The role of milk and fermented milk consumption in stroke risk is unclear. We investigated associations of time-updated information on milk and fermented milk consumption (1997 and 2009) with total stroke, cerebral infarction, and hemorrhagic stroke risk among 79,618 Swedish women and men (mean age 61.3 years). During a mean follow-up of 17.7 years, we identified 9735 incident cases of total stroke, of which 7573 were cerebral infarctions, 1470 hemorrhagic strokes, and 692 unspecified strokes. Compared with an intake of 100 g/day of milk, the multivariable-adjusted hazard ratios  of cerebral infarction were 1.05 (1.02–1.08) for 0 g/day, 0.97 (0.95–0.99) for 200 g/day, 0.96 (0.92–1.00) for 400 g/day, 0.98 (0.94–1.03) for 600 g/day, and 1.01 (0.94–1.07) for 800 g/day. Corresponding estimates for hemorrhagic stroke were 0.98 (0.91–1.05) for 0 g/day, 1.02 (0.97–1.07) for 200 g/day, 1.07 (0.98–1.17) for 400 g/day, 1.13 (1.02–1.25) for 600 g/day, and 1.19 (1.03–1.36) for 800 g/day. No associations were observed between milk consumption and total stroke or for fermented milk consumption and any of the stroke outcomes. Higher long-term milk consumption based on repeated measures of intake was weakly and non-linearly associated with cerebral infarction, and was directly associated with hemorrhagic stroke. Milk is a rich source of several essential nutrients, such as calcium, phosphorous, vitamin D, vitamin B12, riboflavin, and protein. A daily consumption of milk and dairy products is included in many nutritional guidelines to promote health. However, results from previous studies on the relationship between milk and fermented milk consumption and stroke risk are not consistent. An increment of 200 g of milk per day was associated with 8% lower risk of total stroke in recent meta-analysis . These dBesides different types of milk, it is important to separate stroke types, i.e., cerebral infarction and hemorrhagic stroke, which have different pathophysiology and therefore possibly different risk factors . Few stuDietary and lifestyle habits may change during long follow-up periods and there are few studies with repeated measurements of both exposure and confounders. We therefore aimed to investigate potential associations between time-updated information of milk and fermented milk consumption and risk of total stroke, cerebral infarction, and hemorrhagic stroke in a Swedish population with a traditionally high consumption of milk and fermented milk.We acquired data from two population-based cohorts of women born in 1914–1948  and men born in 1918–1952 (Cohort of Swedish Men (COSM)) from central Sweden  [The investigation was approved by the Swedish Ethical Review Authority (Dnr: 2019-03986). Informed consent was obtained from all subjects involved in the study. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this work is consistent with those.Using a validated FFQ, we collected information about usual dietary intake during the past 6 months for FFQ1 (67 food items) in 1987 and during the past year for FFQ2 (96 items) and FFQ3 132 items) in 1997 and 2008/2009 . We summ2 items iMilk consumption was divided into categories <200, 200–399, 400–599, and ≥600 g per day and fermented milk was divided into categories 0, 0–199, 200–399, ≥400 g per day. The cut-offs were selected based on previous studies and represent one glass of milk (200 g) increase for each category ,16.The average energy and nutrient intake per day was calculated by multiplying frequency of each food by the nutrient or energy content in age-specific portions of each food, obtained from the Swedish National Food Agency database .The FFQ has been validated against four 7-day food records completed every third months and the corrected Pearson correlation coefficient for milk intake was approximately 0.7 between FFQ and the food records   by linkage with the Swedish National Patient and Cause of Death Registries. To classify total stroke, we used the International Classification of Diseases (ICD) 9th revision codes 430, 431, 433, and 434 and the 10th Revision codes I60, I61, I63 and I64. Total stroke was divided into cerebral infarction  and hemorrhagic stroke . Those with a total stroke diagnosis before baseline were excluded . In sensIn the main analysis we calculated time at risk, for the pooled cohorts and for each cohort  separately, from 1 January 1998 until the date of the first stroke diagnosis, date of death, or end of follow-up (31 December 2019), whichever came first. When calculating time at risk for cerebral infarction and hemorrhagic stroke we censored for other types of stroke, including unspecified stroke. We updated exposure and covariates with information from the 2008/2009 investigation to account for possible changes during follow-up. Cox proportional hazards regression models with time since baseline as the time scale were used to estimate hazard ratios (HR) with 95% confidence intervals (CI) of total stroke, cerebral infarction, and hemorrhagic stroke according to milk and fermented milk consumption. Non-linear functions of the main exposures were first assessed by restricted cubic spline Cox regression with three knots placed at the 10th, 50th, and 90th percentiles. We used 100 g/d as reference and derived HR estimates based on the spline curve at intakes of 200 g/d, 400 g/d, 600 g/d, and 800 g/d. We then examined associations with pre-specified categories of milk and fermented milk, with lowest category as reference.2; continuous), history of hypertension (yes/no), hypercholesterolemia (yes/no), and diabetes mellitus (yes/no), coronary heart disease, Charlson’s weighted comorbidity index (continuous) [Information on possible covariates was attained from the questionnaires and from the National Patient Registry. Questions on smoking, physical activity, supplement use, hypertension, hypercholesterolemia and diabetes were not included in the first investigation in 1987 and we therefore used information on these variables from the 1997 investigation in sensitivity analyses. We used the directed acyclic graph method  and currTo examine the robustness of the results, several sensitivity analyses were performed. First, we investigated associations in SMC. Time at risk was calculated for each participant from their first examination date in 1987–1989, with time-updated information on exposure and covariates from the investigations in 1997 and 2008/2009, until the date of the first stroke diagnosis, date of death, or end of follow-up (31 December 2019), whichever came first. Then we performed the same analyses as in the main analyses for the three different baselines with start in 1987–1990, 1997 (1 January 1998), and 2008/2009 (4 April 2009) but without time-updated information. We analyzed associations where participants with missing information at baseline on all types of milk  or all types of yogurt/sour milk  were excluded, and associations in complete case analysis. We further adjusted for atrial fibrillation since it is a strong risk factor for cerebral infarction and considered intracerebral hemorrhage and subarachnoid hemorrhage as separate outcomes. The proportional hazard assumption was tested and confirmed graphically using Schoenfeld residuals. All analyses were carried out in Stata 15 .Baseline characteristics according to milk consumption in 1997 are presented in Participants who consumed 600 g of milk per day or more had, on average, higher BMI, higher energy and soft drink intake, lower fruit and vegetable intake, and were less likely to use vitamin- and mineral supplements, further were more likely to have ≤9 years of education, be current smokers and live alone compared with those who consumed less than 600 g of milk per day . ParticiThe pooled analysis with repeat measurements included 79,618 participants without stroke at baseline in 1997. The mean follow-up was 17.7 years  and we identified 9735 total strokes, of which 7573 were cerebral infarctions, 1470 hemorrhagic strokes, and 692 unspecified strokes. The absolute rate (per 1000 person-years at risk) of total stroke was 6.9, of cerebral infarction 5.4, and of hemorrhagic stroke 1.0. For SMC with start in 1987–1989, the mean follow-up was 25.2 years , maximum follow-up was 32.8 years, 60,647 women were included, and 7965 total strokes were identified.The adjusted restricted cubic spline curve for milk consumption and total stroke is presented in The adjusted restricted cubic spline curve for milk consumption and cerebral infarction is presented in Milk intake was directly associated with hemorrhagic stroke  in the pThe same pattern was observed in the stratified analyses; the risk estimates were higher in women than in men, but the confidence intervals were wide . There wThe results for milk intake and risk of total stroke, cerebral infarction, and hemorrhagic stroke were confirmed in sensitivity analyses. For example, the HRs in complete case analysis were the same as in the main analysis but with broader CIs. Additional adjustments for atrial fibrillation did not change the results. HR estimates for milk consumption in categories in relation to total stroke, cerebral infarction, and hemorrhagic stroke, but without time-updated information from 2009, are presented in No associations were observed between time-updated information on fermented milk consumption and risk of total stroke, cerebral infarction, or hemorrhagic stroke in the pooled cohort with baseline in 1997  or in woIn this population-based study of Swedish adults with time-updated information on exposures and covariates, milk consumption showed a non-linear association with cerebral infarction, such as that a milk intake below 100 g/day was associated with a higher risk and intakes between 100–500 g/day were associated with a modestly lower risk, and a direct association with risk of hemorrhagic stroke. There was no clear association between milk intake and risk of total stroke. Fermented milk consumption was not associated with any of the stroke outcomes.The strengths of this study include the population-based and prospective cohort design of the study, including both women and men with a wide range of milk and fermented milk intake, the long follow-up, and repeated measurements. The repeated measurements allowed us to time-update information on exposures and covariates and thus capture diet and lifestyle changes and acquire higher precision and accuracy than with one (baseline) measurement only. As in our study, another Swedish study observed a decreased consumption of milk and an increased consumption of fermented milk over time , reflectUnknown confounders and residual confounding cannot be ruled out, but we had the advantage of adjusting for several possible confounders including education, cohabitant status, comorbidity, physical activity, smoking, and other lifestyle factors. Even though we used repeated measurements of dietary intake, we might not have fully captured the changes in milk and fermented milk intake which could lead to misclassification of exposures. Under- and overreporting of milk and fermented milk consumption is probably limited as these foods are considered neutral in the sense that they are neither associated with being healthy nor unhealthy in the Swedish general population at the time when the study was performed. With self-reported measurements there is always a risk of measurement errors and misreporting. Recall bias is however minimized due to the prospective design of the study. The generalizability of our findings may apply to both men and women, but they may not apply to younger people or children, and those who do not tolerate milk. Furthermore, results from only the lower range of milk intake may apply in populations with low intakes.Like in our study, no association for milk consumption and total stroke was observed in the European Prospective Investigation into Cancer and Nutrition study nor in two Swedish cohorts, with similar range of milk intake as in the present study ,20,22. SOur results for fermented milk are in line with a recent review and meta-analysis where no association between yogurt intake and ischemic stroke was observed . HoweverThe relationship between milk and fermented milk consumption were studied in the present cohorts already in 2012 and no association between milk consumption and total stroke, cerebral infarction or hemorrhagic stroke was observed after a mean follow-up of 10.2 years and 4089 total stroke cases . In the The timing of exposure may be of importance for the different results regarding cerebral infarction and hemorrhage. The main underlying pathophysiology for cerebral infarction is atherosclerosis and thrombosis, where the action of the exposure on the atherosclerotic process may have taken place many years before the carotid plaque ruptures or a thrombus occludes an intracerebral vessel causing a cerebral infarct. For hemorrhage, frail blood vessels in the brain in combination with inflammation of the vessels may have a shorter induction period and the action of exposure, e.g., high blood pressure, may be more recent than for cerebral infarctions. Thus, repeated measurements may capture the time window when exposure has the greatest impact on the disease development.High concentrations of low-density lipoprotein (LDL) cholesterol have been associated with an increased risk of cerebral infarction but with a lower risk of hemorrhagic stroke ,30. An iWe can only speculate about possible mechanisms behind the different results for milk and fermented milk. Although similar in protein and vitamin content, the lactic acid bacteria added to the milk for the fermentation process influence the gut microbiota ,34,35, dResults from this prospective cohort study with time-updated information on exposures and covariates showed that milk consumption is not clearly associated with total stroke, is weakly and non-linearly associated with cerebral infarction, and is directly associated with hemorrhagic stroke. We found no association of fermented milk consumption with stroke. Milk and fermented milk consumption had different impacts on cerebral infarction and hemorrhagic stroke, which highlights the importance of separate analyses for milk and fermented milk. The increased risk of hemorrhagic stroke with high milk consumption is relatively small and therefore there is no need to change present dietary recommendation concerning milk consumption. However, future research is needed with time-updated information on milk consumption and analyses of potential non-linear associations of milk consumption with different stroke types as well as studies focusing on the underlying mechanisms.


Background: Neuropathic pain research has grown impressively in the past two decades, as evidenced by improvements in research quality and increments in the number of research papers. In views of this situation, the use of quantitative measurements to analyze and characterize existing research has become imperative. The aim of this research is to identify and analyze the 100 most-cited papers in neuropathic pain research.Methods: Neuropathic pain-related articles published between 2000 and 2020 were screened from Web of Science (WOS) by using the following subject terms: TI = . The publications were ranked in a descending order on the basis of citation counts, and the top 100 most-cited neuropathic pain papers were determined. Subsequently, we conducted a bibliometric study to determine the authors, journals, countries, and institutions that contributed the most to the top 100 neuropathic pain lists; describe the keywords and hotspots of the top 100 most-cited papers; and explore the factors associated with successful citations.Results: The top 100 most-cited papers were published from 2000 to 2017, and 2003 had the largest number of published papers (n = 16). The mean number of citations per paper was 480.72, with a range of 262–1,569. Forty-four kinds of journals contributed to the top 100 most-cited papers, which were predominantly published in “Pain” (n = 23). The USA was determined to be the leader of neuropathic pain research in terms of quality and quantity.Conclusion: This study provides a comprehensive list of the most influential papers on neuropathic pain and demonstrates the important advances in this field to help understand academic concerns and the directions of technological innovations in neuropathic pain worldwide. With the increase in neuropathic pain prevalence in the past years, neuropathic pain-related research has become a serious concern. Neuropathic pain, which is described as “pain caused by a lesion or disease of the somatosensory system ,” is a mOver the past 20 years, many experts and researchers have struggled to provide new insights into neuropathic pain, and numerous related study results have been published in various journals in the form of articles to clarify the underlying mechanisms and risk factors and explore the therapeutic targets of neuropathic pain –14. WithBibliometrics is a research analysis based on the number and patterns of citations . CitatioThrough a literature search, we found that many papers on neuropathic pain have high citations (citation times > 200), but no analysis of the most influential works in the field of neuropathic pain has been conducted yet. Thus, we performed a bibliometric study to analyze the 100 most-cited papers in this field from 2000 to 2020 on the basis of the Web of Science  and determined the factors related to their successful citations. This task could be beneficial to the paper publication of investigators and design of future research. We look forward to identifying the most promising research directions for neuropathic pain.Ethical approval from an institutional review board was not required because our study was a bibliometric analysis that did not involve human subjects.Neuropathic pain-related articles published between 2000 and 2020 were screened from the Science Citation Index–Expanded (SCI-Expanded) of Web of Science (WOS), which allows access to over 9,381 peer-reviewed journals that have been published in 178 scientific disciplines since 1945. All data were acquired on January 1, 2021, to avoid changes in the online activity of papers, and 9,561 results were produced. In the Science Citation Index–Expanded of WOS, the search terms were created in reference to several academic articles and MESH terms from PubMed, as follows: TI =  AND Language = English, with the period of 2000 to 2020.The publications were ranked in a descending order on the basis of citation counts and reviewed to determine the top 100 most-cited neuropathic pain papers. Papers with fewer than 200 citation times were excluded to reduce the number of papers that require follow-up screening. In terms of literature type, only articles and reviews were included; proceedings papers, conference abstracts, conference presentations, and book chapters were excluded. Two independent investigators reviewed the titles and abstracts and deleted studies that are not related to neuropathic pain. A total of 166 articles remained after reviewing the titles and abstracts. The 100 most-cited papers were then exported to Microsoft Excel 2016 to create tables and figures.Data selection and extraction were performed by two well-trained independent researchers (Huanyu Xiong and XueQiang Wang), and XueQiang Wang was consulted to deal with discrepancies. Related data, including publication date, citation counts, citation per year , author, journal of publication, country of origin (based on the correspondent author's address), institution, document type, research field, and keywords, were extracted from the top-cited papers and used to assess publication quality. In the search results from WOS, we screened several collections, such as journal, date, and study field. Then, we created citation reports and obtained data. The journal impact factor 2020 (IF 2020) and 5-year IF were from JCR 2020. A limitation of this study is that recent papers with high citation potential may not have been included because of the limited timespan since publication. To correct this limitation, we ran the same search within a short period (2018–2020) to select the top 10 most-cited papers.P < 0.05. Descriptive statistics were quantified as the average or counts (percentages) of parameters. The Shapiro–Wilk test was applied to examine the normal distribution of individual variables. Data with a normal distribution were expressed as the mean ± standard deviation. A comparison of means was performed using the Mann–Whitney test was applied when necessary. The Pearson product moment correlation coefficient was employed to test the correlations between IF (2020) and paper counts, IF (2020) and citation counts, as well as correlations between annual citations and total citations. One-way analysis of variance (ANOVA) was performed to test qualitative indicators, including the distribution difference in paper count among country, type of document and open access before and after 2008. CiteSpace V was utilized to analyze and generate a network map of keyword co-occurrence  .We retrieved the 100 most frequently cited papers on neuropathic pain , which r= 1,569) . Since tThe top 100 most-cited papers were published from 2000  to 2017. The majThe largest number of papers published in a single year was 16, and this occurred in 2003. 2005 with 14 papers was the second peak. During this period, except for years 2011, 2012, and 2014, the number of articles was generally higher than the number of reviews . Among tn = 42) were from the USA, and around one-tenth (n = 11) was from Canada. England with 9 papers ranked third, followed by Germany, France, and Denmark that contributed six papers each to the list  and University Klinikum Schleswig Holstein from Germany (n = 9).A total of 259 institutions published at least one top-cited paper, with 139 (53.7%) institutions publishing only one. A small number of institutions accounted for a high proportion of the highest cited papers, similar to the previous findings of lncRNAs studies (n = 23), followed by the Journal of Neuroscience (n = 8), Neurology (n = 7), and European Journal of Neurology (n = 6). With regard to the average citation number per paper, the Journal of Lancet Neurology ranked first with a mean of 993.0 citations per paper, followed by Nature with a mean of 941.5. All journals containing more than two publications are summarized in r = 0.272, P = 0.009) and citation  counts were significantly related to IF.Forty-four kinds of journals contributed to the 100 top-cited papers, which were predominantly published in Pain (n = 15); the total number of citations for his papers was 9,793. Baron R. with 14 papers was the next largest contributor. Attal N. and Cruccu G. were tied at the third place with 10 papers each. Cruccu G. from Sapienza University Rome, Dworkin R. H. from University of Rochester, and Finnerup N. B. from Aarhus University were the first author of the four papers in our list.n = 50), and 46 papers were categorized under “Clinical Neurology.” Many studies were also conducted in other fields, such as “Anesthesiology,” (n = 28) “Medicine General,” (n = 12), and “Multidisciplinary (n = 8).”In n = 0.32) “double blind,” (n = 0.3) “amitriptyline,” (n = 0.24) “management,” (n = 0.19), and “mechanism” (n = 0.17) had a high degree of centrality during this period. n = 30) “double blind,” (n = 22), and “post-herpetic neuralgia” (n = 22) were the most frequently used keywords.CiteSpace V was used to extract the keywords of the top 100 papers on neuropathic pain. A network analysis of the author's keywords or subject words was carried out during the publication time of the article, namely, 2000–2017 . The resP = 0.011), annual citations (P = 0.013), and citation 2020 (P = 0.001) significantly differed among the different document types.In terms of document type, the original articles comprised 69% of the most-cited papers, and the remaining 31% were reviews. Total citations . However, the papers' current citation rate (as measured by citations in 2020) suggested that papers published after 2008 were likely to be cited in recent years. Such a correlation was statistically significant in this study .In this study, a paper's total citation rate was unrelated to the paper's publication date  to the top 100 list.Aarhus University Hospital and Harvard University were the two largest contributors  to the top 100 list. The USA with the most publications and citations ranked first in almost every category and was arguably the leader of neuropathic pain research in terms of quality and quantity. This phenomenon is due to the following reasons:Countries with high gross domestic product (GDP), such as the US, Canada, and Denmark, invest heavily in medical research; a previous study has proven that a country's per capita GDP is weakly correlated with its study results .There's no doubt that native English speakers get more quotes. For example, American authors are more likely to cite local papers than foreign ones, and their papers are more likely to be published in American journals than foreign ones , 39.Mainstream countries dominate publications, and this model has appeared in many medical-related topics, such as lncRNAs , human pIt's worth noting that the citation rate of white and male articles is much higher than that of non-white and female articles .Conversely, we found several papers from Japan and China in Asia, Egypt in Africa, and Australia in Oceania suggesting that neuropathic pain is universal on a global scale. This result again demonstrates that this disease has a research value. Furthermore, this result means that the achievements from these countries and institutions are publicly shared so that numerous people can benefit from them.n = 23) with 13,512 citations in 2020 and whose current IF is 6.961 and the Journal of Neuroscience (n = 8) with 3,070 citations in 2020 and whose IF in 2020 was 6.167. In addition to specific journals in this field, several general medical journals, such as The New England Journal of Medicine (IF2020 = 91.245) and Nature Medicine (IF2020 = 53.44), also played an important role in this analysis.We noticed that two journals exhibited strong professional attributes and contributed almost a third to the 100 most cited papers. These journals were Pain , Baron R. (n = 14), Attal N. (n = 10), and Cruccu G. (n = 10). This study found that three authors  published 12 papers as first authors, and their papers have been cited 7,517 times in total. This finding highlights how a relatively small number of authors can make substantial contributions to the impact of a journal or research field   . The stuAll of these authors must be recognized as important contributors to the neuropathic pain field. However, compared with the top articles in other popular fields, such as cancer, Parkinson's disease, and orthopedics, a gap still exists. Therefore, authors should strive to carry out high-quality research while attempting to increase the quantity of their works.n = 50), clinical neurology (n = 46), and anesthesiology (n = 28). The diagnosis and treatment of neuropathic pain have developed rapidly in the last few decades. Neuropathic pain research is distributed worldwide in neuroscience in the life sciences and neurology in clinical medicine due to the neural basis of the neuropathic pain mechanism. In our study, the topic with the highest frequency of occurrence was the pathology, pathogenesis, and drug therapy of neuropathic pain (n = 71), reflecting the high study interest in this subject in the twenty-first century. Most of these papers involved drug therapy (n = 25), followed by post-herpetic neuralgia (n = 6) and trigeminal neuralgia (n = 4). On the basis of these studies and their results, neuropathic pain has been classified accurately, and several key mechanisms, including abnormal discharging of nociceptive nerves, peripheral, and central sensitization, and impaired inhibitory modulation, have been established (n = 3) was extremely low .ablished . The promely low . NeverthThe citations of a paper often follow a time process. The longevity of papers shows the impact of papers on scientific research. In the last year (2020), 17% of the top-cited papers were cited fewer than 10 times, which might indicate that the content of these papers is no longer the current study hotpots.n = 0.32), double blind (n = 0.3), amitriptyline (n = 0.24), management (n = 0.19), and mechanism (n = 0.17) had high centrality in the past 20 years, indicating that these research directions are essential, which is consistent with the research results on keywords. It is also consistent with the emphasis placed on diagnosis in the 1990s. Several differences were observed between the 2010s and the 2000s. In the 2010s, the keywords that appeared frequently were quality of life, pharmacological treatment, concentration capsaicin patch, and other related words. This observation suggests that researchers have begun to expand their research to several treatments and pay increasing attention to prognostic assessments, new treatment techniques, quality of life, and other aspects of neuropathic pain therapy. Gradually, neuropathic pain research has begun to blossom. These results also indicate that neuropathic pain is still a disease that needs to be solved urgently, and the deficiencies and innovations in this field, such as new drug therapies, pathological mechanisms, and strengthened pain management, must be explored to improve the quality of life.Keywords with citation bursts can reflect the development of a knowledge field. Our keyword centrality analysis showed that allodynia , journals , and most popular research categories. Lastly, we performed statistical analyses to determine the underlying factors that may be related to citation counts.Although our results provide some valuable information, the limitations of this research need to be recognized. First, the search strategy depends only on the relevant search terms contained in the title or keywords. We may have overlooked several popular publications, such as those indexed by “low back pain” . Second,Neuropathic pain research has grown impressively in the past two decades, as evidenced by improvements in research quality and increments in the number of research papers. This bibliometric research is the first to identify and discuss the 100 most-cited papers published in the field of neuropathic pain. Despite the study's limitations, by reviewing these top papers, researchers can immediately understand the significant progress neuropathic pain has achieved over the past two decades, and targeted scientific questions can be selected to fill the gap in research.This analysis provides insights from prominent individuals and institutions who have contributed considerably to neuropathic pain research and identifies characteristics that are relevant to high citation counts. The citation frequency of the top 100 papers published from 2000 to 2017 ranged from 262 to 1,569. IF and country or region were found to be closely associated with citations. Most of the papers had much higher citations in 2020 compared with their annual citations, reflecting the fact that the top 100 papers have received the continuous attention of researchers and may be of potential academic importance in the future. The two-time point analysis indicated that the citation longevity and citation modes of the most-cited papers changed over time. Compared with the early most-cited papers, recently published most-cited papers reached the peak citation rate faster. The exploration of drug therapy, pathophysiological mechanism, diagnosis, and screening tools of neuropathic pain might be the research focus in the future. Overall, the neuropathic pain field appears to be promising. The major contributions of the most influential studies can serve as an important reference for all pain physicians and neuroscientists because their goal is to improve clinical practice and their own scientific findings.The original contributions presented in the study are included in the article/X-QW: conceptualization, investigation, resources, and funding acquisition. H-YX: methodology, software, formal analysis, data curation, writing—original draft preparation, visualization, and project administration. HL: validation. X-QW and HL: writing—review and editing and supervision. All authors have read and agreed to the published version of the manuscript.This work was supported by Fok Ying-Tong Education Foundation of China (161092), the Scientific and Technological Research Program of the Shanghai Science and Technology Committee (Fund number: 19080503100), and the Shanghai Key Lab of Human Performance (Shanghai University of Sport) (11DZ2261100).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.


This study explores the level of physical activity and its associations with sociodemographics and body mass index (BMI) in the Southern Great Plain region of Hungary. A total of 1648 adults  were involved in this study. Their mean age was 43.0 (SD = 15.3), and they were recruited at different face-to-face events from July 2018 to January 2019. The International Physical Activity Questionnaire (IPAQ) was used to assess physical activity, and the participants were asked different questions related to their sociodemographics  and physical attributes . Additionally, a descriptive statistical, chi-square test was used the see the gender differences, and multinominal regression analysis was used to see the associations between gender, age, place of residence, education, income, BMI, and physical activity levels. Our analysis showed that 19.2% of the sample had a low-, 41.1% had a moderate-, and 39.7% had a high level of physical activity. Furthermore, a high and a moderate level of physical activity were associated with gender, age, residence, education, and BMI. We believe this present study helps understand the role of physical activity in health through the example of the Southern Great Plain region of Hungary, which can provide useful information for experts to increase participation in regular physical activity. The benefits of physical activity (PA) has become a leading topic of study in sports and health sciences since physical inactivity is a leading contributor to global mortality rates . The benIn recognition of this vital link between physical activity and health, the member states of the World Health Organization (WHO) agreed to reduce physical inactivity by 10% by 2025 [The European Union (EU) has been monitoring countries’ physical activity levels with the Special Eurobarometer 472 since 2009. The latest data showed that nearly half of Europeans (46%) did not engage in any physical activity or sport, and that proportion has increased gradually in recent years. A similar tendency can be found in the Hungarian population as well. Furthermore, 53% did not engage in any physical activity in Hungary, which is a 9% increase on previous data . In addiThe 2017 Eurobarometer data showed that 57% of the Hungarian population did not engage in vigorous physical activity, which is close to the EU 28’s average . HoweverThe influence of sociodemographic factors on physical activity was proven in previous studies. According to the Hungarian National Population Health Survey , the indIn addition to the impact of sociodemographics on physical activity, there is a clear pattern in physical inactivity in different geographic regions within countries . HoweverThus, it would be interesting to see the level of physical activity in different geographic regions in Hungary. Therefore, our aim is twofold. First, we would like to examine the physical activity on the Southern Great Plain (NUTS2 level) region of Hungary, including three counties . Second, our goal is to explore the relationship between gender, age, education, income, place of living, and BMI with levels of physical activity.The data collection occurred at different face-to-face events from July 2018 to January 2019. A total of 83 events were held in this period. It included 14 major events  and 69 small events . The participants were able to engage in different types of recreational physical activities and completed our paper-based survey voluntarily. Before data collection, ethical approval was sought and obtained from the university’s Institutional Review Board . The survey was voluntary, and it took approximately 5 min to complete. In the survey, the participants were asked about their socioeconomic background and their physical activity. It was ensured that the data were collected anonymously, and that no identifying data, such as names, were collected. After data cleaning, a total of 1648 participants  were involved in this study. Their mean age was 43.0 (SD = 15.3). All of them were inhabitants in a Southern Great Plains settlement.2).Social and demographic data were collected from the participants. The survey included closed-ended questions about age, gender, and educational background . We asked participants about their place of residence. The settlement categories were aligned to official categorization . The income per capita in households of the participants was investigated as well . Furthermore, we asked about participants’ height (m) and weight (kg) whereof BMI was calculated using BMI formula  . The selwww.ipaq.ki.se). We calculated the metabolic equivalent of task (MET), which is used to estimate the energy expenditure of physical activity. The IPAQ scoring protocol suggests the following equation to calculate a participant’s metabolic equivalent:Vigorous MET-minutes/week = 8.0 * minutes of activity per day * days of activity per week;Moderate MET-minutes/week = 4.0 * minutes of activity per day * days of activity per week;Walking MET-minutes/week = 3.3 * minutes of activity per day * days of activity per week.To create physical activity levels, we followed the official IPAQ scoring protocol .After calculating the sample’s MET score, we classified them into low, moderate, and high physical activity levels:2). Multinominal logistic regression with an odds ratio (OR) and a 95% confidential interval (CI) was used to understand more about the relationship between physical activity and sociodemographic. Several potential determinants were included in this analysis, such as gender, age, residential, education, income, and BMI. In the multinominal regression model, low physical activity was used as a reference category, and differences in moderate physical activity and high physical activity were calculated.SPSS for Mac 25.0  was used for statistical analysis. Descriptive statistics, including frequencies (percentage), means, and standard deviation, were used to assess the sample’s characteristics. Significant gender differences were investigated with the chi-square test  39.7% of the respondents belonged to the category of high physical activity and 19.2% to the category of low physical activity; (2) younger men had higher odds of engaging in a high level of physical activity than women in any age group; (3) people living in rural places had lower odds of participating in moderate or high physical activity levels; (4) vocational school qualifications lowered the chance of a high and moderate level of physical activity; and (5) normal BMI was associated with a high level of physical activity.To sum up, we believe that our study provided valuable data on physical activity in Hungary. This study might create the opportunity to continue our project and expand our sample size to improve understanding around what drives people to engage in physical activity in this region. Additionally, this information might help experts to maintain or increase participation in physical activity.


Introduction: Children with Down Syndrome (DS) have nutritional problems with unknown implications besides increased potential for obesity. Their food habits are unknown. We aim to delineate eating and lifestyle habits of DS children attending a multispecialist program to identify the challenges they face and the potential improvements.Patients and Methods: We interacted with 34 DS children  and their families. Food habits, medical conditions and treatments, degrees of development and physical activity, anthropometric and laboratory data were recorded over 6 months and analyzed. A 3-day food diary and a 24-h recall food frequency questionnaire were administered.Results: Twenty-nine (85%) children completed meals, only 11 (32%) received alternative food such as milk. Weaning regularly started in 25 (73%) children. Preschool children introduced adequate calories and nutrients. School children and adolescents did not reach recommendations. All age groups, as the general pediatric population, excessively ate protein and saturated fat, and preferred bread, pasta, fruit juices, meat and cold cuts. Peculiarly, pulses and fish were adequately assumed by preschool and school children, respectively. Five children (15%) were overweight/obese.Conclusions: Dietary excesses commonly found in the general pediatric population are also present in this DS group, proving a narrowing gap between the two. DS group performed better nutritionally in the early years and overweight/obesity occurrence seems contained. DS children may benefit from a practical yet professional care-program in which nutrition education may improve their growth, development and transition into adulthood. Down Syndrome (DS) is a common gene alteration syndrome, mostly consisting in an anomaly in the 21st chromosome. In Europe, DS children are 8% of registered cases of congenic anomalies. Worldwide, prevalence is 23 out of 10,000 births, with a tendency to increase –6. MacroTo fill the gap between diet and health in DS children and to explore potential key factors for improvement, we decided to scour their eating habits, their qualitative and quantitative dietary patterns in relation to their medical conditions and lifestyles, and to compare their food intakes with recommendations for the general pediatric population. Comparisons with what available in the literature for the DS population will also be considered as well as the effects of the participation to this small care program.Thirty-four children, 22 males and 12 females, median age 7 years (range 2–16 years), 31 of Italian nationality and 3 from other countries , were consecutively recruited over a period of 6 months and followed by a dietician while they were participating in their therapeutic sessions at the “Insieme Club” . The Club offers a 360° support consisting of medical care, speech therapy, nutrition counseling, physiotherapy, art and musical education. The children are involved in all the activities, which are tailored to their needs. The sessions, organized in the afternoons, are group-therapy meetings, while others are one-to-one . The children's parents signed an informed consent for inclusion in the study. The study was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the Ethics Committee of the Antoniano Insieme Club (11/10/12). The group reflected the number of children attending the club during the study's timeframe. All the children completed the study.After enrollment, families received a questionnaire and had face-to-face interviews with the dietician. She interacted with all the families at least three times: before handing the questionnaire to explain it including portion-size and food frequencies, after when a concomitant food recall of the previous 24 h was completed, and later when data were presented and discussed. In case of need, further assistance was offered through phone calls and extra meetings. The questionnaire was the same for all children and contained 29 items organized in five sections, as follows:(1) Descriptive data about the child: sex; date of birth; place of birth; weight, height, and body mass index (BMI) as recorded during the interview and converted in percentiles using two different DS-specific charts and one for the general pediatric population –11.(2) Quantity, quality, and distribution of food, physical activity, and relationship with food, as recorded over 3 days (one of which during the weekend). This information was crosschecked with a food recall of the previous 24 h at the time of the interview, to make sure that daily and weekly food intake frequencies were accurately recorded. Data were converted into portions and analyzed using the Scotti-Bassani Food Atlas  to obtai(3) Number of medical and speech-therapist follow-up visits requiring specific nutritional interventions in case of difficulties with specific food texture consumptions. Children were classified based on one or more texture difficulties .(4) Medical and surgical comorbidities .(5) Laboratory tests: lipid profile consisting of total- (TC), high-density (HDL), and low-density lipoprotein (LDL) cholesterol (mg/dL) as well as triglycerides (TG) (mg/dL), hemoglobin (g/dL), white blood cells (/microL), total proteins (g/dL), creatinine (mg/dL), iron (mcg/dL), blood glucose (mg/dL), uric acid (mg/dL), zinc (mcg/mL), magnesium (mg/dL), calcium (mg/dL), potassium (mEq/l), sodium (mEq/l), chloride (mEq/l), vitamin B12 (ng/ml), and folic acid (ng/mL).Data were extrapolated from the questionnaires, validated, and merged with information from the 24-h dietary recall, and then profiled according to some targeted variables which were then compared with benchmark values of the general pediatric populations or people with DS. Direct contacts with the families allowed to verify data in case of discrepancies.Descriptive statistics were expressed as means, percentages, and standard deviations (SD) or ranges. Children were divided into 3 age groups: 2–6 13 patients), 7–12 (16 patients), and 13–16 (5 patients) years. Male and female adolescents were not categorized in two separate groups because pubertal peak growth is generally delayed. Measured nutrient data were compared with the Italian recommendations ; expected intakes for each nutrient were computed in an individualized manner based on age, gender, and anthropometric parameters. Desired daily energy expenditure was computed using the Schofield equation SACN, 14) with the specific four factors  multiplied by the lowest (25 percentile) age-range physical activity factor , bread and derivatives (47%), and sweets (29%) were the favorite food. The least-liked ones were vegetables (34%) or, in specific cases, selected vegetables, followed by sweets (26%) and fruit (18%).Average water consumption was 1 liter ± 400 ml. Children up to 3 years of age were excluded as several of them still used milk as a main source of liquids.Multivitamin supplements were prescribed to 4 (12%) children by the pediatrician. One of them received an additional Omega3 supplement.Most of the children  completed the portion size. Eleven (32%) ate less than half of the amount offered at meals. Alternatives were offered such as salted foods (bread and derivatives) in older children and milk in toddlers. Oral issues may greatly impair childhood nutrition. All children but one were followed by a dentist and 30 (88%) by a speech therapist to improve language and swallowing performances.Most children  practiced a sport outside of school. Of these, the majority devoted at least 2 h per week to physical activity, practicing more than one sport. Overall, the children were very active both at home and at school.In both preschool and school children, average daily time on screens was less than an hour.In 25 children (73%), weaning started at 7.5 ± 2 months of age and continued with a regular progressive insertion of different foods.In the remaining 9 (27%) cases, weaning was delayed due to surgery in the 1st months of life (mostly for cardiac causes) with long hospital stay, forcing families to use milk as a source of primary nourishment. These families (besides two having dairy and eggs intolerance) also reported difficulties with food textures: 4 children consumed only purees until 3–4 years of age, 1 child considered all foods to be too large in size, and 2 children had difficulties with meat, raw vegetables, or fruit. For 45% of parents, the weaning delay was due to a “lazy” chewing action .Sippy cups or glasses were introduced at an average age of 23 ± 16 months; one child waited until 7 years of age. Only 7 children (21%) independently drunk from the sippy cup or glass by the recommended age limit of 24 months.Families were also asked if they had received any nutrition counseling after weaning. The majority (53%) responded negatively, while the remaining 47% seemed to have different sources of counseling such as pediatricians and other health-care professionals, sports instructors, the Internet, and other parents. Those who joined the Club at an early age of the child were those who reached results more quickly.Liquid and puree/mashed foods were easily ingested. As for other consistencies, 52% of parents confirmed difficulties with solid-hard consistencies such as raw vegetables or dried fruit, 48% of children had problems with dual consistencies such as pasta and beans or minestrone with pasta as well as unpeeled fruit or vegetables, and 45% with fibrous, sticky, or smelly foods such as meat, vegetables with high cellulose content, ham, and fish. The most frequent cause of complete weaning delay was lack of appetite and unwillingness to chew (50% of cases), followed by refusal of a despised taste (18%) or hard to chew (18%). In the remaining cases (14%), technical difficulties were likely to be the primary cause .The most frequent medical problems were skin conditions such as recurrent dermatitis and eczema (35%), constipation (29%), cardiac defects (29%), diarrhea (24%), dental cavities (20%), and hypothyroidism (18%). Among drugs, the most prescribed was thyroxine, followed by polyethylene glycol.p < 0.01).Blood values, as summarized in The main results, for the entire sample and by age category, are summarized in Proper nutrition, especially in the 1st years of life, is essential for a prospective healthy life. Some feeding problems in DS children have already been described –6. HowevMost DS families look for help to offer a higher quality of life to their children and often they look outside of hospital settings. Therefore, we recruited a group of DS children with access to a multidisciplinary support in a familiar environment. We collected a wide range of objective data (considering the high level of knowledge these families have) through which we identified some patterns potentially useful for further investigations.Our first consideration relies on an outcome of eating habits: anthropometric measurements. The literature repeatedly reports that DS children are predisposed to obesity , 15, 16.Eating habits are intertwined with neurodevelopment evolution and weaning is a pediatric strategic knot. Our DS children did not show major delays at weaning start. Delays occurred only in those who underwent surgical interventions, especially for congenital heart diseases, during their 1st months of life. Even after a good start, however, the weaning progress was not easy. Sippy cups and glasses were introduced between 12 and 24 months of age in all children but one. However, only one child every five was able to independently drink from the cup or glass by 24 months of age. This threshold should be achieved based on the International Pediatric Association's recommendations to prevent health risks such as overweight . ChewingParents seem to believe that DS children need nutrition supplements because they eat food poor in fibers, vitamins, micronutrients, and antioxidants to bypass the chewing and swallowing difficulties. In a recent survey, almost 50% of interviewed parents gave supplements to their DS children (more than 80% under 2 years of age), and one in five did not inform the pediatrician . This paLooking more specifically at nutrients, eating habits were not despicable either. Our cohort had similar eating patterns as other DS children compared to the general pediatric population , 5, 16. Fruit juices and ready to drink tea were the main sources of simple sugars. Their intakes increased with age and especially in boys, although soft drinks were rarely assumed. It is possible that the observed significantly increasing trend of triglycerides in school age children may depend on fructose intake, largely present in fruit juices and teas. The effects of fructose on liver steatosis and hypertension occurrences have already been confirmed in the literature . Liver uWe are aware that this study has some limitations. First, the group is small and the age range is wide. However, DS is still a rather rare condition and the literature presents studies with similar sample sizes. In addition, the information we collected is less heterogeneous than the age-range would imply, because the parents of these children were all very committed and it was they who provided most of the data. Therefore, the differences we found in the three age subgroups cannot be ascribed to the children's different reporting abilities, and they merit further exploration. Unfortunately, we could not carry out further statistical analysis in the three subgroups due to their small numbers. Second, nutrient intakes were self-reported through food diaries. To limit potential biases, the food diaries were verified through direct interviews with the dietician who had followed the children during the entire time span of the study, offering information and education tools to the families. The dietician also verified and cross-checked the information, combining the 3-day food frequency questionnaire and the 24 h recall. We had the impression that the parents of these children had a good nutrition knowledge, which may have contained biases during data collection. As for the program, there is room for improvements. Meals, for instance, were investigated at the personal level. The next step can be sharing food with other children, instructors, and families. Third, some nutrients such as vitamins D and C were not evaluated, mostly due to the lack of laboratory data. Fourth, the children in this sample reached a level of nutritional, social, and psychomotorial development closer to that of the general pediatric population. Approaches were similar across all patients, offering objective and comparable data and selection bias minimization, both of which enhanced the quality and credibility of the results. However, it is necessary to verify and quantify the potential benefits in the general DS population as well as to confront the benefits of DS childhood participation in different organized support programs.This study offers several practical suggestions. Anthropometrically, the risk of overweight/obesity can be curtailed in DS. Nutritionally, eating habits can reach good standards and future support programs may tailor nutrition patterns with a special attention to saturated fats, simple sugars, fiber, micronutrients and vitamins intakes. Developmentally, the early and late phases of the weaning process are critical and need to be monitored step by step especially in children affected by prolonged hospital stays and surgical operations. Fifty-three percent of the families reported no nutrition counseling when entering the program and 47% relied on different sources including Internet. This may have delayed the weaning process. Therefore, interventions are mandatory and may include sections in which children share meals. This aspect may also prove psychologically useful to parents. Their influence on child eating behaviors has been widely recognized . The revIn conclusion, aspects in lifestyles of DS children attending a small care program seem different than what present in the literature. Nutritionally and developmentally, these children, especially those in early ages, seem to achieve better results. Only few corrections emerged as necessary, such as (i). to contain protein and saturated fat intakes at all ages, (ii). to adjust sodium, calcium, folate, and fiber in children entering adolescence, and (iii). to suggest and start prompt intervention in the early years of DS children. Not only hospital-based approach, but practical yet professional suggestions through a small reality such as this care program may accompany DS children and their families. This small reality may be exported and adapted worldwide.This could make a difference in terms of both treatments and prevention of “future” events. We do hope that this exploratory study may act as a stimulus for larger, multicenter endeavor.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Insieme no profit association. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.GR and MF: conceived, coordinated, and carried out the study including statistical analyses and manuscript drafting. GC, RD, AC, GB, and MS: collaborated in the evaluation. All authors participated in the manuscript and took public responsibility for its content, contributed to drafting, and revising the paper.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.


IDS gene that encodes iduronate 2-sulphatase. As a result, there is an accumulation of glycosaminoglycans—heparan sulphate and dermatan sulphate—in almost all body tissues, which leads to their dysfunction. Currently, the primary treatment is enzyme replacement therapy, which improves the course of the disease by reducing somatic symptoms, including hepatomegaly and splenomegaly. The enzyme, however, does not cross the blood–brain barrier, and no improvement in the function of the central nervous system has been observed in patients with the severe form of the disease. An alternative method of treatment that solves typical problems of enzyme replacement therapy is gene therapy, i.e., delivery of the correct gene to target cells through an appropriate vector. Much progress has been made in applying gene therapy for MPS II, from cellular models to human clinical trials. In this article, we briefly present the history and basics of gene therapy and discuss the current state of knowledge about the methods of this therapy in mucopolysaccharidosis type II.Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder based on a mutation in the  Mucopolysaccharidosis type II , also called Hunter syndrome, is a rare monogenic disease belonging to the group of lysosomal storage disorders (LSDs). The estimated incidence of MPS II is 0.3–0.7/100,000 births . It is iIDS gene, located at Xq28 (OMIM *300823), which encodes iduronate 2-sulphatase. This enzyme is responsible for catalysing the hydrolysis of sulphate groups from dermatan sulphate (DS) and heparan sulphate (HS) molecules. Enzyme deficiency or decreased activity results in the accumulation of glycosaminoglycans (GAGs) in various tissues and organs, leading to dysfunction [Danio rerio, Mus musculus) indicate that a mutation in the IDS gene may also affect the developmental process differently by influencing the signalling pathway of the fibroblast growth factor (FGF) [MPS II is caused by a mutation in the function ,12. Dermfunction . On the or (FGF) . Anotheror (FGF) .The animal models, mainly mouse models, of mucopolysaccharidosis type II significantly contributed to understanding the pathophysiology and the application of therapy in this disease, including gene therapy . The firDespite its heterogeneity, the disease is usually classified into two main forms: attenuated  and severe . Both conditions show signs of many organs’ dysfunction, such as hepatosplenomegaly, coarse facial features, skeletal system abnormalities (dysostosis multiplex), joint stiffness, short stature, carpal tunnel syndrome, heart valve disease, hypertension and other cardiac abnormalities, communicating hydrocephalus and hearing loss. In addition, patients with MPS II also suffer from frequent respiratory infections and decreased exercise tolerance. The severe form of the disease, which accounts for about 60% of cases, is characterized by the child’s normal development until about 2–4 years of age when cognitive functions deteriorate and the process of acquiring new skills is inhibited. Attention difficulties are also common in school-age patients with the severe form of Hunter syndrome. An additional manifestation of CNS damage is frequent epileptic seizures. Patients with deletions, recombination, frameshift mutations, or splicing abnormalities are more likely to develop a severe phenotype. In the case of missense mutations, some may lead to a severe phenotype but most likely predisposed to the attenuated form. Due to the heterogeneity, in many cases, it is difficult to determine the exact genotype/phenotype correlation and predict the nature of development in a particular patient ,17.IDS, but the development of Next Generation Sequencing (NGS) has paved the way to use gene panels to detect the mutation quickly and exclude other lysosomal storage disorders [For the final diagnosis of the disease, biochemical and molecular tests are performed. First, the level of glycosaminoglycans in a 24-h urine collection is determined and then the activity of the iduronate 2-sulphatase enzyme, e.g., in peripheral blood lymphocytes, skin fibroblasts or chorionic cells  is assessed ,13. Sangisorders ,18,19.Patients with mucopolysaccharidosis type II, as a disease with a heterogeneous clinical picture, require multidisciplinary care. Historically, before the pathophysiology of the disease was known, management was limited to symptomatic treatment and palliative therapy. In studies conducted on fibroblasts collected from patients with mucopolysaccharidosis type I (MPS I) and mucopolysaccharidosis type II, normal levels of GAGs were observed. Researchers discovered the phenomenon, so-called cross-correction, i.e., enzyme secretion by some cells and its uptake by others via the mannose-6-phosphate receptor ,21. The ® [Unfortunately, the enzyme itself cannot cross the blood–brain barrier (BBB). Attempts are made to administer the enzyme intrathecally or intraventricularly. These studies were successfully performed in the MPS II mouse model. Higuchi et al.  showed a® . The res®, JR-141, JCR Pharmaceuticals) [®, a system based on J-Brain Cargo® containing 10 mL of pabinafusp alfa administered intravenously. It is the first agent on the market that allowed the enzyme to cross the blood–brain barrier [Another way to bypass the difficulties associated with the blood–brain barrier is to create a system that allows the drug to pass through the barrier. For this purpose, two solutions are tried: receptor-mediated transcytosis and the use of carriers ,27. The uticals)  and an auticals)  enabled  barrier . The res barrier . A clini barrier . Another barrier . An addi barrier ,33.Another method of causal treatment is hematopoietic stem cell transplantation (HSCT). Peripheral blood monocytes can cross the blood–brain barrier and settle in the central nervous system as microglial cells . This prAn alternative to the above therapeutic methods is the constantly developing gene therapy. Mucopolysaccharidosis type II is a monogenic disease with relatively well-known pathomechanisms, making it a good candidate as a target for gene therapy. In addition, it allows avoiding the characteristic difficulties of ERT and HSCT. A comparison of the three main treatments in MPS II is shown in ®, an adenovirus with a human gene encoding p53 protein, introduced in 2004 in China, used in squamous cell carcinoma of the head and neck [®), introduced in 2012. It is an adeno-associated virus (AAV) vector expressing the human lipoprotein lipase gene used in familial lipoprotein lipase deficiency [®) in congenital retinal dystrophy, onasemnogen abeparvovec (Zolgensma®) in spinal muscular atrophy or Strimvelis® in severe combined immunodeficiency caused by adenosine deaminase deficiency [The term “gene therapy” includes the use of nucleic acids to treat human disease. Gene therapy is generally divided into in vivo therapy, with the direct administration of the vector to deliver the correct gene to the cells, and ex vivo therapy, in which cells are collected from the patient’s body, applied with the vector and re-administered to the patient. Vectors are classified as viral  and non-viral  ,36. Geneand neck ,39. The ficiency ,41. Currficiency .IDS complementary DNA (cDNA) under the transcriptional control of a long terminal repeat sequence or cytomegalovirus early promoter. The vector was applied to peripheral blood lymphocytes collected from affected persons. The authors assessed the normal activity of iduronate 2-sulphatase and observed the phenomenon of cross-correction between cells. A similar study using a modified virus was carried out by Hong et al. [IDS was highly expressed in cells. As a result, a clinical trial was opened but was discontinued due to insufficient gene expression and side effects [The first attempts to create gene therapy for Hunter syndrome were made in the 1990s using retroviral vectors. Initially, good results were obtained in preclinical trials using lymphocytes and hematopoietic stem cells CD34+ ,43. Brau effects ,45. The  effects ,47,48. FIDS to mice led to an increase in the activity of iduronate 2-sulphatase and a decrease in the accumulation of glycosaminoglycans in the central nervous system and other organs. In addition, the authors demonstrated a reduction in the accumulation of secondary substances related to autophagy, the p62 protein, and protein and ubiquitin conglomerates in the cells. Neither was there any deterioration in neuronal function [One of the types of gene therapy is the ex vivo method of collecting cells from the patient to be treated. Usually, for this purpose, laboratories use hematopoietic stem cells or so-called induced pluripotent stem cells (iPSCs) obtained from already differentiated somatic cells, e.g., fibroblasts. iPSCs can later differentiate into many cell types, e.g., neurons, myocytes, hepatocytes, etc. ,49. Aftefunction . As discfunction ,54.Parvoviridae family. They contain a 4.7 kbp genome as a single-stranded DNA molecule [Another way to deliver the correct gene to cells is in vivo gene therapy with adeno-associated viruses (AAVs). They are small viruses (25 nm) belonging to the molecule . These vmolecule . Human imolecule ,58. Unlimolecule ,59,60,61One of the first studies on using of AAVs in lysosomal storage disorders, including mucopolysaccharidoses, was carried out at the end of the 20th century by research groups led by Sands  and DaviIDS gene, under the transcriptional control of the liver-specific promoter of the TBG gene. Adult mice were injected intravenously with 1.0 × 1011 virus particles. The mice were evaluated for seven months. The authors showed that iduronate 2-sulphatase activity was restored in plasma and tissues , and the levels of GAGs in urine were normalized. Skeletal abnormalities also improved. A similar study on a mouse model was carried out by Jung et al. [IDS cDNA into the pAAV2-EF-eGFP-WPRE-polyA plasmid as a substitution for the eGFP element. As a result, they created pAAV2-EF-hIDS-WPRE-polyA plasmid, which, in addition to the coding sequence for iduronate 2-sulphatase, contains the human elongation factor 1-a promoter (EF), woodchuck hepatitis virus posttranscriptional element (WPRE) and a signal sequence of bovine growth hormone poly(A) chain. As in the study mentioned above, the viruses were administered intravenously at a dose of 1.0 × 1011 virus particles. The activity of iduronate 2-sulphatase and the concentration of GAGs were assessed 6 and 24 weeks after applying AAVs. Significantly increased activity of the enzyme and decreased concentration of GAGs in the liver, brain, kidneys and spleen have been demonstrated. Additionally, the authors observed a reduction in cell vacuolization in the histopathological examination of the brain [Cardone et al. conducted the first study using AAVs in an animal model (mouse) of mucopolysaccharidosis type II . The autg et al. , who usehe brain .IDS, under the control of the ubiquitous CAG promoter. In this case, a mouse model was also used, but the viruses were administered directly into the cerebrospinal fluid by injection into the cisterna magna at a dose of 5.0 × 1010 vector particles. Four months after the virus administration, the authors assessed the effects of the therapy. Iduronate 2-sulphatase activity in the central nervous system was determined to be 40% of that in wild-type mice. In addition, they showed that most vectors are located in neurons and not in microglia or astrocytes. The concentration of GAGs and the gene expression profile in the brain was normalized. In addition, the vectors permeated the blood and led to the improvement of somatic symptoms and the accumulation of GAGs in peripheral organs such as the liver, lungs and heart. The authors observed an improvement in motor and cognitive functions and increased overall survival compared to untreated individuals [Another type of adeno-associated virus, AAV type 9, was used in subsequent preclinical studies ,68. Motaividuals .Promising results from preclinical studies have opened the way to first clinical trials using AAVs to improve central nervous system function in patients with Hunter syndrome. Two multicentre  phase I/II clinical trials are currently underway on RGX-121 (Regenxbio Inc.), containing a recombinant AAV9 vector with a cassette expressing the human iduronate 2-sulphatase gene (AAV9.CB7.hIDS) ,71,72,7310 genome copies (GC)/g brain mass, 6.5 × 1010 GC/g brain mass, and 2.0 × 1011 GC/g brain mass, respectively [The first study  included patients with the severe form of MPS II, with central nervous system involvement, from 4 months to 5 years of age. In the course of the study, a three-time single administration of RGX-121 to the cisterna magna or the lateral ventricle is planned in increasing doses, 1.3 × 10ectively . The patectively .10 GC/g brain mass. Similarly to the study mentioned earlier, the complete safety and efficacy evaluation will take 104 weeks from the agent’s application [The second study  targeted patients with severe Hunter syndrome aged 5 to 17. In this study, the vector administration route is also intracisternal or intracerebroventricular injection, but only once at a dose of 6.5 × 10Adeno-associated viruses are currently the most optimal choice for delivering the correct gene to affected cells among the viral vectors available. Their ability to provide stable gene expression, deliver it to both dividing and non-dividing cells, and a low risk of mutagenesis make their use in clinical trials likely to increase. The apparent limitation of all viral vectors administered into the body is the risk of developing an immune response against them. However, the above-mentioned benefits of using AAVs outweigh their immunogenicity, which is not major anyway.One way to overcome the limitations of viral vectors in gene therapy is using other vectors that are not based on viral particles. Non-viral vectors include nanoemulsions, the transposon system with the Sleeping Beauty transposase or the electro gene transfer method ,74. TheyIDS in 50 µL solution into quadriceps of mice. Hyaluronidase was injected into the muscle before administration of the plasmid itself to increase the effectiveness. The muscle was electrically stimulated with a current of 75 mA. The procedure proved to be very effective in protein production in the muscle, unfortunately without the proper activity of iduronate 2-sulphatase in the blood. Additionally, the authors observed a strong immune response against the recombinant protein [In research on MPS II using non-viral vectors, preclinical trials have been carried out using only gene electrotransfer, i.e., a method of delivering exogenous molecules into cells through electrical pulses ,76. FrisStaphylococcus aureus and Streptococcus pyogenes [Another promising way to obtain the correct gene into cells is genome editing methods that have been intensively developed in the last decade . Genome pyogenes .IDS gene in place of the first intron of the albumin gene locus in mouse hepatocytes. They applied AAV type 2/8 administered intravenously in increasing doses, 2.5 × 1011 vector genomes (vg), 5.0 × 1011 vg, and 1.5 × 1012 vg, respectively. The authors demonstrated normal activity of iduronate 2-sulphatase in blood and other tissues, as well as normalization of GAGs concentration in urine and organs. Additionally, they found that the applied therapy prevented the deterioration of neurocognitive functions in the examined mice [Laoharawee et al. conducted a preclinical study in a mouse MPS II model. They used zinc finger nucleases (ZFNs) to insert the human ned mice .IDS gene, and the AAV type 2/6 vector. The target site for IDS is the albumin locus in hepatocytes. The drug is administered intravenously in increasing doses. The study includes nine patients aged five years and older, divided into three cohorts depending on the dose. Currently, the recruitment for the trial is closed [Thanks to the positive results of research on model organisms, the first multicentre phase I/II clinical trial was launched in patients with MPS II  using in vivo gene therapy based on genome editing . This sts closed . Sixteens closed . locus and provide long-stand gene expression. Genome editing systems, like their vectors, AAVs, are exogenous particles, and immune response may be developed against them. However, the benefits of using these precision genetic modification tools outweigh the potential limitations. A summary of the methods of gene therapy in mucopolysaccharidosis type II is presented in Genome editing methods are gaining significant interest due to their precision in reaching the target site in the genome. It seems that they will gain an advantage over other forms of gene therapy as they do not randomly integrate the correct gene but directly repair the defective sequence in its Gene therapy for mucopolysaccharidosis type II has made tremendous progress since the first studies on cell lines in the 1990s. However, there are still a few limitations that researchers dealing with this topic must face. First, many animal models differ from the in vivo conditions in the human body. The beneficial effects of the applied therapies in the mouse model, especially on the central nervous system, may not necessarily be reproduced in human studies due to the therapeutic threshold, which differs between species.Another limitation is the risk of generating an immune response that mainly affects viruses but also elements of genome editing systems. AAVs seem to be the most optimal viral vector so far. However, neutralizing antibodies have been reported, and they lead the way to reduce the effectiveness of the entire therapy . AdditioThe use of retroviral and lentiviral vectors is likely to be abandoned in the near future due to their random integration into the genome and the risk of mutagenesis. AAVs seem to be the most optimal vectors. Combined with the precise genome editing method, they are currently the best form of gene therapy and the only one so far used in human clinical trials. It is possible that in the next few years, new clinical studies will be opened, and this process would be significantly accelerated by the positive results of phase I/II trials with RGX-121 and SB-913.It is also worth mentioning additional therapy that may play a role in improving organ functions and quality of life in patients with Hunter syndrome soon. The process of autophagy, which is disturbed in lysosomal storage disorders, may become a potential therapeutic target. Maeda et al.  analysedIDS gene to target cells and maintain a high expression level far exceeds the need for repeated intravenous administration of the enzyme. ERT applied intravenously is a heavy burden on the healthcare system and partially improves somatic symptoms without improving central nervous system function.Mucopolysaccharidosis type II, as a monogenic disease with relatively well-known pathophysiology, is an optimal example of the possibility of applying gene therapy. Over the past two decades, this technology has been advanced enough to open up clinical trials using the most promising methods—genome editing and adeno-associated viral vectors. Thus far, it has not been possible to come up with an ideal therapeutic agent. Additionally, gene therapy has some limitations: the immunogenicity of vectors and transgenic elements and the relatively high cost of developing this technology. However, comparing gene therapy to the current standard of treatment for Hunter syndrome—enzyme replacement therapy is essential. A single administration of a vector that will deliver the In conclusion, the further development of molecular engineering techniques and the ongoing clinical trials in humans will likely contribute to the broader use of gene therapy in mucopolysaccharidosis type II. Additionally, this will lead to faster achievement of therapeutic goals and improvement of patients’ quality of life.


The possibility of systematically implementing IGRAs such as the QFT-Plus assay in centers that perform LTBI screening has been accelerated by the decreased availability of the tuberculin skin test (TST) in several countries. Nevertheless, the process to implement IGRA testing in routine clinical care has many gaps. The study utilized the expertise acquired by the laboratory teams of the Regional Prospective Observational Research in Tuberculosis (RePORT)-Brazil consortium during study protocol implementation of LTBI screening of tuberculosis (TB) close contacts. RePORT-Brazil includes clinical research sites from Brazilian cities and is the largest multicenter cohort of TB close contacts in the country to date. Operational and logistical challenges faced during IGRA implementation in all study laboratories are described, as well as the solutions that were developed and led to the successful establishment of IGRA testing in RePORT-Brazil. The descriptions of the problems identified and resolved in this study can assist laboratories implementing IGRAs, in addition to manufacturers of IGRAs providing effective technical support. This will facilitate the implementation of IGRA testing in countries with large TB burdens, such as Brazil.The interferon gamma release assay (IGRA) has emerged as a useful tool for identifying latent tuberculosis infection (LTBI). This assay can be performed through testing platforms such as the QuantiFERON-TB Gold Plus (QFT-Plus) assay. This IMPORTANCE The IGRA has emerged as a useful tool for identifying persons with LTBI. Although the implementation of IGRAs is of utmost importance, to our knowledge there is scarce information on the identification of logistical and technical challenges for systematic screening for LTBI on a large scale. Thus, the descriptions of the problems identified and resolved in this study can assist laboratories implementing IGRAs, in addition to manufacturers of IGRAs providing effective technical support. This will facilitate the implementation of IGRA testing in countries with large TB burdens, such as Brazil. Mycobacterium tuberculosis . Between 5% and 10% of individuals with LTBI, if not treated with tuberculosis preventive therapy (TPT), can progress to active tuberculosis (TB) during their lifetime  estimates that one-quarter of the global population is infected with rculosis , 2. Mostlifetime , 4. ThusMycobacterium bovis BCG vaccination or infection with nontuberculous mycobacteria and false-negative reactions in persons with immune suppression (M. tuberculosis-specific antigens that drive production of interferon gamma (IFN-γ) by specific T lymphocytes, enabling the development of more specific assays based on cellular recall responses to identify LTBI , which consists of intradermal inoculation of the TB purified protein derivative (PPD) and evaluation for cutaneous induration. However, this test has several limitations, such as false-positive reactions in persons with pression , 6. Addipression , 8. Receify LTBI , 10.in vitro tests have been incorporated into several guidelines worldwide and have recently been considered by the WHO as equivalent for the diagnosis of LTBI  has emerged as a useful tool in identifying persons with LTBI. This assay can be performed through two distinct testing platforms, such as the enzyme-linked immunosorbent spot assay (ELISPOT) (T-SPOT.TB) or the QuantiFERON-TB Gold Plus (QFT-Plus) assay. These  of LTBI , 10. Of cificity –13. The The advantages of the IGRA over the TST indicate that this immunoassay may be a reliable alternative to the TST. The possibility of systematically implementing IGRAs such as QFT-Plus in centers that perform LTBI screening has been accelerated by the decreased availability of TST in several countries, including Brazil . ImplemeAlthough the implementation of IGRAs is of utmost importance, to our knowledge there is scarce information on the identification of logistical and technical challenges for systematic screening for LTBI on a large scale. The present study was designed to fill this gap and provide information that would improve the effectiveness and efficiency of IGRA-based LTBI screening. The operational and logistical challenges faced during IGRA implementation in all four study laboratories are also described, as well as the solutions that were developed and led to the successful establishment of IGRA testing in RePORT-Brazil.The IGRA/QFT-Plus implementation process started with the checklist provided by Qiagen Corp. The four laboratories proceeded with the acquisition of equipment , reagents (ultrapure distilled water and cleaning solutions), consumables , QFT-Plus tube kits, and QFT-Plus enzyme-linked immunosorbent assay (ELISA) kits. Subsequently, laboratory technicians took GCLP training and were trained on the steps of collecting, transporting, incubating, and processing samples for laboratory tests. In addition, QFT-Plus ELISAs were performed, and a pilot test was carried out, with the objective of including improvement strategies to initiate the study protocol .During the IGRA implementation process, several gaps were identified, even after following all of the recommendations by the manufacturer of the QFT-Plus assay. Initially, the laboratories identified problems related to acquisition of equipment, reagents, and consumables. The equipment had to be purchased through the sites, generating additional costs. Furthermore, the microplate washer had problems at the beginning of the ELISAs, requiring corrective maintenance. QFT-Plus tube kits and QFT-Plus ELISA kits were imported, which generated logistical problems regarding delivery to the sites. To resolve this problem, delivery of the QFT-Plus tubes and ELISA kits was directed to one of the clinical sites in Rio de Janeiro, which then distributed the supplies to the other laboratories.The five clinical sites started recruiting patients and collecting samples under routine conditions. Prior to starting, the collecting station was organized, accounting for the environment and the tubes to be used in the QFT-Plus test. These tubes had to be stored between 4°C and 25°C and taken out for immediate use only. In addition, the tubes had to be collected in a specific order, following the manufacturer's recommendations, with the nil tube being collected first, followed by the TB1, TB2, and mitogen tubes. The tubes containing the samples went through a homogenization process that consisted of several inversions, in which all of the biological material had to come into contact with the inner surface of the tube (L-motion with exactly 10 inversions). Finally, QFT-Plus tubes were packed in boxes with temperatures ranging from 17°C to 27°C for transfer to the processing laboratory .After collection at the clinical sites, samples were transported to the four RePORT-Brazil laboratories in two different settings, as follows. Setup A (pertaining to sites 1 and 2) was characterized by performing the collection and processing of samples in the same place, with the collection room and laboratory within the same site. Thus, the study staff responsible for collecting and organizing the samples could transport the samples to the laboratory without requiring a vehicle  . Setup BUpon arrival at the laboratories, samples had to pass a quality check regarding transport time, temperature, tube identification (ID), volume, and the presence of hemolysis or clots. Finally, the samples were processed and incubated at 37°C for 20 h, aliquots were prepared, and the plasma samples were frozen at −20°C until the QFT-Plus ELISA was performed.During the QFT-Plus implementation process, several problems were detected, generating nonconformities, such as samples without an ID, transport with temperatures outside the established standard (temperature deviation), sample leakage, and other issues . The proportions of nonconformities are shown in P < 0.001). These results were likely due to conditions related to the collection and transportation of samples, since setup B required sample transport by vehicle. In addition, these gaps generated learning opportunities for the teams, which over the years of implementation decreased the proportions of reported nonconformities. Of note, setup B also presented significantly higher occurrences of transport box change, coagulated samples, and lack of minimum transport conditions, characterized in Table S1 as “Others” (P < 0.001).The temperature deviation and other nonconformities were noted particularly among setup B sites , compared to setup A sites, in all years analyzed  . There wP < 0.001), influencing the temperature variation, which was also significantly less in comparison to setup B (P < 0.001).The distance between the collection site and the sample-processing laboratory directly influenced the time and the temperature variation during transportation . Setup ASetup A had higher temperatures at the time of specimen shipment, compared to setup B, but the two setups had similar temperatures at the time of specimen receipt/arrival.  and B. AOver the years of implementation, the variation in time between sending and receiving samples tended to decrease until 2018, and then there was an increase in the variation in 2019. The decrease in time variation was possibly because study and transport  became more efficient with the implementation of the test at the sites. Although we observed this increase in 2019, it is noteworthy that there was an increase in the number of samples analyzed that year.versus sending time and the variation in temperature. In this analysis, it was possible to note that the temperature variation was directly correlated with the time variation  and that this variation was particularly seen in setup B  in the study influenced the results of the QFT-Plus assay. Setup B had a higher percentage of indeterminate and positive results, probably due to the longer time between the collection and processing of the samples, in addition to other variables evaluated here . It is important to ensure that, in the analytical phase, the laboratory technicians involved in these steps are comfortable with performing the QFT-Plus assay, including simple tasks such as controlling the temperature of the shipping boxes, identifying nonconformities, minimizing errors, and identifying problems in the results generated. It is recommended to include in the QFT-Plus implementation planning a period of adaptation and short retraining directed at the critical stages of sample collection, transport, and processing, with the aim of gradually minimizing or eliminating nonconformities and preanalytical errors. This learning can assist in the quality control of test results and performance, since the reproducibility of IGRAs can be influenced by these factors .Biological samples can have different performance or results with respect to the quantity of analytes used and, based on this, we must seek to identify points that cause variation of test results. The laboratory routine can have different components that can cause variability in results, such as different processing rates, variations in processing rates throughout the month, and variations of factors related to the environment (such as temperature) throughout the year .The reproducibility of the test results depends directly on the training of the team, since variation in the operator for the collection, homogenization, and performance of the test can impact the results. Therefore, the standardization of quality control in the clinical and laboratory spheres is essential so that there are no significant effects on the result. In addition, structural variations between different laboratories can influence the proportions of indeterminate results , 19.in vitro, delays in the start of the incubation of study samples can interfere with the quantification of IFN-γ levels, leading to a decrease of up to 0.24 IU/ml after 6 h  between 2016 and 2019. It was conducted in five research centers located in four Brazilian cities, namely, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD) in Manaus-Amazonas, Instituto Brasileiro para Investigação da Tuberculose (IBIT) in Salvador-Bahia, Secretaria Municipal de Saúde de Duque de Caxias (SMS-DC) in Duque de Caxias-Rio de Janeiro, and Instituto Nacional de Infectologia Evandro Chagas (INI) and Clínica da Família Rinaldo Delamare, Secretaria Municipal de Saúde do Rio de Janeiro (SMS-RJ), in Rio de Janeiro . The fivStandard operating procedure (SOP), good clinical practice (GCP), GCLP, and other trainings were carried out by the project staff prior to initiation of the study. In addition, all sites provided up-to-date equipment maintenance certifications to ensure test quality and to minimize risks for laboratory technicians who processed biological materials daily.Initially, the QFT-TB Gold In-Tube (QFT-GIT) assay was implemented in the laboratory routine of the RePORT-Brazil consortium; subsequently, it was replaced by the QFT-Plus assay. Every laboratory received training on sample collection and processing by the Qiagen Corp. . Figure S2 in the supplemental material summarizes the QFT-Plus steps. Briefly, venous blood for testing was collected in four tubes  and incubated at 37°C for 20 h. After incubation, samples were stored at −20°C until the ELISA was performed within 2 weeks. IFN-γ levels  were quantified with a 4-point standard curve. QFT-Plus analysis software was used to generate the results according to the manufacturer’s recommendations . The sofLaboratory information from the QFT-Plus implementation process was evaluated by the teams from all RePORT-Brazil laboratories. Data were obtained from team training, equipment maintenance, and preanalytical evaluations, such as type of sample collection, place of sample collection and processing, tube ID, transport quality control , presence of hemolysis or clots, and volume of the samples, as well as qualitative results . All data were entered into the Research Electronic Data Capture (REDCap) platform, reviewed by data managers for quality control, and subsequently approved for the study analyses.P values of <0.05 were considered statistically significant.Descriptive analyses were performed to characterize the study laboratories and quality control measurements. Categorical variables were displayed as frequency and percentages and compared using a two-sided Pearson's chi-square test (Yate’s correction) or the Fisher's two-tailed test in 2 × 3 or 2 × 2 tables, respectively. Continuous variables were displayed as median and interquartile range (IQR) and tested for Gaussian distribution using the D’Agostino-Pearson test. Comparisons of values between two groups of data were performed using the Mann-Whitney U test. The Spearman rank correlation test was carried out to assess relationships between variables. Data analyses were performed using R (version 3.6.3) and the Hmisc (version 4.4.1), compareGroups (version 4.4.3), ggplot2 (version 3.3.2), and ggcorrplot (version 0.1.3) R packages. All analyses were prespecified. Differences with The original contributions presented in the study are included in the supplemental material, and further inquiries can be directed to the corresponding author.