation and analysis. CL and WM wrote the manuscript. WM provided constructive comments to help perform the analysis and participated in the revision of the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Staphylococcus aureus is one of the main bacterial agents responsible for cases of mastitis in ruminants, playing an important role in the persistence and chronicity of diseases treated with antimicrobials. Using the multilocus sequence typing technique, network approaches and study of the population diversity of microorganisms, we performed analyzes of S. aureus (ES-GPM) isolated from goats with persistent mastitis (GPM). The most strains of ES-GPM were categorically different phylogenetically from the others and could be divided into two lineages: one with a majority belonging to ES-GPM and the other to varied strains. These two lineages were separated by 27 nuclear polymorphisms. The 43 strains comprised 22 clonal complexes (CCs), of which the ES-GPM strains were present in CC133, CC5 and a new complex formed by the sequence type 4966. The genetic diversity of some alleles showed be greater diversity and polymorphism than others, such as of the aroE and yqiL genes less than glpF gene. In addition, the sequences ES-GPM to the arc gene and glpF alleles showed the greatest number of mutations for ES-GPM in relation to non-ES-GPM. Therefore, this study identified genetic polymorphisms characteristic of S. aureus isolated from milk of goats diagnosed with persistent mastitis after the failed treatment with the antibiotic enrofloxacin. This study may help in the future to identify and discriminate this agent in cases of mastitis, and with that, the most appropriate antibiotic treatment can be performed in advance of the appearance of persistent mastitis caused by the agent, reducing the chances of premature culling and animal suffering. Staphylococcus aureus is a major opportunistic pathogen in humans and one of the most important pathogenic Staphylococcus species in veterinary medicine1. It appears on the world stage as a challenge in disease chronicity and persistence in antimicrobial treatments3. S. aureus is part of a small group of bacteria that can be named as 'persisters' in some diseases4, since administration of antibiotic treatments based on correct protocols allows the survival of a distinct form of bacterial subpopulation in an infection5. This bacterial agent is one of the most commonly found pathogens in clinical mastitis in several livestock species1, such as goats6. However, it can cause subclinical, clinical, persistent, gangrenous and difficult-to-treat intramammary infections7.8, and not as an antimicrobial persistence, which can results in the cure failure due to the performance of only the exchange of the antimicrobial, and consequently in premature culling or replacement of incurable animals. In chronic goat’s mastitis cases, the mammary gland can atrophy due to severe damage to the glandular tissue leading to fibrosis and total loss of the ability to produce milk9 and can result in the disposal of the animal.This type of persistent infection could also be identified in the types of mastitis, but it is often just referred to as chronic10, and mainly occurs due to the selection of different pathogens capable of evading the immune system5, which are genetically prepared to activate the series of stress responses for long-term survival4. Persistent mastitis in ruminants12 raises concerns because it renders ineffective treatments with antibiotics such as enrofloxacin12. Enrofloxacin is a fluoroquinolone exclusively developed for use in veterinary medicine13 and presents itself as an appropriate choice in some cases of mastitis in goats14, being active against major pathogenic bacteria (both Gram-positive and Gram-negative)15. Therefore, despite the best possible antimicrobial treatment, failures of bacteriological cure can often occur in mastitis treatments, especially for S. aureus mastitis, and antimicrobial resistance is considered one of the reasons for low cure rates16.Persistent infections are typically multifactorial and may have mechanisms for different classes of antibiotics, such as β-lactams and fluoroquinolonesS. aureus to characterize it as persistent during mastitis may be a solution in provide the barcode that can be used to track and identify strains with the same or similar polymorphisms, and thus increase the chances of providing a treatment appropriate antimicrobial. Thus, multi-locus sequence typing (MLST), which use phylogenetic procedures on the nucleotide sequences of allelic locus used in MLST are being widely used for identification and determination of phylogenetic relationships between isolates17. On the other hand, the haplotype network presents itself as an alternative representation of the clonal genealogy of bacterial isolates18 and complementary to the phylogeography of S. aureus. In addition, reconstructing bacterial haplotypes is required when choosing the right treatments for diseases caused by specific haplotypes in a population19.Consequently, identifying the nuclear polymorphisms of 20. Therefore, it was the aim of this study is to investigate the genetic diversity of S. aureus (ES-GPM) isolated from goats with persistent mastitis (GPM) through MLST scheme and compare with other sequence of S. aureus isolated from different other types of mastitis (non-ES-GPM) and hosts that are deposited in the PubMLST database21, using phylogeographical, phylogenetic, and diversity approaches.Population analyses of bacteria based on data from different loci using MLST schemes can provide important epidemiological results, especially if confronted with different methods involving genealogical typinghttps://pubmlst.org/organisms/staphylococcus-aureus/); these are shown in supplementary Table https://pubmlst.org/organisms/staphylococcus-aureus/) referring to animal mastitis in Brazil up to 09/28/2019. The MICs ranged from 0.125 to 16 μg/mL in the isolates prior to treatment, whereas in the isolates after treatment with enrofloxacin antibiotic, the antimicrobial MICs ranged from 0.19 to 16 μg/mL, with only samples of IDs 33784 and 33785 showing 16 μg/mL, as previously reported22.A total of 43 isolates were analyzed in this study. Within these 43, 18 (ES-GPM) isolates from the milk of animals were diagnosed with GPM at Capril UFV, named Minas Gerai—LDBAC Table . In addiS. aureus isolates are characterized, relating the phylogeny of the strains to the isolation sites of the sample, which showed little association between the origin of the isolate and the phylogenetic structure found. Despite the apparent small number of sequences as shown in Fig. S. aureus that infected dairy animals. However, strains related to ovine and caprine mastitis apparently had isolates from bovine mastitis as common ancestors. In addition, ES-GPM-related strains were highly associated in clades or clusters, which may be because few different strains causing GPM to circulate among animals within the same goat herd.In Fig. On the other hand, strains related to bovine mastitis demonstrated phylogenetic ramifications along the branches of the phylogenetic tree Fig. , regardlS. aureus  was higher  was formed by haplotypes H5, H9, H20 and H22. With this, H22 was positioned exactly where the two different clusters/strains are separated Figs.  and 5, bOf the 22 haplotypes found, only 13% were goats and H20 was the most common , formed by ES-GPM collected at UFV, Minas Gerais. The H20 haplotype (Minas Gerais—LDBAC) from goat strains proved to be a common ancestor of the H22 (Minas Gerais—LDBAC), H9 (Pernambuco), and H5 (Rio de Janeiro) haplotypes  was formed by the other haplotypes, with cattle representing 82.35% of this line Fig. . The numS. aureus sequences, was the haplotype with the greatest dispersion among the states, presenting itself in the states of the southern region (Santa Catarina and Rio Grande do Sul) and in the southeast region (Rio de Janeiro). In addition, H16 presented itself as a common ancestor and divisor of the haplotypical network, branching out several haplotypes, such as H13 and H12 (Pernambuco) and H1 (Rio de Janeiro). In the entire haplotype network referring to the different Brazilian regions  to 0.01119  content, the number of polymorphic sites, the ratio of non-synonymous (dN) to synonymous (dS) substitutions and nucleotide diversity (π) were calculated and are shown in Table tpi) to 0.432  and glpF (60%) alleles showed the greatest number of mutations for ES-GPM in relation to non-ES-GPM  at position 111 of ST 2988 (bovine); aroE  at positions 274 and 57, STs 742 and 3258, respectively (bovine); glpF (CAA → TAA) at positions 363 in ST 1728 (ovine); yqiL  at positions 170 in ST 1730 and at positions 495 for the STs 1730 (ovine), 744 (bovine), 1728 (ovine), 1730 (ovine) and 5 (goat). Interestingly, nonsense mutations were not identified in the new ST 4966.Six nonsense mutations were unexpectedly found in four distinct alleles and, interestingly, only in the lineage L2, being denoted in phi test is a rapid statistically efficient test for recombination. The P-value generated from the phi test for all 22 STs was p = 4.053E−5  by the program used, which suggested that there was no significant evidence of recombination for L1 or, if not, partly since there are very few informative sites. Furthermore, the phi test for each AT did not demonstrate a significant result.The phi test did not indicate that there would be evidence, as previously reported in the text. The IAs values were 0.0052 (P =  < 1.00E10−04) and 0.0014  value for the 22 STs was 2.16E01 Table , suggest23, being persistence a potential critical trigger for therapeutic failures4. Overall, phylogenetic analyses resulted in the establishment of two lineages of cases of bovine, ovine and caprine mastitis in different states of Brazil, and highly clonal ES-GPM unresponsive to the antibiotic enrofloxacin, in a single herd of goats. The 43 isolates that formed the 22 CCs provided further evidence that geographic isolation was not the primary factor leading to moderate genetic differentiation of S. aureus ES-GPM and Non-ES-GPM.In recent years, persistent infections have shown an important role in the relapse and recalcitrance of infections, moreover they are likely to help spread antibiotic resistanceS. aureus isolates from animals are commonly assigned to host-specific CCs24, and CC133 was the main group of characterized isolates belonging to ES-GPM in this study, in addition to the presence of the CC5 complex. In previous studies, CC133 has been associated with small ruminants25 and has been specifically assigned to goat, ovine and bovine isolates in several different countries24 and in Brazil26. According Aires-de-Sousa et al.27, the CC133 of S. aureus may have adapted to small ruminants, with human ancestry, due to the adaptive diversification of the genome resulting from allelic variation, the loss of genes or horizontal acquisition of mobile genetic elements.S. aureus (MRSA)28. CC5 has also been characterized in bovine mastitis29, in buffalo milk30 and also isolated in foods such as samples of milk and dairy products31. Consequently, the persistent S. aureus lineage, may be silently acting on persistent infections along with CC133 and CC5 in other cases of mastitis or even in other clonal complexes not studied here.The CC5 was also identified as belonging to the ES-GPM association but in a separate lineage from CC133. In addition, CC5 is recognized as a common clonal complex and generalized to several hosts, and is among the most prevalent clones that cause hospital infections in humans and causes methicillin-resistant 32 suggests that the prevalence of a limited number of clones is strictly related to mastitis in different herds and HOEKSTRA et al.33 demonstrated that the same genotype of S. aureus can cause clinical and subclinical mastitis in goats and ovine. However, diversity analyses implemented using different techniques indicate that there is a difference between sequences by type of mastitis, such as ES-GPM. This indicates that questions of agent selection or challenge3 can trigger the persistence34 of mastitis, and with this, questions related to the history of choosing certain classes of antibiotics in treatments and adaptations of the agent must be taken into account in the analysis of MLST in mastitis, as in cases of MRSA or MSSA (methicillin-susceptible S. aureus). As the mutations that lead bacteria to persist to enrofloxacin and other fluoroquinolones are still being determined37, more complete studies, with total genome sequencing38 and biocomputational analyses should be better implemented in the future to elucidate these issues.RABELLO et al.S. aureus in small regions of Brazil are related to low genetic variability and a small clonal population27. Furthermore, a greater number of sequences could better demonstrate the genetic diversity, patterns of distribution and evolutionary history of this agent in dairy animals. On the other hand, in cases with a large number of isolates collected from different regions of Norway, the strains were closely related genetically, and their clonal population was responsible for most cases of mastitis by S. aureus in domestic ruminants39. The low diversity of S. aureus in milk samples in studies with ruminants40 may be related to its later diversification from S. aureus associated with humans through a combination of foreign DNA acquisition and gene decay41 and also that their strains, in general, are young in relation to the species38. However, in this study the strains related to bovine mastitis demonstrated greater nucleotide polymorphism possibly associated with evolutionary pressures under the pathogen due to factors related to adaptation of a species to optimize the process of infection, escape host immune response, and also as possible adaptation to a different environmental niches and use of antibiotics that impacts on the evolution of certain core genes42.As was reported above, we did not find high genetic variability in this study, but nevertheless, it has been reported that cases of animal mastitis by S. aureus from invasive disease the r/m per allele parameter is approximately 1:143, which means that the isolates would have the same probability of diversifying in their large population by recombination as by point mutation. However, in humans, the genetic variability of S. aureus may be mainly associated with point mutation, since alleles are up to 15 times more likely to change by point mutation than by recombination44, similar to the results of our study. Conversely, in S. aureus adapted to cattle, it was found by MLST alleles that a nucleotide substitution was more likely to be due to recombination than to point mutation, and equally likely in humans45. Nevertheless, there is an extensive difference between the S. aureus genomes associated with cattle and those isolated from humans41. These differences between genomes and their r/m ratios demonstrate that there are gaps in the understanding of the diversification behavior of S. aureus between different hosts. In addition, subtle changes in strain due to single non-synonymous point mutation in S. aureus may be involved with persistence to antibiotics46 as diagnosed in GPMs unresponsive to treatment with enrofloxacin.In general, for isolates of S. aureus in studies of animal-associated strains of ovines, bovines, and poultry38, due to the performance of mobile genetic elements (MGEs), which generate a landscape of hotspots in the core genome38. Furthermore, there are concerns about the exchange of S. aureus CCs between animals and humans48, in which strains of this agent have been previously described in the literature as being capable of causing disease in animals and humans49. Therefore, the probability of widespread homologous recombination between S. aureus from different hosts may be high, and the potential of this agent to infect both humans and animals may indicate the chances of a greater or lesser degree of recombination or point mutation not having a noticeably clear pattern in S. aureus, unless characteristics other than the infection chain are intrinsically defined in the analysis metadata, such as those of ES-GPM.Nevertheless, there is evidence of widespread homologous recombination in the core genome of S. aureus may be associated with adaptive mutations due to response to environmental changes or a switch in host species, since this agent presents tropism to several hosts and in particular, antibiotic exposure50. In addition, housekeeping genes are genes associated with metabolic maintenance, which demonstrates that adaptations in their genomes may be affecting metabolism in response to distinct nutrient availability50. Of all genes, yqiL and aroE showed the highest values of non-synonymous substitutions and mutations. The yqiL locus in S. aureus demonstrated a potential signature of recombination45 compared to the other six gene fragments38, but the role of this locus in infections is poorly understood. However, a high number of non-synonymous substitutions may suggest that the removal of deleterious mutations by purification selection should be relatively slow. Gene aroE has also been shown to contribute to the chronicity of S. aureus infection, such as the invasiveness and cytotoxicity of the agent, with an increase in the load of intracellular bacteria51, and because of that, this same locus can play an important role in the persistence of infection such as ES-GPM in goats. Moreover, the aroE gene locus is shown to be in a region of the genome with an excess of homologous recombination, most likely in MGEs38 and with that it can confer an increased capacity to colonize and infect ruminants52.The greater diversity and polymorphism of some alleles of glpF sequence, among all others, was shown to be more conserved, with the lowest haplotypic diversity and the highest average G + C content, and also presenting low numbers of mutations, even though it is present in a region of the genome with excess homologous recombination38. The highest average G + C content may be linked to the pathogenic potential for the host genome53. However, we can emphasize that the glpF gene has a role in the formation of the L form in bacteria54, which is directly involved in antibiotic tolerance or persistence55 in S. aureus, such as to ampicillin or norfloxacin3. This demonstrates that glpF sequence, even though it is a member of the metabolic housekeeping genes and is present in the core genome, it is directly linked to persistence in infections; therefore, a high glpF sequence polymorphism may not be advantageous for colonization of the host as in ES-GPM for enrofloxacin and thus is shown to be more conserved within the other six alleles.Interestingly, the S. aureus isolated from goats with persistent mastitis) that may help in the future to discriminate S. aureus in cases of persistent goat mastitis (GPM). In addition, in 22 CCs that we found, CC133, CC5 and a new ST 4966 were specifically related to ES-GPM. We describe polymorphisms in specific alleles arcC and glpF genes, that showed a greatest number of mutations for ES-GPM in relation to non-ES-GPM. Furthermore, the identification of S. aureus and these polymorphisms genetics in persistent bacterial infections together with the MLST, can assist in decision making for the appropriate choice of protocol and of an antibiotic for the treatment of mastitis persists in goats. We hope that future studies can better clarify the persistence of this agent in certain antibiotic treatments.In this study we identified 27 specific genetic mutations for strains of ES-GPM , as well as changes in the mammary gland, such as an increased volume and body temperature, and the presence of pain, redness during forestripping performed at the milking parlour, in the presence of a veterinarian. After antibiogram results, these animals were treated with enrofloxacin (KINETOMAX – Bayer). Minimal inhibitory concentrations (MICs) for enrofloxacin were performed using the Etest method . However, due to acute mastitis and the need for rapid treatment, only after completion of seven days of enrofloxacin treatment were these MIC results available. Twenty-one days after the completion of treatment, these animals continued to have clinical mastitis. New milk samples were collected, and S. aureus was isolated again. Therefore, the diagnosis of persistent mastitis (GPM) was based on the detection of the same species of agent in more than one consecutive sampling59. Thus, 18 isolates of S. aureus were obtained (nine before treatment and nine after treatment) and were grouped in this study as ES-GPM.Briefly, the animals selected were examined and diagnosed with clinical mastitis caused by S. aureus isolates recovered from milk samples, these being selected from mastitis cases obtained from ovine and bovine isolates from the MLST online database (https://pubmlst.org/organisms/staphylococcus-aureus/). The search terms used were: country (Brazil), source (Milk) and disease (Mastitis), totaling 43 isolates from seven Brazilian states , shikimate dehydrogenase (aroE), glycerol kinase (glpF), guanylate kinase (gmk), phosphate acetyltransferase (pta), triosephosphate isomerase (tpi), acetyl coenzyme A acetyltransferase  and sequence type (ST), obtained from the PubMLST database 21. Any ST or allele not previously described were submitted to the database and assigned new AT and ST numbers.The ST, allelic profile and CC were confirmed by the E-MLST database (63. “HKY + I” was selected as the best evolution model of the MLST genes by the jModeltest 2 program64. The phylogenetic tree was inferred by the Markov Chain Monte Carlo (MCMC) method using MrBayes v3.1.265 and phylogenetic trees were calculated in two runs with 1,000,000 (one million) generations and a sampling frequency of 100 (one hundred). The parameter convergence was analyzed in Tracer version 1.6 (http://tree.bio.ed.ac.uk/software/tracer) and 25% of the trees generated were burned to produce the consensus tree. The phylogenetic tree and geospatial information was visualized together with associated metadata using Microreact Web server version 5.93.066 and by Figtree software version 1.4.467.Phylogenetic reconstructions by Bayesian Inference (BI) and Maximum Likelihood (ML) require the setting of a DNA evolution model to calculate the probabilities of nucleotide changes68. The average non-synonymous/synonymous rate ratio (dN/dS) was calculated with KaKs Calculator Version 2.069 to infer the direction and magnitude of natural selection. The ratio of non-synonymous and synonymous substitutions (dN/dS) measures the level of selection in a protein-coding gene. Further, the dN/dS ratio indicates purifying selection if dN/dS < 1, positive selection if dN/dS > 1, and neutral evolution if values are close to 117.The G + C content, variable sites (Ss) or mutations (Ms), number of polymorphic sites, average pairwise nucleotide, number of singleton (SNPs) and difference per site (π) were calculated with DnaSP Version 6.12.0370, as implemented in the software PHYLOViZ71 to cluster the STs into CCs based on the most stringent definition. Global optimal eBURST implemented by PHYLOViZ was used to cluster STs, generating a multi-locus sequence tree (MS tree) to visualize possible evolutionary relationships between STs. The pairwise homoplasy index (phi) test72 implemented in SplitTree473 for recombination was performed, and a P-value of < 0.05 indicated that recombination existed.Strain relationships were analyzed using the goeBURST algorithm74 implemented in Recombination Detection Program (RDP) v.4.9775 was used to calculate the per-site ρ/θ ratio based on the concatenated sequences of seven loci with 1,000,000 MCMC updates. The parameters ρ and θ represent the rates of recombination and mutation, respectively. Linkage disequilibrium from allelic data was evaluated by calculating the standardized index of association (IAs) using LIAN v3.73576 in web interface (http://guanine.evolbio.mpg.de/cgi-bin/lian/lian.cgi.pl/query). The null hypothesis of complete linkage equilibrium  was tested by using Monte Carlo methods with 10,000 iterations on allelic profile17. If there is linkage equilibrium because of frequent recombination events, the expected value of IAs is zero, which suggests no association between alleles at different loci; if IAs are statistically significant different from zero, alleles are suggested with genetic linkage77.The LDhat programS. aureus isolates in the Brazilian dairy milk were predicted following the methodology described by Vidigal et al.78. To reconstruct the network, sequences of AT and ST genes were grouped into haplotypes using DnaSP v668. Following this, the network was constructed using the network 4.6.1.0 (http://www.fluxus-technology.com) and the Median Joining algorithm (MJ)79.Genetic networks present an alternative view of genealogies represented by bifurcated structures of phylogenetic trees, and the possible dispersal routes of The experimental protocol was approved by the Ethics Committee (Comissão de ética no uso de animais - CEUA) of the Federal University of Viçosa, according to the protocol number 43/2016. The methods were carried out in accordance with the approved guidelines. In addition, this experiment was conducted by Bacterial Diseases Laboratory (LDBAC) and the Molecular Biology Laboratory (BIOMOL) located at the Veterinary Department (DVT) of the Universidade Federal de Viçosa (UFV), Viçosa, Minas Gerais.Supplementary Information.


The negative impacts of spousal bereavement on the emotional health of the elderly  have been revealed. However, whether widowhood impairs emotional cognition among the elderly is less known. The purpose of this study is to reveal the emotional cognitive deficits among the widowed elderly.In this study, we recruited 44 widowed elderly (WE) and 44 elder couples  and examined their emotional cognition including attention and visual working memory, which were measured by the visual search task and delayed-match-to-sample task, respectively. Three kinds of emotional faces  were adopted as the attentional or mnemonic targets.It revealed that WE had a general deficit in search efficiency across emotional types, while they showed mnemonic deficits in negative faces but not positive faces. Furthermore, the modeling analysis revealed that the level of depression or state anxiety of the elderly moderated the effects of widowhood on the deficits of mnemonic processing, i.e., the deficits were only evident among WE with the high level of depression or state anxiety.These findings reveal the attentional deficits in sad, angry, and happy faces and the mnemonic deficits in sad and angry faces among elderly who suffer from widowhood and point out the important role of emotional problems such as depression and state anxiety in modulating these emotional cognitive deficits. Spousal bereavement is a common negative event in later life that negatively influences the mental and physical health of the elderly. The widowed elderly reported increased anxiety, grief, depression, emotional loneliness, and social loneliness . These nFirst of all, emotional cognitions could change with aging. From the perspective of socioemotional selectivity theory, aged ones are motivated to derive emotional meaning from life and adopt an adaptive emotion regulation strategy to maximize the positive affect and minimize the negative affect . EvidencAnother factor that may affect emotional cognition could be the emotional state. According to the cognitive-behavioral model, individuals with high anxiety/depression have distorted or dysfunctional cognitive structure-labeled schemas that lead to distorted or biased information processing, which plays a central role in the development and maintenance of depression/anxiety . It has Although the effects of aging and emotional state on emotional cognition have been well studied, the effect of widowhood on emotional cognition among the elderly was rarely examined. On one hand, according to the socioemotional selectivity theory, the elderly might prefer positive emotion. It thus can be hypothesized that the widowed elderly might have cognitive biases toward happy faces. In contrast, according to the cognitive-behavioral model, it can be hypothesized that the widowed elderly might have biased cognition to negative emotions, as the level of anxiety and depression may be higher among them. To reconcile this discrepancy, we conducted behavioral measurements of emotional cognitions between widowed elderly and elder couples. Both attentional processing and working memory processing were examined. Moreover, we explored the moderating roles of depression and anxiety in these cognitive deficits.Regarding the attentional processing, we adopted the visual search paradigm in which participants were asked to detect, locate, or identify a target among distractors as quickly as possible. The results reveal how attention suppresses irrelevant distractors as well as shifts/orients to the target. The visual search paradigm is a well-established approach in measuring visual attention, which has been pervasively adopted to test emotional attention across adults of different ages . BesidesRegarding the working memory processing, we adopted the delayed-match-to-sample (DMTS) paradigm, which is one of the most common tasks used to study visual working memory. The DMTS task consists of three phases, namely, a sample (encoding) phase, a delay (maintenance) phase, and a test  phase. It is mainly used to examine the accuracy and capacity in encoding and maintaining visual stimuli. Previous studies showed stable test-retest reliability in the DMTS task  and stabTaken together, this study was designed to reveal the emotional cognitive deficits among widowed elderly, by comparing attentional bias and mnemonic bias to emotional stimuli between widowed and non-widowed elderly. Furthermore, we aimed to examine the role of the negative emotional state induced by widowhood in the cognitive deficits among widowed individuals. The results of this study may help test and develop existing theories such as the socioemotional selectivity theory and the cognitive-behavioral model and help develop interventions on the emotional problems among widowed elderly.d = 0.8 with a statistical power of 0.9. The result showed that two samples of 34 subjects, at least, were required. The distribution of sex was not significantly different between groups . However, the age of the WE group  was significantly larger than that of the NWE group  . Therefore, age was treated as a covariate in all the analyses. In addition, although the sex distribution was not significantly different between groups, to fully exclude the influence of sex, we also treated sex as a covariate in all the analyses. The study was approved by the ethics committee for human research at Zunyi Medical University, and informed consent was obtained from all participants.Forty-four widowed elderly  and 44 non-widowed elderly  were recruited from southwestern China. The inclusion criteria for the WE group were: (1) older than 55 years; (2) currently widowed and has no partner; and (3) able to understand the contents of the questionnaires and to complete the behavioral tasks. The inclusion criteria for the NWE group were: (1) older than 55 years; (2) currently in marriage and lives with the partner; and (3) the couple could both understand the contents of the questionnaires and complete the behavioral tasks. All of the participants were right-handed and had normal or corrected-to-normal vision and normal color vision. We performed a power analysis to determine the sample size using the G*Power 3.1.9.7 software . To findAll the participants completed two scales before they participated in behavioral experiments.The Geriatric Depression Scale  = 351.92, p < 0.001, ηp2 = 0.959]. Post-hoc tests with Bonferroni adjustment revealed that the valence of happy faces  was higher than that of sad faces  and angry faces  , while the valence was not different between sad and angry faces (p = 0.760). The difference of arousal among the three categories of emotional faces was non-significant .Sixteen sad faces, sixteen angry faces, and sixteen happy faces were selected from the Chinese Facial Affective Picture System (CFAPS) as search targets. Fifty-eight neutral faces selected from the CFAPS were treated as distractors. Half of the faces were women, and the other half were men. The hair, ears, and necks of all faces were removed using the Photoshop software. We then laid each picture on a black background and cropped it into a uniform size (130 × 150 pixels). All photos were then grayscaled and matched for brightness and contrast using the MATLAB software. The valence and arousal of each face were evaluated on 9-point scales. Valence was significantly different among three categories of emotional faces . Post-hoc tests suggested that the valence of happy faces  was significantly higher than those of angry  and sad  faces (ps < 0.001), while the difference was not significant between sad and angry faces (p = 0.490). In addition, no significant difference in arousal was found .Seventy-two emotional faces, namely, 24 sad, 24 angry, and 24 happy faces, were selected from CFAPS, half of which were female faces. Images were processed in the same way as Experiment 1, except that the image size was set to 185 × 200 pixels. Repeated measures ANOVA showed that the valences were different among emotional faces . The simple effect analysis showed that WE responded more slowly than NWE when the set size was 8 (p = 0.003), while the difference was non-significant when the set size was 2 (p = 0.406). Furthermore, the interaction between the emotional type and set size was significant . The simple effect analysis showed that the effect of the emotional type was marginally significant when the set size was 8 (p = 0.092), while it was non-significant when the set size was 2 (p = 0.800). Specifically, when the set size was 8, RTs to happy faces were faster than those to sad (p < 0.001) and angry (p < 0.001) faces. In addition, there were significant main effects of set size  and group , indicating faster responses at set size of 2 and in the NWE group. Other interactions and main effects were non-significant .Next, the same 3 × 2 × 2 mixed ANCOVA was performed on the RTs. There was a significant interaction between the set size and group, indicating lower search efficiencies among WE. Furthermore, the main effect of the emotional type was significant . Post-hoc tests showed a marginally significant difference between the slopes for happy and sad faces (p = 0.093). The interaction between the emotional type and the group was non-significant .Finally, a 3  × 2 (group) mixed ANCOVA was performed on the search slopes. We found a significant main effect of group . The simple effect analysis revealed that WE had lower accuracies in memorizing sad (p = 0.071) and angry (p = 0.050) faces than NWE, while the difference was non-significant for happy faces (p = 0.559). The main effects of emotional type and the group were non-significant .We first performed a 3  × 2 (group) mixed ANCOVA on the accuracies. Results showed that the interaction effect was significant . The simple effect analysis revealed lower discriminability among WE in memorizing sad (p = 0.071) and angry (p = 0.037) faces than NWE, while the difference was non-significant for happy faces (p = 0.544). The main effects of the emotional type and the group were non-significant .Finally, a 3  × 2 (group) mixed ANCOVA on the b = 1,605.76, t = 3.51, p = 0.001, 95%CI ), while the difference was non-significant among low state anxiety individuals .First, regarding the visual search task, we found significant between-group differences on the RTs at a set size of 8, as well as the difference on the search slopes. Therefore, six moderation models with depression as moderators and six moderation models with state anxiety as moderators were constructed. As we described in methods, group and behavioral performance were set as the independent variable and dependent variable, respectively. The level of depression or state anxiety was set as the moderator. Age and sex were set as the covariates. The moderation effects of depression and state anxiety are summarized in d′ for sad and angry faces. Therefore, four moderation models with depression as moderators and four moderation models with state anxiety as moderators were constructed. Results are summarized in b = –0.12, t = –3.40, p = 0.001, 95%CI ; accuracies for angry faces: b = –0.12, t = –3.25, p = 0.002, 95%CI ; d′ for sad faces: b = –0.74, t = –3.25, p = 0.002, 95%CI ; d′ for angry faces: b = –0.74, t = –3.39, p = 0.001, 95%CI ) but not among low-depressive individuals. Similar results were found on the effect of state anxiety  but not among low state anxiety individuals.Next, regarding the DMTS task, we found significant between-group differences in the accuracies and  anxiety . NWE shoIn this study, we examined the effect of spousal bereavement on the emotional state and emotional cognition among the elderly. First, we found that WE indeed showed a higher level of depression and state anxiety than NWE, consistent with previous studies . Second,In general, our results revealed cognitive deficits among WE, consistent with previous findings S.H. . After cn-back task showed greater activations elicited by negative emotional stimuli in left DLPFC among patients with depression (Although the general deficit was found on emotional cognition, the effects of the emotional valance on these deficits may be different between specific tasks. For the attentional processing, the deficits seemed not dependent on the emotional valence. It seemed to support neither the socioemotional selectivity theory nor the cognitive-behavioral model. According to the socioemotional selectivity theory , all eldpression . TherefoHowever, the working memory for negative faces among WE was less affected by their emotional problems compared with NWE, which was inconsistent with the cognitive-behavioral model. This result may stem from the impairment of working memory among WE. First, widowhood might impair working memory. Studies revealed that spousal bereavement was associated with declines in working memory among the elderly . Second,Although the effects of age and sex were controlled statistically in this study, we still know less about the impact of age and sex on our results. First, studies with stricter control are required. Second, as there was evidence showing moderation effects of sex and age on the impact of spousal bereavement on memory , the intBesides, the faces adopted in this study were mainly the faces of young adults. A previous study found that the elderly showed attentional bias to own-age face cues regardless of the emotional valence, which was similar to our results . HoweverWidowed elderly had a general deficit in search efficiency across emotional types, while they showed mnemonic deficits in negative faces but not positive faces. Furthermore, the level of depression or state anxiety of the elderly moderated the effects of widowhood on the deficits of mnemonic processing, i.e., the deficits were only evident among WE with a high level of depression or state anxiety. These findings reveal the attentional deficits in sad, angry, and happy faces, and the mnemonic deficits in sad and angry faces among elderly who suffer from widowhood, and point out the important role of emotional problems such as depression and state anxiety in modulating these emotional cognitive deficits.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee for Human Research at Zunyi Medical University. The patients/participants provided their written informed consent to participate in this study.TB: conception, design, acquisition of data, analysis of data, interpretation of data, funding acquisition, writing–original draft, and writing—review and editing. HK: conception, design, data acquisition, data analysis, data interpretation, funding acquisition, and writing—review and editing. YK: conception, design, acquisition of data, and writing—review and editing. BS: conception, design, and writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dietary lutein was associated with slight improvements in cognitive performance in complex attention , executive function  and memory , but its effect was not significant. Change-from-baseline analysis revealed that lutein consumption could have a role in maintaining cognitive performance in memory and executive function. Although dietary lutein did not significantly improve cognitive performance, the evidence across multiple studies suggests that lutein may nonetheless prevent cognitive decline, especially executive function. More intervention studies are needed to validate the role of lutein in preventing cognitive decline and in promoting brain health.Emerging literature suggests that dietary lutein may have important functions in cognitive health, but there is not enough data to substantiate its effects in human cognition. The current study was intended to determine the overall effect of lutein on the main domains of cognition in the adult population based on available placebo randomized-controlled trials. Literature searches were conducted in PubMed, AGRICOLA, Scopus, MEDLINE, and EMBASE on 14 November 2020. The effect of lutein on complex attention, executive function and memory domains of cognition were assessed by using an inverse-variance meta-analysis of standardized mean differences (SMD) (Hedge’s  Lutein and its isomers, zeaxanthin and meso-zeaxanthin, are xanthophyll carotenoids found commonly in green leafy vegetables, avocados and eggs which play significant roles in human health, particularly the health of eyes and brain, due to their antioxidant attributes ,2,3,4. AA significant body of literature in the past two decades has evaluated the effect of dietary lutein on human health. Strong evidence has been built on the protective functions of lutein in the eye, particularly in reducing the risk of age-related macular degeneration (AMD) and cataracts ,9,11,12.Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition defines these domains as perceptual-motor function, language, executive function, learning and memory, complex attention and social cognition [Cognitive function, in clinical neuropsychology, is conceptualized as several domains of cognition ,15. The ognition .Lutein, as the antioxidant of the brain, is proposed to not only protect cognitive function but also improve cognitive performance ,19,20,21Despite the advancements, there are no conclusive effect sizes for dietary lutein on global cognitive performance nor individual domains of cognition. In addition, there are currently no dietary recommendations for lutein despite its significant roles in human health. The current study was aimed at evaluating the strength of the effect of lutein on improving specific domains of cognitive function in adults. The study was conducted on the adult population since more studies are available in the literature in comparison with infant or adolescent demographic population. To the best of our knowledge, this randomized-controlled trial (RCT) meta-analysis is the first investigation on lutein and cognitive functions.Five databases  were searched from 2000 to November 2020 for relevant published articles. Each relevant article’s references were also searched for additional publications. The keywords in the search were as follows:  AND (Brain OR cognition OR memory OR attention OR language OR executive function OR processing). The searches were limited to randomized-controlled trials, human studies and English publications. All retrieved studies were assessed based on a set of inclusion and exclusion criteria by two reviewers . The maiThe total number of studies and their extrusion and inclusion criteria are presented in After qualifying for inclusion in the meta-analysis, a set of information that defines each study, e.g., name of the study, year of publication, demographics, type of intervention and dosage (milligrams of lutein per day), type of control, source of intervention, cognitive outcome measurements and duration of the study, was summarized . The quag SMD was calculated for each individual study and inverse-variance pooling was utilized for the overall effect estimate [2I statistic and Chi-square parameters. Forest plots were used to display the overall effect size of dietary lutein on cognitive performance for each domain. The overall effect of lutein is presented based on Hedge’s g SMD value. For the tests where their lower scores indicate cognitive improvement, the SMD scale was adjusted by multiplying the original mean by −1 to show the improving impact on the positive side for all measurements. Finally, potential publication bias was assessed by using funnel plots assessing the standardized mean difference of each study versus its standard error [The R-Studio version 1.4.1103 and version 3.0.1  were used to analyze the data. Due to the variety of scales used in assessing cognitive performance, a Hedge’s estimate . A separestimate . Additioestimate . Nonetheestimate . The intrd error .Funnel plots examining publication bias are presented in X2) and heterogeneity estimate (I2). The executive function domain showed high values of X2 and I2, indicating a high degree of heterogeneity among studies primarily due to the use of various tests and the absence of common or standardized tools to measure executive function. On the other hand, there are at least two common tests for assessing complex attention or memory domain. Forest plots for complex attention, executive function and memory domains are shown in g value was 0.02 with a 95% confidence interval (CI) of −27:0.31. The Hedge’s g value for executive function was 0.13 with a 95% CI of −0.26:0.51 and 0.03 with a 95% CI of −0.26:0.32 for memory domain. As observed in g value of −0.06 and a 95% CI of −0.22:0.11 . Only inconsistency was rated “serious” due to the variety of tools used to assess the three cognitive domains of complex attention, executive function and memory. .22:0.11 a, while .34:0.46 b. OveralThe changes from baseline were retrieved from studies directly if available or were calculated by using the paired sample t-test, and the results are presented in In the current study, we evaluated the overall effect of lutein on the three cognitive domains of complex attention, executive function and memory in adults who have consumed dietary supplements or foods rich in lutein in RCT interventions . The comp = 0.002). In the latter study, the treatment group had similar improvements in the Flanker, a test of response inhibition (p < 0.01). For memory, the treatment group of the Lindbergh et al. [p = 0.856), whereas the placebo group’s performance showed substantial decline (p = 0.084). For complex attention, the treatment group improved from the baseline significantly (p < 0.01), whereas the placebo group underwent no significant changes (p = 0.91) [Our results suggest that dietary lutein and its isomers could maintain cognitive functions and brain health, but it did not significantly improve cognitive function in complex attention a, executh et al.  study di = 0.91)  Table 3p = 0.002Lindbergh et al.  reportedInterestingly, the impact of lutein in the form of pills on executive function was not positive nor statistically significant, as shown in Many studies including those used in the meta-analysis reported improvements in serum lutein ,46 or MPThe role of lutein and zeaxanthin in protecting the eye by filtering harmful blue light and maintaining healthy retinal structures and in reducing the risk of ocular diseases such as age-related macular degeneration (AMD) and cataracts has been well documented ,11,12,13Several factors varied among the studies included in the meta-analysis ranging from cognitive performance assessment tools to population characteristics to intervention form and dosage. This undoubtedly affects the outcomes of the meta-analysis, yet the beneficial role of lutein is evident , and sigOverall, the meta-analysis based on the available literature indicated that dietary lutein does not consistently improve cognitive function and performance in randomized controlled trials as indicated by the statistically insignificant overall SMD values for the complex attention, executive function and memory domains of cognition. On the other hand, there is evidence in the data from the intra-study treatment/control group changes-from-baseline that dietary lutein may nonetheless be effective in maintaining cognitive function. Thus, large clinical studies with proper designs in terms of intervention duration, dosage and population should be considered to support the role of lutein in brain health.The seven RCT studies that met the inclusion criteria have shown insignificant effects for lutein on complex attention, executive function and memory cognitive domains, but slight improvements in the three domains were observed. The studies used different intervention dosages and periods which could influence the overall effect of lutein. Lutein has been found, in many cases, to improve cognitive performance and prevent further cognitive decline. Moreover, dietary lutein affects executive function domain more positively and in terms of greater magnitude than compared to complex attention or memory domain. This suggests that the executive function domain may be more closely related to lutein status, which warrants further investigation and validation. No preference was observed for lutein absorption and accumulation consumed either in dietary pill or food form. Due to the fact that the mechanisms of lutein on neurocognitive processes are unclear, future studies need to consider morphological brain changes in response to dietary lutein over the course of a trial. Additionally, future research should also focus on the possible protective effect of lutein on executive function and other under-evaluated cognitive domains in the literature such as language, social cognition and perceptual-motor function.


Rabies is considered one of the most relevant public health problems owingto its high fatality rate and the high number of deaths worldwide.  We included patients with human rabies who attended a reference hospital inthe state of Ceará during 1976-2019.  Data were available for 63 out of 171 (36.8%) patients. Of these patients,48 (76.2%) were attacked by dogs. In recent years, wild animals have beenthe most common aggressor species (marmosets and bats). Only 39 (70%)patients were initially correctly suspected with rabies. Bites were the mostfrequent exposure , most commonly on the hands  and thehead . Only 14 (22%) patients had sought medical assistance beforethe onset of symptoms, and only one completed post-exposure prophylaxis. Themost prevalent signs and symptoms included aggressiveness/irritability , fever , sore throat/dysphagia , and myalgia. Hydrophobia was present in 17 patients (22.0%).  Most cases of human rabies in Ceará occurred due to the failure to seekmedical assistance and/or the failure of the health system in initiatingearly post-exposure prophylaxis. There is a need for specific informationand education campaigns focusing on the cycle of sylvatic rabies as well asprevention measures. Health professionals should undergo refresher trainingcourses on the signs and symptoms of rabies and on the specificepidemiological features of the disease in Brazil.  It isa vaccine-preventable disease; however, its course is practically irreversible afterthe onset of signs and symptomsDesmodus rotundus) is themain transmitter of rabies virus to humans in Brazil, and the variant maintained andtransmitted by this species is commonly found in human cases where other species,mainly dogs and cats, are aggressors,In Brazil, a significant reduction in mortality rates due to HR has been achieved inthe last 30 years after the implementation of canine vaccination campaigns and theintensification of the provision of post-exposure prophylaxis. Currently, there areonly sporadic cases of HR transmitted by dogsDesmodusrotundus), and other wild species are responsible for maintaining andtransmitting variants of the rabies virus to humans-Callithrix jacchus) (26%), bats (8.7%),and raccoons (2.2%)--In the state of Ceará in Northeast Brazil, dogs, hematophagous bats Desmodusrotundus,Post-exposure prophylaxis after aggression by a rabid animal is highly effective andprevents disease and death. Early diagnosis and the initiation of post-exposureprophylaxis are essential. However, physicians generally have limited knowledgeabout signs, symptoms, and prophylaxisThis study describes the clinical and epidemiological characteristics of 63 patientsaffected by human rabies in the state of Ceará over a period of 44 years. Desmodusrotundus) occurred in 2016. The study was carried out in the state of Ceará in Northeast Brazil . All registered rabies cases were treatedat this hospital, as this is the only reference hospital for infectious diseases inthe state. The analysis included all patient charts encountered in the hospitalarchives.The variables available and analyzed in this study included epidemiological andclinical data, such as aggressor species, type of aggression/exposure to the virus,location of lesion, post-exposure prophylaxis, origin of the animal, animalsituation, follow-up on animal, animal observation, incubation period, signs andsymptoms, initial diagnosis, aggression dates, onset of symptoms, and death.Data entry, processing, and analysis were performed using Microsoft Excel 2010software within a database. The virus incubation period was calculated as thedifference in days between the aggression date and the date of symptom onset. Thepatients' lifetimes were calculated based on the difference in days between the dateof death and the date of the onset of clinical signs.The study was approved by the Ethical Review Boards of the Federal University ofCeará (CAAE number: 13466719.6.0000.5054), of the State Health Secretariat (CAAEnumber - 13466719.6.3001.5051), and of the Reference Hospital for InfectiousDiseases São José (CAAE number: 13466719.6 .3002.5044).Sixty-three patient charts were included in the analysis, representing 36.8% of the171 patients who attended the reference hospital during the study period. The most prevalent signs and symptoms included changes in behavior(aggressiveness/irritability), followed by fever and pain in the throat/dysphagia. HydrophOnly 39 (70%) of the patients had an initial diagnosis of rabies when they sought thehealth unit after presenting the clinical signs of the disease; the other cases werefirst diagnosed as acute encephalitis  and meningitis , followedby a single diagnosis of respiratory infection, poliomyelitis, acute abdomen, acutepolyencephalitis, pneumonia, nervousness/pneumonia/meningitis/rabies, head trauma,intestinal obstruction, dengue/meningitis, and Guillain-Barré syndrome. Desmodus rotundus 33.5 (14-68), and for the patient beingbitten by a cat, it was 118 days.The mean time of the incubation period, in days, for patients being attacked by dogswas 49.2 (amplitude: 13-149), by marmosets 44.8 (20-61), by hematophagous batsIn general, dogs were the main transmitters, with six and seven cases in 1979 and1980, followed by six cases in 1982 and 2003, respectively. Six cases transmitted bymarmosets were recorded in 1980, 1981, 1991, 2008, 2010, and 2012. Of the five casestransmitted by bats, two occurred in 1985, and single cases occurred in 1983, 1995,and 2016. A single case transmitted by a cat occurred in 1979 .The mean lifespan of patients after being attacked by dogs and cats was 5.8 and 3.0days, respectively. After the attack by marmosets and bats, the mean lifespan was9.7 and 9.4 days, respectively.This is the first systematic study on the epidemiological and clinicalcharacteristics of human rabies in the state of Ceará, covering a 44-year period. Inthe first few years, dogs were the main transmitters of the virus and wereresponsible for 75% of all cases. In recent years, wild animals have become the mostimportant aggressor species. Most victims were not aware of the importance andnecessity of seeking assistance after being attacked. Hands were the most commonbody parts affected through bites. Health professionals often failed to diagnoserabies and did not prescribe post-exposure prophylaxis over time.The clinical signs presented were mostly related to behavioral changes, such asaggressiveness and irritability, but the initial diagnosis of the disease wasdismissed in 30% of cases. This reinforces the importance of assessing any mammalexposure and suspecting human rabies whenever a patient is seen in the health caresystem with a sudden onset of neurological symptoms. During an outbreak of humanrabies in the state of Amazonas in Brazil, most victims presented similarneurological disorders such as paraparesis (87.5%), aggression and/or agitation(37.5%), mental confusion (25%), and tetraparesis (18.8 %)A single patient was attacked by a cat with a longer incubation period, presenting a118-day interval that does not match the rabies incubation period commonly observedin human beings, which is, on average, 45 daysThe lifespan of patients counting from the onset of clinical signs until death waswithin the period of 10 days, consistent with the lifetime of a patient affected byrabies described in the Brazilian Health Surveillance GuideDespite attempts to cure the last four patients since 2010 in the state of Ceará,none has had a successful response. The affected patients were mostly treated withantibiotics and antivirals. After the onset of clinical signs, the chances ofsurvival are remote and evidence was restricted to a few case reports,-,Programa Nacional de Profilaxia para a Raiva - PNPR),based on mass vaccination campaigns of dogs and cats, an action that is consideredof great importance for the control of the disease in the country, has not yet beenestablished in the state of Ceará. Implementation started in 1973, with roll-out inall Brazilian states during the four years thereafterDogs, especially stray dogs, are the most common aggressor species in manysettingsThe cases involving wild mammals in this study have been reported since the 1980s. Inparticular, marmosets are responsible for transmitting the rabies virus to humans.In Ceará, it is common for the rural population to capture and raise non-humanprimates, such as pets, including marmosets. Because these rural populations areusually unaware of the rabies virus transmission cycle, they do not seek medicalassistance after being attacked by a wild animal. Callithrix jacchus) has been the maintransmitter of the rabies virus to humans in recent years in Ceará. It harbors aunique and exclusive variant, identified for the first time in Ceará, and thus far,this variant has been restricted to Northeast Brazil. Marmosets are the onlyterrestrial wild species responsible for human cases through direct aggression inthe country,,-In fact, the marmoset , marmosets, and raccoons (Procyoncancrivorus), as confirmed by the state laboratoryOnly one case was transmitted by a cat in 1979, in Fortaleza, the capital city ofCeará. To date, there have been no records of rabies cases with cats as transmittersin the state's database. Although canine rabies is under control in Ceará,surveillance in domestic species still requires attention, as treatment for diseasetransmitted through dogs accounts for more than 70% of post-exposure treatments inthe state, after attacking humansOnly 22% of victims had sought medical care after exposure. There is still a need foractions to alert the population of Ceará on the main aspects of rabies, especiallyon prevention, and the awareness that post-exposure prophylaxis is the only form ofprevention after being attacked by a diseased animalSistema Único de Saúde). In other settings, limited access to the health care system is another key factor.For example, in South Africa, the main reason for not initiating post-exposureprophylaxis was the lack of health professionals,Another Brazilian study on 82 cases of human rabies transmitted by wild animals,mostly bats, reported that post-exposure prophylaxis was not prescribed for 72% ofpatients,,Several patients abandoned post-exposure prophylaxis, compromising the body’s immuneresponse and favoring disease development. In Mozambique in 2014, similarobservations were made, and many patients did not receive full treatment to developthe diseaseEven though canine rabies is controlled in Ceará, there is a high risk that dogs andcats might be contaminated by wild animals and then the virus transmission mayrestart to humans. To control rabies maintained and transmitted by dogs and humanswith virus transmission by wild species, the state of Ceará performs surveillanceactions in an integrated and continuous manner, in collaboration with differentinstitutions. The state develops strategies that consider the local situation and conditions,providing the availability of supplies in a timely manner to ensure a betterresponse and the non-occurrence of human cases. To increase effectiveness, rabies control requires a multidisciplinary, one-healthapproach. The disease is associated with all four determinant groups described byOne Health as causing diseaseThis study had several limitations. We analyzed the secondary data, and some medicalcharts did not provide complete information. In addition, it was possible to includeonly 37% of registered rabies cases during the study period because the other chartscould not be encountered in the archive. The information contained in the oldestmedical records, such as data of type of exposure, lesion location, initialdiagnosis, and origin of the attacking animal, was not complete. Despite theselimitations, this study provides information of great relevance to human rabies. It can be concluded that most human rabies cases in Ceará occurred due to the failureto seek assistance and due to the failure of the health care system to initiate andcomplete early post-exposure prophylaxis. There is a need for specific informationand education campaigns focusing on the sylvatic rabies cycle and preventionmeasures. Health professionals need refresher training courses on the signs andsymptoms of rabies and on the specific epidemiologic features of the disease inBrazil.