To determine the fecal contamination level, fecal coliforms were quantified using the most probable number (MPN) method and the source of fecal contamination was determined by Microbial Source Tracking (MST) using the Cryptosporidium and Giardia genotyping method. Separately, to determine if antimicrobial resistance bacteria (AMB) were present in the rivers, Escherichia coli and environmental bacteria were isolated, and the antibiotic susceptibility profile was determined. Fecal coliform levels in the Maule and Maipo Rivers ranged between 1 and 130 MPN/100-ml, and 2 and 30,000 MPN/100-ml, respectively. Based on the MST results using Cryptosporidium and Giardia host-specific species, human, cattle, birds, and/or dogs hosts were the probable sources of fecal contamination in both rivers, with human and cattle host-specific species being more frequently detected. Conditional tree analysis indicated that coliform levels were significantly associated with the river system (Maipo versus Maule), land use, and season. Fecal coliform levels were significantly (p < 0.006) higher at urban and agricultural sites than at sites immediately downstream of treatment centers, livestock areas, or natural areas. Three out of eight (37.5%) E. coli isolates presented a multidrug-resistance (MDR) phenotype. Similarly, 6.6% (117/1768) and 5.1% (44/863) of environmental isolates, in Maipo and Maule River showed and MDR phenotype. Efforts to reduce fecal discharge into these rivers should thus focus on agriculture and urban land uses as these areas were contributing the most and more frequently to fecal contamination into the rivers, while human and cattle fecal discharges were identified as the most likely source of this fecal contamination by the MST approach. This information can be used to design better mitigation strategies, thereby reducing the burden of waterborne diseases and AMR in Central Chile.Freshwater bodies receive waste, feces, and fecal microorganisms from agricultural, urban, and natural activities. In this study, the probable sources of fecal contamination were determined. Also, antibiotic resistant bacteria (ARB) were detected in the two main rivers of central Chile. Surface water samples were collected from 12 sampling sites in the Maipo ( Fecal pollution in water is associated with negative health impacts, economic losses , and pathogen contamination of raw foods . The WorCryptosporidium spp. and Giardia spp. can be enhanced by climate conditions, water availability, increasing illegal usage of raw wastewater, night soil, and manure amendment for crop soils  in discharges from areas with large cattle herds during precipitation events.The quality of many water sources has been greatly affected by fecal pathogens from anthropogenic activity such as urban and animal protein production . In geneop soils . For exaop soils . Lewis eE. coli O157:H7 was associated with upstream livestock pasture density, with 20% of the detections located where cattle had access to the waterway. Land use is a classification that provides information on the type and intensity of human activities occurring in a given geographical area  infects a variety of mammals, including humans  of G. duodenalis and species, each with specific hosts. Therefore, identifying the genotype of Cryptosporidium spp. and assemblage for Giardia duodenalis can provide information on the host source  of origin of the fecal contamination and its zoonotic potential or if they are infective to productive animals such as cows.Many of the programs monitoring water quality only focus on measuring Fecal Indicator Bacteria FIB; . These Forldwide . The preorldwide . Despiteorldwide . Researcorldwide . Among tcificity . For insus hosts , a situaemblages . Giardiag humans  and willCryptosporidium and Giardia, respectively, to determine the probable source of fecal contamination and detect the presence of antibiotic-resistant bacteria (ARB) in two main rivers of central Chile based on land use . Finally, we evaluate the association between the presence of fecal coliforms, Cryptosporidium spp., Giardia duodenalis, and ARB with environmental factors. This study provides information regarding the land uses and sources that contribute the most to rivers’ biological contamination with fecal material and ARB, consequently providing information to create strategies to manage and reduce loads of this fecal contamination to the rivers.Chile provides an ideal condition to study the water quality of rivers because of the geography it presents. In addition, it is a narrow country, allowing an entire river that crossed different land use zones to be sampled on the same day. Water quality is particularly important for Chile as the rivers are the main source of irrigation and drinking water source. Therefore, the objective of this study was to determine the fecal contamination levels using fecal coliforms as indicators, identify the genotypes/assemblages of 6 fecal coliforms/100 ml in past reports  and discharges in the studied site in the Maipo River, and the Claro River receives the urban influence of the capital and major city in the Maule region  and discharges in the area sampled in the Maule River  were also recorded at the time of sampling.Water samples were collected from each river every 3 months between June 2017 and April 2019. The timing of sampling was selected to ensure sampling during each season: summer (December–March), fall (March–June), winter (June–September), and spring (September–December). Fall and winter sampling events correspond to the rain event, while spring and summer sampling events correspond to the dry season. All sampling sites were georeferenced using Google Maps. Water was collected in a sterile plastic bottle at each sampling site, which was rinsed three times in the river before collecting the sample. The sample was collected by submerging the bottle at least 10 cm below the surface. Immediately after sample collection, bottles were placed in a cooler and transported on ice. Samples were all processed within 24 h. Physical parameters of pH, water temperature (°C), conductivity (μS or mS), salinity (ppm or ppt), and total dissolved solvents (ppm or ppt) were measured Water samples (15 ml) were used for fecal coliform enumeration following the Chilean Regulation NCh2313/22:1995 protocol for the detection of fecal coliforms in wastewater using EC medium  to deterCryptosporidium spp. oocysts and Giardia duodenalis cysts in the ultra-filtrated water were quantified according to the United States Environmental Protection Agency (EPA) method 1623  immunomagnetic separation (IMS). IMS was conducted following Cryptosporidium spp. and Giardia duodenalis beads to remove metallic sediments. Levels of each target were stained using Waterborne kit reagents  per the manufacturer’s instructions and enumerated according to Environmental Protection Agency (EPA) method 1623  hemodialysis filters following the methodology described by hod 1623 . Cryptoshod 1623 . Brieflyhod 1623 . Cryptosal forms . Giardia μm long . The samA modified version of a previously published method  was usedCryptosporidium PCR was based on molecular characterization of the 18S rRNA gene using nested primers that amplify ~825 bp product (Giardia PCR was based on molecular characterization of the glutamate dehydrogenase (GDH) and the SSU rRNA genes. GDH confirmation amplifies a ~432-bp fragment using a semi-nested-PCR protocol and primers  infects a variety of mammals, including humans and livestock , each of them having specific hosts. Thus, assemblage A can infect humans, other primates, livestock, dogs, cats, rodents, and other wild animals; similarly assemblage B is specific to humans and other primates, dogs, some species of wild mammals, assemblage C and D  are specific to dogs and other canids; assemblage E  to cattle and other hoofed livestock; assemblage F  to cats; and assemblage G (or G. simondi) to rats  of origin of the fecal contamination, its infective potential to productive animals (cattle), and its zoonotic potential.Therefore, in our study the idnetifying the genotype of G. duodenalis and Cryptosporidium subtypes associated with specific hosts and, therefore, fecal sources, three custom databases  were built by searching the NCBI-nr database and publicly available literature. DNA sequences were aligned using ClustalW-2.1 , and phylogenies were built in Molecular Evolutionary Genetics Analysis (MEGA) version 10.2.5 software  are a type of modified classification and regression tree (CART) to address fundamental limitations of traditional CART algorithms . It is important to note that this is a multivariable and not a multivariate analysis and is a standard approach used in environmental microbiology literature, including by water-focused studies throughout the past decade . Ctrees Giardia spp. were detected by PCR (Yes/No); tree analyses were implemented using the mlr and party packages and visualized using the partykit as previously described (Giardia detection versus non-detection), SMOTE resampling was performed as part of tuning  fecal coliform levels log transformed , (ii) whether fecal coliform levels exceeded the Chilean water quality standard of 1,000 fecal coliforms/100-ml (Yes/No), and (iii) if escribed ,c. Brieff tuning .Giardia, coliform levels and if coliform levels met the Chilean water standard  were also considered explanatory factors. Separately, nonparametric Spearman’s one-tailed tests were conducted to assess the correlation between continuous outcome variables  to enable comparison of the findings reported here with previous studies . In both analyses (Spearman’s correlation and GLM), differences with p < 0.05 were statistically significant. All statistical analyses were performed using RStudio v1.1.456 or R v3.6.3  tuned the maxdepth parameter, (ii) limited the upper bound of maxdepth to 6, and (iii) used a mincriterion of 0.95. Conditional forests analysis was used since it is robust to correlation and missingness in explanatory factors (by enabling surrogate splits), can handle both categorical and continuous outcomes, and can capture complex, hierarchical relationships  between explanatory factors. The environmental features considered were the river system ; predominant land use at the site ; season ; if feces, garbage, or livestock were present at the sampling site; pH; water temperature; total dissolved solids; salinity; if it rained during the 24 h before sampling; and weather conditions at the time of sampling . For es e.g., . A generR v3.6.3 .E. coli were present in water samples, a subset of samples  were tested . Briefly, 1 ml ultra-filtered samples were enriched in 5 ml of Buffer Peptone Water and incubated at 37°C for 24 h. Following incubation, 100 μl of the samples were streaked onto plates with MacConkey agar supplemented with Ciprofloxacin (1 mg/L), Cefotaxime (1 mg/L), and Tetracycline (4 mg/L) and incubated at 37°C for 24 h. Colonies were selected according to morphology using a magnifying glass. This selection was made according to standard patterns of color (pink), as described previously  compared to the Maule River  compared to the Maule River  collected downstream of wastewater discharges  were positive for G. duodenalis assemblages A or B  and lowest in samples collected from all other sites during the wet season  but were not significantly correlated with Cryptosporidium counts . However, the lack of a correlation between coliform and Cryptosporidium spp. levels may be due to the large number of Cryptosporidium-negative samples. According to GLM analysis, neither G. duodenalis  or Cryptosporidium spp.  detection were associated with fecal coliforms levels being compliant with the Chilean water quality standard of 1,000 CFU of fecal coliforms/100-ml.Spearman’s correlation analysis indicated that coliforms counts (MPN/100 ml) significantly, and positively correlated with E. coli were isolated, and their resistance profile was characterized. Twenty-eight out of 48 samples analyzed (58.3%) contained Gram-negative bacteria resistant to at least one of the antibiotics tested . We obtained 66 isolates; 26 (lactose positives) isolates were characterized at the species level by MALDI-TOF MS. Finally, we identified eight E. coli isolates and three of them showed a multi-drug resistant profile  compared to the Maule River (approx. 1 million) basin . Based on our findings efforts to mitigate fecal contamination in Central Chile should focus on the Maipo River, and specifically on sources of contamination in urban and agricultural regions. Further studies are needed to identify specific sources of contamination in urban and agricultural reaches of the Maipo River.The contamination of water with fecal material represents a problem for public health. Determining the source of fecal material could be useful to reduce the load of fecal material in rivers to designing mitigation measures that provide water with adequate quality for agriculture and livestock. In this study, fecal contamination levels were determined, and the probable sources of contamination were identified through an MST analysis, together with antimicrobial resistance, in two main rivers of central Chile based on land use. The main finding of our study suggests that: (i) microbiological water quality is affected by environmental conditions, particularly land use and season, and (ii) identification of n) basin . Indeed,n) basin . For exaGiardia levels, there was correlation between fecal coliform and Cryptosporidium levels, which may be driven by the small number of Cryptosporidium-positive samples in the present study. Indeed, a review that assessed relationships between FIBs and pathogens in water hypothesized that the failure to find associations between FIBs and the target was due to insufficient data , a positive association with pathogens was reported in two-thirds of them (n = 23), and the remaining studies did not support statistical analysis , C. meleagridis , and C. andersoni (in two samples from urban land use), which are specific to dogs, humans, and birds, and cattle, respectively  and free-ranging dogs, among others, inhabit the natural sections of the river systems studied  associated with rats and/or mice was detected in the livestock/forestry area in the Maule River  as the rivers in Chile naturally have high concentrations of metallic sediments  are ineffective , protozoa levels, and ARB presence in river water depended on the interaction of environmental factors such as river, land use, and season. In our study, the Maipo River had higher levels of coliforms , protozoa detection, and ARB than the Maule River. The land use that contributed most to the fecal and protozoa contamination of river water was after the discharge of wastewater treatment plants (urban treated), urban land use, and agriculture. Therefore, the environmental factors of land use, river, and season should be considered when designing studies to evaluate water contamination as the microbial contamination varies according to the human activity in the area of interest.Cryptosporidium spp. and assemblages of Giardia duodenalis indicate that humans, cattle, birds, rats/mice, and dogs are the most probable source of origin of the fecal contamination. Furthermore, these protozoa are known to be zoonotic and therefore may pose a potential public health threat if this water is directly or indirectly consumed. Therefore, based on our results, we can conclude that Cryptosporidium spp. and G. duodenalis detection and land use can be used as MST tools to provide information on the possible source of fecal contamination. Finally, we can say that the highest levels of fecal coliforms were detected in both the summer season and the land use classified as urban, and the largest amount of environmental ARB was isolated, increasing the probabilities of mobilization and dispersion of genetic elements involved in AMR. These data could aid in implementing measures to mitigate the dispersion of coliforms and ARB, thus reducing risks to public health.The presence of different species of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/CD-G, CB, LV, MK, AM-S, JO-P, and ADA designed the study, planned the field work logistics, and wrote the manuscript. DW and AA ran the statistical analysis, while CD-G constructed the phylogenetic trees. CD-G, CB, MS-C, EE, AA, MK, and ADA conducted experiments. DW, MS-C, EE, WS, MK, AM-S, JO-P, and ADA designed experiments and critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.We acknowledge the financial support received from the FONDECYT 11160116 and 1221536 to ADA, FONDECYT 1181167 to AM-S, and ANID Millennium Science Initiative/Millennium Initiative for Collaborative Research on Bacterial Resistance, MICROB-R, NCN17_081 to ADA, AM-S, and JO-P. Data analysis was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health (NIH) under award number T32Es007271.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.


Fatal and non-fatal youth (ages 0–17) injuries in U.S. agriculture continue to be a significant public health concern. Despite sustained work and attention from federally supported research programming, we continue to observe an unacceptably high number of life-altering and life-ending traumatic injuries to youth in agricultural environments. Likewise, there is still a gap in stringent systematic agricultural injury and/or illness surveillance at the federal level. This paper will provide an updated review of child agricultural injuries from U.S. news media reports, expanding upon this author team's initial 2018 report.Data collection from 2016 to 2021 occurred as part of the AgInjuryNews initiative, and data were coded according to the Farm and Agricultural Injury Classification (FAIC) system and the Occupational Injury and Illness Classification System (OIICS). The AgInjuryNews system primarily contains news media reports. Categorical variables were analyzed and compared using a chi-square test. In addition, the Jonckheere-Terpstra test for trend was used to test the yearly change in the number of youth injuries.We observed a general decrease in agricultural injuries compared to the original 2015–2017 dataset. Younger children (<5 years-old) and males were more often injured and more fatally injured than older children and females, respectively. Males and older victims were more likely to suffer an occupational-related injury compared to females and younger victims, respectively. Vehicles remained a major source of injuries, with tractors comprising 28%, and ATVs/UTVs comprising 26% of all injuries. Roadway incidents involving tractors and UTVs were less often fatal compared to non-roadway incidents, while ATVs were more fatal on roadways.AgInjuryNews.org. These data identify emerging issues within the industry and further inform national and international planning committees' work.This updated review shows childhood agricultural injuries and fatalities continue to be a major public health concern within the US. It is unclear if the trend downward in injuries is due to reporting, data capture methods, or a true decrease in injuries. These data continue to be of interest to stakeholders in academia, public health, government, and private industry—user groups who regularly and consistently seek this type of information, often from multiple data sources, including as registered users on  Agriculture remains one of the most dangerous occupations in the US and is the only occupation where children are legally allowed and often expected to actively engage on the worksite , 2. As oDespite this, there is currently no central data repository for youth agricultural injuries or fatalities in the U.S. Even within the most respected information sources of occupational fatalities, the Bureau of Labor Statistics' (BLS) Census of Fatal Occupational Injuries and Illness (CFOI), gaps exist—particularly for working and non-working youth in agriculture . These gIn a recent study of U.S. emergency department admissions, the authors uncovered 62,079 people treated for agricultural related injuries between 2015 and 2019, of which 30% of those injured were youth (age 0–17) . ApproxiThe key aim of this paper is to provide an updated review of youth (age 0–17) agricultural injuries by analyzing data harvested from U.S. news media reports. Our previous review, published in 2018, was an initial exploratory look at the first national dataset of its kind . News meGiven the ever-evolving field of production agriculture and limited availability of national agricultural injury surveillance data, an updated review is presumably welcomed, even necessary, for agricultural safety and health stakeholders in academia, government, and private industry—user groups who regularly and consistently seek this type of information, as evidenced by a recent report in safety .Data were collected as part of AgInjuryNews operations; detailed collection methods are further described in separate papers , 12. Conα = 0.82)  system and the Occupational Injury and Illness Classification System (OIICS) , 14. The = 0.82) . Finally = 0.82) . Inciden = 0.82) .Descriptive and summary statistics were determined for all cases in AgInjuryNews from 2016 to 2021. Geographic distributions were mapped using Tableau. We compared demographics, incident year, and FAIC and OIICS codes across fatal and non-fatal injuries, occupational vs. non-occupational, tractor, and ATV/UTV injury cases, mirroring the initial review. Categorical variables were analyzed and compared using a chi-square test. In addition, the Jonckheere-Terpstra test for trend was used to test the yearly change in the number of youth injuries. Statistical analyses were conducted in IBM SPSS Statistics version 27.0.n = 66) followed by October (n = 65) and July  between 2016 and 2021.  n = 63; .n = 53), Iowa (n = 43), and Pennsylvania (n = 42).Agricultural youth injury cases occurred in 43 states  and the n = 132, 28.8%). The <5 age group had proportionally higher fatal injuries than the other age groups . A total of 327 victims were male (73.6%), and the proportion of fatal injuries to male victims was significantly higher than fatal injuries to females (P < 0.001).Almost one-third of the victims were under 5 years old . There was no significant association between occupational relatedness and injury outcome . Non-occupational injuries mostly occurred as a result of exposure to farm equipment, tools, and product hazards . A total of 95 youth occupants of other vehicles were injured in agricultural roadway incidents (FAIC-9). Other injuries were related to farm structures and landscapes  and non-occupational animal-related injuries . Males and the 15–17-year age group had higher proportions of occupational injuries.Characteristics of occupational and non-occupational injuries are given in n = 31) and youth drivers (n = 15).Tractor-related incidents were associated with 153 injuries, and 77 of these happened on roadways . Similarvia ATV/UTV-related incidents, and more than half of these injuries were fatal . A total of 143 victims sustained injuries This study of U.S. news media reports expands upon current known trends from 2015 to 2017, providing further insight into how the landscape of child agriculture injuries is changing. We continue to observe an overrepresentation of male injuries across all variables, notably seeing an increase in the proportion of males suffering occupational injuries vs. non-occupational injuries compared to females. Adult supervision is one known strategy for reducing injuries, however, some studies have found that supervision alone is not always effective in the agricultural environment, especially for young children, who lack the developmental abilities to remain safe in this environment , 17. GivTransportation continues to be the largest source of injuries to youth on and around farms. While tractors remain a significant injury agent, ATV/UTV injuries appear to trend downward over time. The cause for this is difficult to identify, as we have seen regulations surrounding these vehicles change on a county by county basis throughout several states , 19. ThiAs mentioned, tractors remain a consistent injury agent in these incidents, including youth operators, passengers, or bystanders. Over 50% of youth tractor injuries occurred to children under 10 years-old; however, age did not appear to have a significant impact on the fatality of an injury. Roadway incidents continued to be more fatal than non-roadway incidents, often involving non-farming victims in passenger vehicles. This emphasizes the need for tractor safety courses aimed at youth operators, as well as bystander and non-working youth safety in areas where tractors are operating. Some U.S. states require youth to be at least 16 years-of-age before operating a tractor; however, some states allow children as young as 12 to be certified for public roadway operations, and there are many exceptions for those remaining on privately-owned land, non-work related activities, and more , 21.AgInjuryNews.org system. This is an excellent finding from this updated review, and suggests that injury prevention efforts with agricultural youth are having a positive impact. However, there may be other explanations for the downward trend. It is possible that some news media are shifting away from reporting on agricultural injuries, or instead focusing more on fewer, more serious cases. There is also the ongoing consolidation of farms across the U.S. and more corporate operations buy-out of smaller farms. These larger operations can be less willing to employ young workers, particularly young children under 14 that may have otherwise performed work duties on a family-owned operation. The most recent, comprehensive data on the number of youth agricultural workers is from 2014, making this determination difficult.Overall, we observed a slight downward trend in injuries reported through news agencies and captured by the AgInjuryNews.org. While the system utilizes many avenues of data collection with comprehensive search terms, significant changes in the wording and content of news reports can result in a lower capture rate. Finally, it is unknown what effect the COVID-19 pandemic had on agricultural hazard and risk exposures involving youth. Some studies suggest that youth did spend more time in the workplace, particularly in 2020  this represents actual counts of traumatic agricultural injury in the U.S. or (2) this decline will be sustained. While news media remain one of the few supplemental data sources for non-fatal agricultural injuries, continued monitoring of cases and, preferably, a more rigorous, centralized child agricultural injury surveillance program would be ideal. Expanding and simplifying injury reporting for small and non-employing operations can further improve capture rates of agricultural injuries.We observed a slight downward trend in agricultural youth injuries reported through news media from 2016 to 2021, though we recommend continual monitoring of injuries therein. We also suggest further research and policymaker attention on public roadway safety , tractor operations and limiting extra riders, ATV/UTV usage and regulation, and an emphasis of removing young children from the worksite. Each of these priority areas should be of concern for youth injury prevention.AgInjuryNews.org system continues to provide a hypothesis-generating information and a strong foundation for collaborations and partnerships throughout agricultural health and safety, including international expansion.Media monitoring remains a powerful tool for agricultural injury surveillance, particularly fatalities, and youth cases. Digital reports, such as news media, obituaries, and social media posts, are valuable data sources in an industry lacking a comprehensive injury surveillance system. The The variety and vastness of our nation's farms and ranches pose serious challenges, with limited formal safety regulations, incentives, or enforcement. However, that should not dissuade us from doing what we can—taking steps toward creating a safer, more sustainable industry and workforce. Continued improvement of our surveillance and injury monitoring methods are essential to ensure accurate and actionable agricultural injury data, to better inform health and safety guidelines, resources, and public policy.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.All authors participated in the conception or design of the work, the acquisition, analysis, or interpretation of data for the work, drafting the work and revising it critically for important intellectual content, final approval of the version to be submitted/published, and all agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.1025545. Funding for the AgInjuryNews program has been made possible, in part by the National Children's Center for Rural and Agricultural Health and Safety through the National Institute for Occupational Safety and Health Cooperative Agreement U54 OH009568, a cooperative agreement award for the Centers for Disease Control and Prevention/National Institute for Occupational Safety and Health. Funding support was also provided through a safety grant from the Agricultural Safety and Health Council of America (ASHCA), the University of Wisconsin Clinical and Translational Science Award (CTSA) program, through the NIH National Center for Advancing Translational Sciences (NCATS), grant UL1TR002373, and through collaborators, donors, and friends of the National Farm Medicine Center, part of the Marshfield Clinic Research Institute.Funding support for this work was provided through the National Farm Medicine Center and Marshfield Clinic Research Institute and the USDA National Institute of Food and Agriculture: Project FLA-ABE-006052, Accession number 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.


Background: Two-dimensional speckle-tracking echocardiography (2DSTE) has been present for years. However, it is underutilized due to the expertise and time requirements for its analysis. Our aims were to provide strain values in a paediatric Spanish population and to assess the feasibility and reproducibility of a new strain software analysis in our environment. Methods: A cross-sectional study of 156 healthy children aged 6 to 17 years. Longitudinal strain (LS) analysis of the left ventricle, right ventricle, and left atrium was performed. Feasibility and reproducibility were assessed. The associations of clinical and echocardiographic variables with strain values were investigated by multivariate analysis. Results: Mean age was 11 ± 3 years . Feasibility of LS measurement ranged from 94.2% for left ventricle global LS (LVGLS) to 98.1% for other chamber strain parameters. Strain values were 26.7 ± 2.3% for LVGLS; 30.5 ± 4.4% and 26.9 ± 4% for right ventricle free wall LS (RVFWLS) and four chambers view LS (RV4CLS) respectively; and 57.8 ± 10.5%, 44.9 ± 9.5%, and 12.9 ± 5.5% for left atrium LS reservoir phase (LALSr), conduct phase (LALScd) and contraction phase (LALSct), also respectively. Body surface area (BSA) and age presented a negative correlation with strain values. Higher values were found in females than in males, except for LALScd. Excellent intra- and inter-observer reproducibility were found for right and left ventricular strain measurement, with intraclass correlation coefficients (ICC) ranging from 0.88 to 0.98, respectively. In conclusion, we described strain values in a healthy Spanish paediatric population. LS assessment by this new strain analysis software by semi-automatic manner was highly feasible and reproducible.  The assessment of cardiac function requires continuous advances. There is great interest in finding a reliable, reproducible and easily accessible tool to assess both systolic and diastolic function . MyocardCurrently, its most widespread use is the assessment of longitudinal strain by 2DSTE . HoweverMyocardial strain evaluation in the paediatric population is of growing interest. Normal strain values have been reported by different authors for the left ventricle ,10,11,12Most data have been reported in an independent manner for each cardiac chamber, using various methodologies, different equipment, and even different parameters in their report; this is understandable since most papers are prior to the publication of the current recommendations ,21. Our The research was structured as a prospective, cross-sectional descriptive study. It was conducted in a locality of 2864 inhabitants in the south of Spain between March 2017 and April 2018. Participation was offered freely in all the educative institutions of the city and to all its students.. The main exclusion factors were pre-existing cardiopulmonary or systemic diseases, including congenital diseases and such cases detected during the study; first-degree family history of sudden cardiac death (SCD) or diagnosed cardiomyopathy. The age limit was 17 years old. Anthropometric measurements and blood pressure were taken prior to the echocardiographic study. The Haycock formula was used for body surface area . ParticiAll studies were performed with the same echocardiography equipment iE33  with the S5-1 probe 1–5 MHz. The images were taken in parasternal long and short axis, apical, subcostal and suprasternal views, as well as in non-conventional views when necessary. The images were digitally stored as raw data and analysed off-line in Q Station 3.7 for conventional measurements. These analyses included dimensions, volumes and systolic and diastolic function and followed the current recommendations for studies in paediatric populations ,25. For the study of myocardial deformation, digital acquisition of at least three cardiac cycles was performed in the apical two-, three- and four-chamber views for the left ventricle. A focused apical view for the right ventricle and left atrium was also acquired for both. Equipment settings, such as sector width and depth, were optimized to obtain an image frequency of at least 60 Hz. All images were acquired by two expert echocardiographers (CAD and JFC).Strain analysis was performed with the new Q-station, version 13.0 of Qlab , which integrates the TOMTEC auto-strain software and has been designed according to the new recommendations for the assessment of longitudinal strain. Whilst the longitudinal deformation, except for LALSr, gives negative values, in order to a better understanding of the data, we will express the measurements in absolute numbers, as proposed by Flashkampf, FA et al. , partingLeft Ventricle: The three left ventricle apical windows were selected according to the vendor specifications, and the left ventricle auto-strain function was activated. After the labelling of the phases and tracking, which was performed automatically by the software, manual correction was added when appropriate A.Right Ventricle: A focused image of the right ventricle was selected. Software analysis was performed specifically for this chamber. The right ventricle auto-strain function was activated. After automatic tracking and analysis by the software, the borders were manually corrected when necessary, and two results were provided: an average free wall view and a four-chamber view, including the septum segments B. For leLeft atrium: An apical four-chamber window focused on the left atrium was used. As for the right ventricle, the analysis software is specific to this chamber. The left atrial automated strain was activated, and the system provided the direct analysis of the three phases of the atrium: reservoir, conduit and atrial contraction, which were manually corrected if necessary C. For thp < 0.01. The feasibility of the strain measures was expressed in percentages, estimated as a ratio of the number of interpretable cases to the number of assessed cases, multiplied by one hundred.The normality of the data distribution was assessed using the Kolmogorov-Smirnov method. Continuous variables were expressed as mean and standard deviation (SD) or as median and interquartile ranges for variables with a non-normal distribution, whereas proportions data were expressed as frequencies and percentages. For the comparison of subgroups, ANOVA was used for quantitative variables with Bonferroni or Games-Howell post hoc analysis as appropriate, and the chi-square test was used for qualitative variables. The associations of strain values with the different anthropometric, echocardiographic and sex variables were evaluated by univariate regression analysis and multivariate models for those with a significance https://www.ibm.com/analytics/spss-statistics-software, accessed on 2 June 2022), and a p < 0.05 was considered significant.The intra and inter-observer variability assessment was performed by combining the intraclass correlation coefficient and Bland-Altman plots. To assess intra-observer variability, the main analyst (CAD) randomly repeated the analysis on 20 random cases three months after the previous analysis and blinded the previously obtained values. As for the establishment of the inter-observer variability, an additional 20 different cases were randomly selected, and another echocardiographer (JFC) independently analysed the data in a blinded manner with respect to the values obtained by the main analyst. SPSS for Windows, version 21.0  was used for the analysis,  were females. The mean age was 11 ± 3 years. Significant differences were found between males and females in systolic blood pressure (SBP), basal right ventricular diameter, left atrial volume, left ventricular end-diastolic volume and other conventional echocardiographic parameters, although all values were within normal limits. The samples were analysed by age group using terciles, which proved to be the most homogeneous form of distribution for our sample. As expected, significant differences were found in most of the anthropometric and echocardiographic variables, except for left ventricular ejection fraction (LVEF), mitral A velocity and TEI index. The feasibility of all cardiac chamber strain analyses was studied by age group and sex. For the entire sample, it ranged from 94.2% for the left ventricle to 98.1% for several strain parameters of the right ventricle and the left atrium. In the categorized analysis, it ranged from 87.5% for the left ventricle in the age group 10 to 12 years and up to 100% for all variables except for the left ventricle in the age group 6 to 9 years. Average strain values were significantly higher in female than male subjects for all variables except for LALScd . We alsoFinally, higher BSA was inversely related to mean strain values for most variables in bivariate correlations , even th2: 0.091) was observed for the model. The results are summarized in In addition to age, sex and BSA, LVGLS was significantly associated with SBP, left ventricle mass, left ventricle diastolic diameter (DDLV), end diastolic volume and left ventricle ejection fraction (LVEF). In the multivariate regression model, only EDV and LVEF were independent predictors of LVGLS. However, an extremely low coefficient of determination  basal, right ventricle diastolic area (RVDA) and TAPSE. 2: 0.276) was also observed. In the multivariate regression model, only BSA, LV mass and sex were independently associated with RVFWLS. A poor coefficient of determination (R2: 0.227) for the final model. The results are summarized in Regarding the RV4CLS, other variables were significantly associated within the univariate analysis in addition to age, sex and BSA: LV mass, DDLV, EDV, RV basal, A vel and left atrium volume (LA vol). In the multivariate analysis, only age and sex were independent predictors, with a low coefficient of determination .The variables that were significantly associated with LALScd in univariate analysis were LV mass, DDLV, EDV, RV basal and LA vol. The only independent predictor in multivariate analysis was LV mass, also with a very low coefficient or determination for the model (R2: 0.067). The results are summarized in Finally, LALSct was associated with sex, RVDA, A vel, tissue doppler velocity (TDV) A septal and E/A ratio. Only A velocity was independently associated with this variable in the multivariate regression model, also with a low coefficient or determination for the model , in agreement with the values reported by Cantinotti et al., who have the largest sample and developed their study using the Philips platform; our values are practically the same as theirs in the comparable age groups. This strongly suggests that there are still inter-vendor differences, at least with the main samples reported in this age group.Our research aim is to show the possibility of assessing strain in the paediatric population in a user-friendly way and a replicable manner using currently available methods. We are convinced that the strain values that we have reported can add value to the already growing information on strain in children and adolescents that is currently available. The findings can encourage other groups to make use of this or similar methods and include them in their daily use of strain cardiac function a reality.To the best of our knowledge, our data represent the first report of strain values in school-aged paediatric population in our environment obtained with this new software according to the latest strain analysis recommendations.®). However, at the time of this study, there was no other commercial platform available with specific analysis software for each cardiac chamber apart from the one we used in this study. Recently, other commercial vendors have made available similar tools to the one we used, and comparisons between them should be the subject of future study.Given the free nature and the participating centres, the sample contains a small number of subjects from a single geographic region and common ethnicity. Our sample did not include children under 6 years of age. No neonates or young children were included. We did not make any exclusions based on ethnic origin, which differs from other studies that have done so. We used only one vendor for the analysis (PhilipsIn addition, even though we acquired short-axis images for circumferential and radial strain analysis, to date the platform does not have specific semi-automatic or automatic software for this purpose. Therefore, it was decided not to include this in the study.Another limitation is that left atrial strain could only be performed in the apical four-chamber view in the version used, although this is in agreement with most previous studies and is considered adequate under current recommendations .We did not compare with nuclear magnetic resonance, given the limited availability of the technique in our centre and the large number of subjects.We recognise that a comparative analysis between the three different methods, fully automatic, semi-automatic and manual, could be of interest. However, this has already been done previously . In addiFinally, the correlation R-values are very low for most comparisons. In addition, much of the data, although they show few statistical differences, are not clinically important; given the weak relationship we found between the correlation coefficient strain values and age and BSA, we decided not to perform Z-score analysis for nomogram construction as other studies have done.We report that the analysis of the left ventricle, the right ventricle and the left atrium is highly accurate and feasible using this new software in semi-automatic mode. In a discrete manner, the strain values for all cardiac chambers present relationships with age, BSA, and sex, especially for the right ventricle. Left atrial strain values are less replicable than the ventricular strain values and are related to atrial volumes and parameters affecting left ventricular diastolic function.


Drosophila melanogaster. Some glutathione transferases are tissue-specific, others are specifically underexpressed in some tissues, and some appear expressed at the same level in all the tested tissues. All glutathione transferases present a specific pattern that does not depend on their sequence proximity. This study allows us to better understand the potential role of these enzymes by analyzing their expression profile and evolutionary links.Glutathione transferases are key enzymes found in all living species. In insects, two classes of these enzymes are specific: the Delta and Epsilon classes. These classes of glutathione transferases are generally considered supports during the evolution for the adaptability of the species of an insect. We measured the expression levels of the 25 enzymes forming these two classes in different organs of Drosophila melanogaster, Delta and Epsilon are the largest classes, with 25 members. The Delta and Epsilon classes are involved in different functions, such as insecticide resistance and ecdysone biosynthesis. The insect GST number variability is due mainly to these classes. Thus, they are generally considered supports during the evolution for the adaptability of the insect species. To explore the link between Delta and Epsilon GST and their evolution, we analyzed the sequences using bioinformatic tools. Subgroups appear within the Delta and Epsilon GSTs with different levels of diversification. The diversification also appears in the sequences showing differences in the active site. Additionally, amino acids essential for structural stability or dimerization appear conserved in all GSTs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the transcripts corresponding to these two classes are heterogeneously expressed within D. melanogaster. Some GSTs, such as GSTD1, are highly expressed in all tissues, suggesting their general function in detoxification. Conversely, some others, such as GSTD11 or GSTE4, are specifically expressed at a high level specifically in antennae, suggesting a potential role in olfaction.Glutathione transferases (GSTs) are ubiquitous enzymes that catalyze the conjugation of glutathione to various molecules. Among the 42 GSTs identified in  Drosophila melanogaster [D. melanogaster, 42 GSTs belonging to six different classes have been identified: Theta, Omega, Sigma, Zeta, Delta, and Epsilon. The Delta and Epsilon classes appear to be specific to arthropods, including insects and, thus, D. melanogaster. All solved structures of Delta and Epsilon GSTs are homodimeric, similar to most GSTs. The GST numbers are variable among the different insect species [Apis mellifera has only one GST member in the Delta class and not any GST member in the Epsilon class. In contrast, more than half of the GSTs belong to these two classes in D. melanogaster or Anopheles gambiae. This variation has been proposed to play a key role in the adaptation of insects in response to environmental selection pressures [D. melanogaster, 25 belong to the Delta and Epsilon classes , representing the largest classes. All 25 GSTDs and GSTEs present glutathione transferase activity and can potentially be involved in the detoxification process; however, only GSTD3 does not exhibit this activity [Glutathione transferases (GST) are ubiquitous enzymes that are present in all kingdoms of life. They represent a key family of enzymes involved in various processes, including detoxification. GST members constitute the GSTome  and can nogaster . The micactivity .D. melanogaster provides significant protection against isothiocyanates found in cruciferous species [GSTs are known to belong to the phase II enzymes of the general detoxification process. This process can be divided into three phases, including a first phase with enzymes involved in the activation of exogenous molecules, and a second phase where different enzymes can act, including GSTs, which is dedicated to the enzymatic addition of a polar group. The third and last phase occurs when the first two phases act inside the cells and consist of the elimination of the polar molecule formed from the cell. Drosophila GSTs have also been shown to be active toward endogenous toxic compounds such as 4-hydroxynonenal, which is highly reactive and can affect protein function . The cat species . HoweverD. melanogaster organs using quantitative polymerase chain reaction (qPCR) analysis. After an analysis of the Delta and Epsilon GST amino acid sequences, the expression level of the corresponding coding mRNA will be measured in the different organs of the fly. Then, this localization will be discussed with respect to the knowledge of its biological function.Because of the redundancy of GST Epsilon and Delta functions, some of these GSTs have potentially evolved to support new functions in different insect species. Moreover, some organs are more prone to be in contact with xenobiotics than others. Thus, one can wonder if GST expression differs depending on the organ. GST exhibiting a specific function could be expressed in a specific organ, whereas GST with a generalist detoxification function could have ubiquitous expression in the body. This work aims to decipher the GST Delta and Epsilon amino acid sequences and their localization in different https://flybase.org/ (accessed on 20 May 2022)) [https://bioedit.software.informer.com/ (accessed on 20 May 2022)).The amino acid GST sequences were downloaded from the flybase website (y 2022)) . Sequenchttps://flybase.org/ (accessed on 20 May 2022)) and aligned using the online software MAFFT version 7 using the progressive method G-INS-I [http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 20 May 2022)).For the phylogeny, amino acid sequences from the Delta and Epsilon clades were retrieved from flybase , and the carcass  were collected from 5-day-old adult males. In detail, after separating the heads from the bodies, the labella were dissected manually and the heads that still have the antennae and maxillary palps were placed in liquid nitrogen. Frozen heads were placed on a device comprising a tube surmounted by meshes and vortexed to separate olfactory appendages from the head. Finally, the gut was dissected from the rest of the body without the wings and legs. For the appendage, head without appendages, gut and carcass RNA extraction, we used about 1000 chemosensory appendages, 10 heads, 20 guts and 15 carcasses, respectively. Total RNA was extracted using TRIzol reagent  and treated with RNase-free DNAse  to avoid genomic DNA contamination. Total RNA was reverse-transcribed using the iScript cDNA Synthesis Kit . The qPCRs were carried out on a Step One real-time PCR system  using the Takyon Low Rox SYBR . Each reaction was performed in triplicate with at least three independent biological replicates, and all results were normalized to the Rpl18, Rps20 and Rpl32 mRNA levels and calculated using the Pfaffl method . SpecifiD. melanogaster harbor the Ser residue, contrary to the Delta class. Indeed, GSTD3 has a shorter sequence with a missing N-terminal part, explaining the absence of Ser. GSTD3 did not present any activity with the tested substrates [https://services.healthtech.dtu.dk/service.php?SignalP-5.0 (accessed on 12 May 2022)); however, it cannot be ruled out that it is a noncanonical signal peptide. Delta and Epsilon Drosophila GSTs also share common features in their amino acid sequences. First, the glutathione-binding site consists of residues from the thioredoxin fold, including the conserved cis-Pro of the β3 strand and the first two residues N-terminal (Glu/Asp-Ser) to the α3 helix, also including a His residue from a conserved beta turn that joins helix α2 and the β3 strand. Second, the α3 helix and α4 helix are fairly conserved in both classes due to their importance for dimerization. The conserved residues further include those involved in dimerization motifs, such as the “lock-and-key” residue, which is a Tyr/Phe for the Delta GST class (named ‘clasp’) and a His for the Epsilon class (named ‘wafer’) [Sequence alignment of Delta and Epsilon GSTs reveals the presence of some conserved amino acids  ,35,36,37‘wafer’) . Third, ‘wafer’) . The ideD. melanogaster . Phylogenic analysis of the Delta and Epsilon GST protein sequences allowed us to observe two separate groups, one for each class (D. melanogaster. Epsilon GSTs are organized into three subgroups: one resulting from an ancient separation, including GSTE11, GSTE12, GSTE13 and GSTE14. The two other groups of Epsilon were formed a second time. The diversification of the last two groups was scaled on the largest period of time compared to the Delta GSTs. Compared with the other Delta GSTs, GSTE13 and GSTE14 have higher evolutionary rates, as mirrored by the relatively long branch lengths in the Delta clade of the phylogeny.The Delta and Epsilon classes specifically found in arthropods appear with a large number of members in ch class . The DelTo support the potential functions of the 25 Delta and Epsilon GSTs, the transcript abundance of the corresponding genes was measured in the antennae, labellum, head, gut and body . SensoryGSTE4, GSTD1, GSTE12 and GSTD4 were the most highly expressed in antennae. Some of them are highly specific to antennae compared to the other organs tested . For exaGSTD4, GSTD1 and GSTE12 appeared to be most highly expressed in the proboscis . These tAs in the labellum, the three most expressed GSTs in the gut, head without appendages, and carcass are GSTD4, GSTE12 and GSTD1. However, they are expressed at a lower level compared to the labellum, and the lowest expression is detected in the head . InteresGSTs are ubiquitous enzymes; six classes are found in insects, including the Delta and Epsilon classes, which are insect-specific. Delta and Epsilon GSTs have been shown to be involved in different functions, such as insecticide resistance and ecdysone biosynthesis. Phylogenetic analysis revealed the diversity of the Delta and Epsilon classes, supported by the different subgroups emerging within these classes in the phylogenetic tree. Sequence analysis additionally shows amino acid differences in the sequence forming the active site as well as the amino acid sequence involved in the dimerization motif. The length of the GST sequences also diverges between sequences. GSTD11b appears with an additional N-terminal part, and GSTD3 appears shorter. Additionally, in this study, qPCR analysis of mRNA-encoding GST Delta and Epsilon was performed. First, the results on Delta and Epsilon GSTs support a previous RNA sequencing analysis conducted on other organs . Both ap


Background: Although vascular risk factors have been found to be closely related to the development of benign paroxysmal positional vertigo (BPPV), the relationship between BPPV and cerebral small vessels diseases (CSVDs) has rarely been discussed in literature. This study set out to investigate the efficacy of repositioning therapy and prognosis among BPPV patients with CSVDs.Methods: We enrolled 553 BPPV patients who had undergone brain MRI, and categorized them into two groups based on the presence or absence of CSVDs. After controlling for other confounders using a propensity score matching (PSM) approach, we compared the incidence of recurrence and residual dizziness (RD). Then, we analyzed the recurrence rate and RD incidence in 176 BPPV patients with CSVDs, and assessed potential risk factors.Results: White matter hyperintensity  and lacunar infarction  were the two CSVDs that were present in the highest proportion among the BPPV patients. The incidence of RD in patients with CSVDs was significantly higher compared to subjects without CSVDs. Patients with RD  were older, had more severe WMH, and had a higher incidence of brain atrophy; age and higher Fazekas score were independent risk factors. Among the recurrent patients , the ages were older, the Fazekas score of WMH was higher, and number of LIs was increased; age was the sole independent risk factor.Conclusion: BPPV patients with a combination of CSVD comorbidities, especially elderly patients with WMHs, are more likely to develop RD, which needs to be paid more attention. Benign paroxysmal positional vertigo (BPPV), a peripheral vestibular disease, refers to repeated episodes of vertigo that are triggered by a change in head position In recent years, some studies have shown that vascular risk factors are closely related to the occurrence and prognosis of BPPV Brain MRI is not been routinely conducted for patients with BPPV. However, the pathogenesis of acute vertigo can sometimes be very complicated, especially in middle-aged and elderly patients with more basic diseases and a complicated past medical history. Therefore, sometimes we may need an MRI to check whether such patients have other brain diseases Although great progress has been made in the diagnosis and treatment of BPPV in recent years, there are different prognoses for the same treatment method This is a retrospective study that was granted approval by the Ethics Committee of the First Affiliated Hospital of China Medical University, and was granted a waiver of written informed consent based on the study's retrospective design and anonymous data collection. The study was performed in accordance with the declaration of Helsinki.Patients who were diagnosed with BPPV and treated at the Department of Otorhinolaryngology, the First Affiliated Hospital of China Medical University from December 2016 to May 2020 were enrolled retrospectively. All patients had undergone brain MRI examination for complaining of acute-onset dizziness at emergency department or neurology department before been transferred to the vertigo clinic of ENT department. The inclusion criteria are as follows: 1. recurrent episodes of transient vestibular vertigo induced by gravity-related head position changes; 2. Dix-Hallpike test or Roll test can induce vertigo and characteristic nystagmus; 3. the patients were clearly conscious and able to cooperate in the canalith repositioning procedure (CRP) treatment, with effective results; 4. MRI was performed within 1 week after onset; 5. denied a previous history of dizziness.The exclusion criteria were as follows: 1. insufficient medical records; 2. patients who failed complete follow-up; 3. history of head trauma or head surgery; 4. intracranial tumor; 5. inner and middle ear diseases including Ménière's disease, vestibular migraine, sudden deafness, vestibular neuritis, otitis media; 6. use of ototoxic drugs; 7. patients with bilateral or multiple semicircular involvements; 8. cerebrovascular diseases due to cardiac and large vessel obstruction; 9. progressive multifocal leukoencephalopathy; 10. history of atrial fibrillation, myocardial infarction, valvular heart disease, infective endocarditis, myocarditis, mitral stenosis, or aneurysm; 11. severe extracranial and intracranial large blood vessels diseases; 12. evidence of known vasculitis; 13. multiple sclerosis, or other metabolic or toxic leukoencephalopathy; 14. osteoporosis.The eligible subjects were separated into two groups based on presence of CSVDs. Age, gender, brain MRI results, features of BPPV , duration of BPPV, times of CRP, as well as the recurrence and RD of BPPV after successful treatment were recorded. We used a propensity score analysis to match CSVDs patients with non-CSVDs patients at a ratio of 1:1.Patients with posterior canal (PSC) BPPV were treated using the Epley maneuver. The Barbecue maneuver was performed for treatment of geotropic horizontal canal (HC) BPPV. The Gufoni maneuver was used for apogeotropic HC BPPV. Details of the Epley, Gufoni, and Barbecue maneuvers were described in clinical practice guidelines of BPPV established by the AAO-HNSF (American Academy of Otolaryngology-Head and Neck Surgery Foundation) RD was defined as a feeling of unsteadiness and/or light-headedness and/or dizziness in absence of true vertigo and nystagmus, despite successful repositioning. The recurrence was defined as further BPPV episodes similar as the primary attack and the positive result of positional nystagmus test during follow-up. Three days after CRP treatment, BPPV patients returned to clinic for evaluation of efficacy. A successful CRP was defined as an absence of both positional vertigo and positional nystagmus A 3-T system scanner  was utilized for MRI. Parameters of spin-echo T1-weighted imaging (T1WI) included repetition time (TR) of 2000 ms, echo time (TE) of 17 ms, slice thickness of 5 mm, and slice distance of 1.5 mm. Parameters of spin-echo T2-weighted imaging (T2WI) included TR of 3500 ms, TE of 95 ms, slice thickness of 5 mm, and slice distance 1.5 mm. Parameters of fluid attenuated inversion recovery (FLAIR) sequences included TR of 8000 ms, TE of 104 ms, slice thickness of 5 mm, and slice distance of 1.5 mm. Susceptibility weighted imaging(SWI) included TR of 27 ms, TE of 20 ms, slice thickness 5 mm, and slice distance of 1.5 mm. The field of view was 230 mm × 230 mm, while the construction matrix was 192×256, and standard T2 and FLAIR for T2WI and FLAIR.The MRI features of vasogenic WMH are high-intensity signal on T2 weighted (T2WI) images and Flair sequences, which showed punctate, patchy or confluent high intensity signal in dots, patches or fusion, and iso-signal or low signal on T1 weighted (T1WI) images. We further graded WMH according to the Fazekas LI is manifested as a well-defined lesion with a diameter of 3-15 mm, T1WI with low intensity signal and high intensity signal on T2WI, and needs to be distinguished from EPVS . We determined and counted the total number of LIs in each patient.The imaging definition of CMB is on a SWI sequence, which shows a uniform, round, well-defined boundary, low-signal focus/lesion with a diameter of 2-5 mm and no peripheral edema. It is necessary to exclude the vascular flow void and calcification without hemorrhage.The EPVS manifests as a gap consistent with the course of the blood vessel. In the vertical plane of the blood vessel, there is often a round or oval cerebrospinal fluid signal with a diameter of less than 3 mm. The signal intensity is low on T1WI images, high-signal on T2WI and low-signal on Flair images.Brain atrophy is characterized by a reduction in brain volume, which is reflected by an enlargement of ventricles and sulcus.All brain MRI were reviewed and evaluated by an experienced radiologist.P<0.05 at univariate test statistics were regarded as covariates and included in multifactorial logistic regression model as potential predictors. The odds ratio (OR) and 95% confidence interval (CI) were calculated. A difference was considered statistically significant if P <0.05.Software SPSS 25.0 was utilized for statistical data analyses. We used a propensity score analysis to match CSVDs patients with non-CSVDs patients at a ratio of 1:1 with a match tolerance 0.02 and based on age, gender, laterality, previous BPPV history, duration of BPPV, number of CRPs and involved canal. Categorical variables were represented as either counts or percentages. For comparison among groups, the Chi-square test, Fisher exact test and McNemar test were used. Independent-samples t-test and paired t-test were used for continuous variables which were normal distribution and described by mean ± standard deviation (SD). The Mann-Whitney U and Wilcoxon test were used for continuous variables with an abnormal distribution and described by median . A binary logistic regression model helped determine risk factors for recurrence and RD of BPPV. All variables with Overall, 689 patients who met the inclusion criteria were initially enrolled in the study. According to the exclusion criteria, 136 patients were then excluded at screening. Finally, a total of 553 patients were found to be eligible. Among them, 176 BPPV patients whose brain MRI showed the presence of CSVDs, including 74 males and 102 females with an age range of 31-91 years (median: 68). The distribution of CSVDs among 176 BPPV patients with CSVDs is shown in Figure After matching the two groups using propensity score analysis based on age, gender, laterality, previous history of BPPV, duration of BPPV, CRP times and involved semicircular canal types, we found a statistically significant difference in RD. Although there were more cases of 1-year recurrence in the CSVDs group, the difference was insignificant (Table As WMH 72.2%) and LI (65.9%) are the two CSVDs with the highest proportion in BPPV patients Figure , we furt.2% and LIn multivariate logistic regression analysis for RD, when other factors remained unchanged, for every one point increase in Fazekas score of WMH, the probability for RD occurrence was 1.078 times higher compared to if the Fazekas score did not increase . Similarly, for every year increase in age, the probability for RD occurrence was 1.397 higher times compared to if the age did not increase . Hence, it can be concluded that increased severity of cerebral white matter lesions and age are risk factors for RD. Among the multivariate logistic regression analysis for recurrence, we found the increased age was an independent risk factor .CSVDs are common among middle-aged and elderly people, and often are accompanied by no obvious clinical symptoms et al. found that about 70% of patients had WMH et al. also noted that stroke can be an independent risk factor for BPPV The concomitant CSVDs among BPPV patients in this study include WMH, LI, EPVS, CMB and brain atrophy. Among them, WMH was the most common CSVD, which is consistent with the findings of Cha and others. Through investigation of the brain MRI of BPPV patients in the emergency department, Cha Pathological changes, including cerebral small vessel stenosis or occlusion, are mostly chronic, and there are no obvious stroke events in clinic Frequent recurrence of BPPV seriously affects the quality of life and makes patients anxious. Hence, the early identification and prompt management of BPPV is very important. We excluded patients with a history of head trauma, Meniere's disease, migraine, osteoporosis, simultaneous bilateral or multiple canals involvement and other factors that could lead to the recurrence of BPPV Several studies have identified that non-rotational dizziness and walking instability continues to exist after a successful repositioning of BPPV, with an incidence rate of 31.1% to 61%, and is termed as residual dizziness (RD) et al. found a higher percentage of RD in BPPV patients with central neurologic disorders, a result which needs to be verified in a larger case-control study et al., who found that the presence of WMH is a predictor for chronic RD among patients with previous vestibular neuritis et al. found that the severity of WHM is higher among patients with unexplained dizziness compared to those with specific causes  In this study, we determined that the incidence of RD in the CSVDs group is higher compared to the non-CSVDs group, after matching on the propensity score; the differences are statistically significant. This suggests that BPPV patients with CSVDs are more likely to have RD. Chen et al. found that brain atrophy was related to long-lasting dizziness after BPPV treatment and speculated that there may be a relationship between brain atrophy and otoneurologic diseases In addition, some studies have shown that RD may be related to the duration of vertigo prior to CRP Therefore, it is of great clinical significance that we identify BPPV patients who are prone to RD and recurrence, as well as to intervene with treatment. In addition to CRPs, vestibular rehabilitation exercises have been shown to be effective at controlling RD symptoms and preventing BPPV recurrence We investigated the type and proportion of CSVD in BPPV patients with CSVDs and focused on determining the prognosis of BPPV patients with CSVDs, including recurrence rate and RD after successful CRP. WMH and LI were found to be the most prevalent types of CSVD in among BPPV patients. After controlling for other confounders using a propensity score matching approach, we found that patients with CSVDs were more likely to have RD. In addition, logistic regression analysis also indicated that increased age and WMH level were independent risk factors for RD. Accordingly, we conclude that older BPPV patients with WMHs are more likely to develop RD, and therefore need to be paid more attention. Perhaps, for BPPV patients with CSVDs, we should actively treat CSVDs at the same time as CRP treatment in order to reduce the risk of RD. We will expand the sample size in future studies and design controlled trials to further validate our findings.


Objective: Cardiorespiratory fitness (CRF) reference data for Tibetan (Zang ethnicity) children and adolescents at high altitudes in Tibet of China are lacking. The present study aimed to develop sex- and age-specific 20mSRT norms for Chinese Tibetan children and adolescents at high altitudes. Method: A total of 4667 participants from Lhasa (3650 m), Nagqu (4500 m), and Amdo (4700 m) were selected by a stratified random cluster sampling method in two stages. The 20 m SRT test was used to estimate cardiorespiratory fitness. The 20 m SRT norms were developed by the lambda, mu, and sigma method (LMS). Results: The 20 m SRT laps, completed stages/minutes, and the speed at the last complete stage of Chinese Tibetan children and adolescents aged 7–18 years increased with age. Conclusion: Given the importance of CRF for children and adolescents’ health, the government should strengthen the monitoring of the CRF of Tibetan children and adolescents in high-altitude areas, strengthen physical education curriculum reform, and increase the level of physical activity in order to improve the level of CRF in children and adolescents. Substan 0.0001) .2max in children and adolescents  [The 20-m shuttle run test (20-m SRT), a simple and cost-effective test, can test large groups of children simultaneously ,10. It w < 0.05) . Additio < 0.05) . Hence,  < 0.05)  and has  < 0.05) ,15.Assessing CRF from an early age may help to identify the target population for primary prevention as well as be helpful for health promotion policies. The key to scientifically and accurately assessing CRF lies in developing normative values. International sex- and age-specific 20 m SRT norms based on 1,142,026 children and adolescents from 50 countries were established for health and fitness screening and monitoring . FurtherKnown as the “roof of the world”, the Tibet Plateau has a typical plateau climate and is home to about 5 million Tibetans, with 66% of them living at altitudes above 3500 m, and with ancestors having lived at similar altitudes for more than 3000 years . To adapGiven the large Tibetan population living in Tibet, China, the present study aimed to develop sex- and age-specific 20mSRT norms for Chinese Tibetan children and adolescents at high altitudes.From October to December 2019, the research was conducted in Tibet, China. Participants meeting the following requirements were included in the study: (1) had family that lived in Tibet for two generations or more; (2) both the students and their parents are Tibetan (Zang ethnicity); (3) provided written consent from themselves and their parents; (4) had no physical or mental illness.A stratified random cluster sampling method was used for the selection of participants in two stages. In the first stage, according to geographical distribution and population distribution, Lhasa (3650 m), Nagqu (4500 m), and Amdo (4700 m) were selected, and 21 public schools were selected from the three cities. In the second stage, according to the school size, 2–8 classes were randomly selected from each grade in the 21 public schools. Students in the selected classes meeting the inclusion criteria were recruited as participants. A total of 4982 Tibetan children and adolescents aged 7–18 were included in this research. After excluding 315 (6.32%) data because of missing information, 4667 data were included in the present study.This study was approved by the Human Subjects Protection Committee of East China Normal University (HR0782020). To protect the privacy of the participants, the names of participants were numbered, and the data was strictly managed.The 20mSRT test was conducted according to the FitnessGram protocol . The mea−1) were calculated according to laps.Completed stages/minutes and speed at the last complete stage , which is a widely accepted method in reference centile curves, was used to develop 20mSRT norms  for Chinese Tibetan children and adolescents ,29,30. CFor the speed at the last completed stage, differences in means between (a) age-matched Chinese Tibetan boys and girls ; (b) sex-matched Tibetan children of different ages  were expressed as standardized effect sizes . In age-p = 0.05. The age was calculated based on the last birthday .All statistics were processed and analyzed using SPSS 25.0  software and Graph Pad Prism 8, and the significance level was set at −1) at the last complete stage for Chinese Tibetan children and adolescents living at high altitude in Tibet, China. In general, the 20 m SRT performance of Chinese Tibetan children and adolescents aged 9–17 years is lower than the Chinese and international values, especially in the younger age groups.Using a representative sample, the present study provided up-to-date age and specific reference data of the number of laps, number of completed stages/minutes, and speed (km hThe CRF of Tibetan children and adolescents aged 9–17 in high-altitude areas of China increases with age, and boys showed higher performance levels than girls, which is consistent with the results of many previous studies. There are many reasons for this result. First, there are congenital genetic differences between Tibetan boys and girls. Compared with girls, boys’ muscle strength and physical activity levels are higher, which is an important reason for boys’ higher CRF than girls . Second,The advantage of this study is that, firstly, this study adopts the international common CRF test method for testing, which is convenient for international horizontal and vertical comparison. Secondly, this study is the first to study the reference value of CRF in high-altitude Tibetan children and adolescents, which provides a reference for the CRF evaluation of Tibetan children and adolescents in high-altitude areas, which is of great interest. However, this study also has some limitations. On the one hand, this study is a cross-sectional study. If we understand the gender and age changes of CRF in Chinese Tibetan children and adolescents, we need to conduct a longitudinal cohort study. On the other hand, although the participants were given positive encouragement in the test performed in this study, there may be deviations between the CRF test value and the real value due to the influence of each participant’s personal subjective will and psychological condition.This study developed, for the first time, CRF reference data for Chinese Tibetan children and adolescents in high-altitude areas. Considering the importance of CRF for children and adolescents’ health, the government should strengthen the monitoring of CRF in Tibetan children and adolescents in high-altitude areas, strengthen the reform of the physical education curriculum, and increase the level of physical activity, with the aim is to promote the health level of Chinese Tibetan children and adolescents in high altitude areas.


When patients complained of symptoms compatible with relapses, they attended an extra in-person visit. Results: We enrolled 155 patients with RMS, and 88.38% of patients were treated with disease-modifying therapies. In the dataset, 126,381 total patient days, 78 relapses, and 1202 infections were recorded over a period of about 2 years. No increased risk of relapse after clinically manifest infections was found in the short-, medium-, or long-term period. No correlation was found between all infections and the number of relapses (p = 0.212). The main analyses were repeated considering only those infections that had at least two of the following characteristics: duration of infection ≥ 4 days, body temperature > 37° Celsius, and the use of drugs , and no significant associations were observed. Conclusions: No associations between infections and relapses were observed, likely suggesting that disease-modifying therapies may protect against the risk of relapse potentially triggered by infections.Background: Many factors are believed to be positively associated with the incidence of relapses in people with multiple sclerosis (MS), including infections. However, their role is still controversial. We aimed to investigate whether symptomatic infections in people with MS increase the risk of relapse in the short, medium, or long term. Materials and Methods: We enrolled consecutive patients with relapsing MS (RMS) from October to December 2018. From enrolment up to September 2020, an online questionnaire investigating the occurrence of infections was sent via WhatsApp Multiple sclerosis (MS) is a chronic neurological disease and the most common cause of non-trauma-related disability in young adults . Most ofMany factors are believed to be positively associated with the incidence of relapses, including smoking, pollution, low levels of serum vitamin D , non-traMany autoimmune disorders, such as MS, are associated with vitamin D deficiency, and several studies have shown that vitamin D has anti-inflammatory effects by suppressing the innate and adaptive immune systems . In the Between the 1990s and the 2000s, several studies highlighted a risk of exacerbations after infections; furthermore, in some of these studies, the authors concluded that infections contributed to an increase in clinical disease activity, without impacting the severity of relapses ,14,15. MDespite several studies on this topic, the effect of infections on relapse incidence in people with MS is still controversial ,18,19,20Previous studies highlighted a positive correlation between upper respiratory tract infections and monthly relapse rates and a negative one with ambient ultraviolet radiation expressed as solar hours ; howeverIn this study, we aimed at investigating whether symptomatic infections in an MS outpatient population increase the risk of relapse in the short, medium, or long term, accounting for confounding factors.This was a cohort study. Consecutive patients with a diagnosis of relapsing MS (RMS) attending the MS outpatient clinic of the University of Campania “Luigi Vanvitelli”  from October to December 2018 were offered to take part in the study. Ethical approval was obtained from the ethical committee of the University of Campania “Luigi Vanvitelli” (prot. 593/2018). Signed informed consent was obtained from each patient before enrolment in the study in accordance with the Declaration of Helsinki.We included patients with MS diagnosis according to the revised McDonald criteria , with a ®  and MS history , and EDSS at enrolment) were collected.https://www.google.it/?client=safari&channel=iphone_bm) contained items to detect the presence of infection and to identify the occurrence of relapses; in detail, the questionnaire inquired about the presence of an infection and its duration, the presence of fever with body temperature measurement, the type of infectious symptoms , and the use of symptomatic drugs and/or antibiotics and/or antivirals , solar hours, and daylight hours in Naples on each day of the study period, the data were retrieved from the National Oceanic and Atmospheric Administration (NOAA) using a method for sun ephemerides calculations . Changes in DMT were also collected.Based on previous studies ,18,19,20We aimed to enroll 155 MS patients. The sample size was calculated based on previous studies ,20.The characteristics of the study population are presented using descriptive statistics. Mean or median and (interquartile range (IQR) values were calculated for continuous variables and frequencies for categorical variables.For the primary analysis, a generalized linear mixed model adjusted for the patient (as a random effect), hours of daylight, and DMT (at the time of relapse) as fixed effects was used to evaluate short-, medium-, and long-term association between all infections and relapses. A simpler model without DMT was also explored. A variable indicating whether a date was subject to restrictions due to the COVID-19 pandemic, which occurred during the observation period, was also used in supplementary analyses as a potential confounder due to its obvious effect on infections and unknown role in relapses. These analyses were also performed by restricting the definition of exposure to severe infections, as previously defined.To evaluate the association between the number of relapses and the number of infections and between the total number of relapses and infections with body temperature higher than 37 °Celsius, nonparametric Kruskal–Wallis tests were performed. The same analysis was used to evaluate the single association between organ-specific infections  and relapses. A test of coefficient of correlation (rho-Spearman’s) was calculated for each analysis.Given the data collection methods, missing data almost exclusively affected the infections. The main analysis was conducted under a missing not at random (MNAR) assumption that no response to the online questionnaire indicates no infection in that month. Sensitivity analyses were conducted under a missing completely at random (MCAR) assumption, considering that the absence of response is completely uninformative regarding the occurrence of infection. A generalized linear mixed model was conducted both adjusting for patient, hours of daylight, and DMTs and without adjusting.p-values were rounded to four decimal places.Given a relevant true difference in risk between “AR” and “not AR” days, to determine the power of our study  to detect it, a simulation approach was used, using our same dataset for all covariates but simulating the relapse (0/1) column based on real risk increases of 1.5, 1.75, 2, 2.5, and 3. The main scenario to interpret power was prespecified at 2×. For all comparisons, the level of α = 0.05 was assumed, and Out of 209 patients screened for eligibility, 155 patients with RMS signed the informed consent forms, answered the online questionnaire at entry, and were enrolled into the study, of whom 125 had RMS without progression and 30 with progression, and 54 patients did not sign the informed consent and were not enrolled.The mean age of the participants was 35.4 years, and the median baseline EDSS was 3. The average time from diagnosis to study initiation was 13.25 years, and 113 patients were female .In our study, at the enrollment, 88.38% of patients were treated with DMTs, 41.93% were on first-line DMTs , and 46.45% were on second-line DMTs  . In the In the dataset, 126,381 total patient days were registered. The dataset included 78 relapses and 921 infections; frequencies of infections and relapses are shown in A total of 78 relapses were experienced by 49/155 patients; 27 patients experienced 1 relapse, 15 experienced 2, and 7 experienced 3, averaging 0.50 relapses per patient over the whole study period (range: 0–3). Sensory symptoms were found in 38.46% (30/78) of relapses, motor symptoms in 39.74% (31/78), brainstem or cerebellar symptoms in 15.38% (12/78), and optic neuritis in 6.41% (5/78).A total of 921 infections were registered, averaging 6 infections per person over the study (range: 0–23). Overall, 17/155 (11%) patients did not report any infections; 138 patients reported a total of 921 episodes of infections including 134 severe cases. The majority of the infections were related to upper respiratory tract symptoms, followed by infections with urinary tract symptoms and gastrointestinal symptoms .p = 0.212)  .p = 0.591)  .p = 0.459, p = 0.317, and p = 0.215, respectively). The main analyses were repeated considering only severe infections, and no association was observed with the risk of relapse in the short, medium, or long term , and for the basic model . Given that the simulation did not include any real effect of the patient, seasonality, or treatment, a higher power for the basic model was expected when considering the association in the short-, medium-, and long-term .No association was found between clinical infections and subsequent relapses.Several lines of evidence have shown a link between the development of an infection and the onset or the exacerbation of autoimmune diseases such as MS . While sOne of the largest studies on this topic was conducted by Sibley et al., which enrolled over 8 years 170 untreated MS patients and 134 healthy controls and showed that annual exacerbation rates were almost threefold in AR periods, defined as two weeks before to five weeks after the onset of a clinical infection, and also that 27% of exacerbations were related to infections .Andersen and colleagues in their prospective study recording upper respiratory and gastrointestinal infections with serological diagnosis in 60 untreated RRMS patients, showed a relative risk of 1.3 to have relapses in the so-called AR period, four weeks after a clinical infection .In the same period, Panitch led a prospective study on 30 RRMS patients, divided into three groups treated with either a high dose of interferon-B 1b (IFN-B lb) (8 MIU), low dose IFN-B lb (1.6 MIU), or placebo and concluded that mild infectious illnesses, largely of nonspecific viral origin, were responsible for most of the exacerbations. He found a strong relationship between MS attacks and upper respiratory tract infections in the AR period  and pointed out that treatment with a high dose of IFN-B 1B reduced the attack rate .p = 0.004) relative risk of clinical relapse for upper respiratory tract infections and a higher relative risk of relapse in the AR period (from 14 days before until 14 days after the first symptom) compared with the “not AR” periods . Their patients were participating in a double-blind placebo-controlled trial of IFN-B 1a, and, at the time of publication, they did not know which patients were on active treatment; for this reason, a subanalysis of treatment was not performed as in Panitch’s study [A few years later, Edwards et al.  in a pro’s study .The above-mentioned studies ,19,20 usThese studies highlighted two fundamental points: (i) all the studies except for that of Sibley et al. had a small sample size and, in some cases, included heterogeneous populations by clinical phenotype, and (ii) the AR periods had different time frames.In any case, all these studies, although conducted in different ways, point towards a positive correlation between mild infections, in most cases of viral origin, and relapses. These studies were conducted in the 1990s, mainly on untreated patients. Panitch was the first author to observe in the cohort of his treated patients that the number of relapses was lower compared with untreated patients, regardless of the number of infections.Nearly 80-85% of people with multiple sclerosis experience a relapsing course, and if left untreated, they develop disability over time . Over thAt the start of our study, 88.38% of patients were treated with DMTs, whereas after one year 92.90% of patients were on DMTs. This could be one of the reasons why we did not observe any correlation, as the effect of DMTs is currently so strong that external factors such as mild or moderate infections do not influence disease activity.Given that, our study has adequate power to detect strong associations ; we trusGiven the correlation between solar hours and infectious risk and relapse, we adjusted results for solar hours to add strength to our analysis .Unlike our study, Tremlett et al. , consideOf note, we have to underline that this cohort of patients is extremely different from ours, as in our cohort almost all patients were treated with DMTs: 88.38 % at the enrollment and 92.90% one year after the study started, with a stronger impact on disease activity, possibly overcoming the detrimental effect of infection in triggering relapses.One of the major limitations of our study is the modality of the infection recording. This was performed through the administration of an online questionnaire that was sent monthly to the RMS patients of our MS Center. Subsequently, they were not asked to confirm infection by more in-depth investigations.Unfortunately, vitamin D levels were not available for all the patients, representing a further limitation.Moreover, this was a single-country, single-center study that may limit generalizability to the MS patient population.In addition, there were missing data (about 11%) as patients received the questionnaire monthly over a long period (two years). However, the presence of missing data was managed through statistical analysis. The analyses were conducted both considering that the absence of response meant the absence of infection (MNAR) and not attributing any value to the absence of response (MCAR). If the probability of being missing is the same for all cases, then the data are said to be missing completely at random (MCAR), while MNAR means that the probability of being missing varies for some reason and we assume that the reason is the absence of infection. Moreover, this was a monocentric study, and patients with underlying conditions increasing the risk of infections were excluded, and the results might not generalize to that subgroup or in other contexts.We recognize that these types of studies may suffer from variations in MS severity, infection type, and severity and that pooling all the cases  and performing the correlation analysis may lead to suboptimal sensitivity for investigating the correlation between infection and relapse. Studying subtypes of infections or patients with different severities of MS, might have improved the statistical power for the correlation, but this would require a larger sample size to be explored. Considering these aspects, we tried to overcome this limitation through the statistical model by adjusting for the patient as a random effect and for sunlight and treatment as fixed effects.In conclusion, in our cohort of MS patients, most of them taking DMTs (He or ME DMTs), mild or severe infections were not associated with an increased risk of relapse. Although associations between mild infections and relapse rate in the AR period had been described in the past, we speculate that, currently, the use of effective DMTs may protect against the risk of relapse potentially triggered by infections. Future studies are needed to confirm this hypothesis.