Food frequency questionnaires (FFQ) is an easy and inexpensive tool that can be used to evaluate nutrient and dietary trends of groups and individuals. Few studies in the East Mediterranean region tailored FFQs to describe dietary intakes of older adults. The purpose of the study is therefore to assess the validity and reproducibility of a FFQ, designed for use with older adults living in a Mediterranean Arabic speaking country, Lebanon.The FFQ is composed of a list of 90 food items, commonly consumed by adults above 60 years of age. Validity of the FFQ was tested using the mean of two 24-hours dietary recalls (24HDR), and reproducibility, by repeating the questionnaire within a one-month period, along the second dietary recall. Our study included 42 and 76 participants, for the repoducibility and validity analysis respectively. Subjects were randomly selected from 2 of the 8 governorates in the country.FFQ reproducibility showed a mean relative difference of 1.03% without any significant difference between all paired components of nutrients. Intra class correlation (ICC) showed good and excellent reliability for caloric intake and all macronutrients, moderate to good reliability for all remaining nutrients, except for poly-unsaturated fatty acids, vitamins A, B12 and fibers. Correlation coefficients for all nutrients were fair to strong. Both administrations of the FFQ showed good internal validity. Validation of FFQ showed a mean relative difference between FFQ and mean 24HDR at 19.5%. Agreements between the 2 methods, for classifying individuals in the same or adjacent quartile, for nutrient intake and nutrient adequacy, were 80 and 78.2% respectively. Mean Kappa coefficient was 0.56 and energy-adjusted correlations were within the recommended values for all items except for vitamin A and B12. Adjusting for nutrient-dense food intake improved the agreement for theses 2 vitamins to 0.49 and 0.56, respectively.The proposed FFQ can be considered a valid tool to help describe nutrient intake of older individuals in an Arabic speaking Mediterranean country. It could serve for possible use in the East Mediterranean region for the evaluation of regular dietary intake of community-dwelling older adults.The online version contains supplementary material available at 10.1186/s12937-022-00788-8. Understanding the impact of dietary factors on the development of chronic diseases in elderly, is important for better prevention and treatment –6. ChoosFrequently used direct retrospective methods are diet records (DR), 24 hours dietary recalls (24HDR) and food frequency questionnaires (FFQ). They can be self-reported or interviewer- reported depending on the needs of the study. To reflect chronic and habitual intakes, it is recommended that DR and 24HDR tools be repeated over several days. These recommendations might be difficult to apply, expensive and time consuming particularly in the setting of lack of literacy and old age, especially in large scale population-based studies –9.FFQ is easier and less costly and can be administered to large cohorts. It usually can categorize and rank individuals by their usual frequency of food consumption. FFQ allows for comparison of food and nutrient intake across populations and countries with similar cultures and food availability , 10–13 aEach FFQ is constructed based on specific study objectives and target populations and needs to be validated to serve other groups and other study designs. For more reliable results, adaptation should therefore be made to the age-group targeted , 16. TheNon-institutionalized older adults in Lebanon are susceptible to a higher risk of malnutrition and insufficient dietary intakes due to inappropriate or inexistant retirement plans, limited access to health care services and social welfare programs, as well as inadequate living conditions , 18. ThiQuestionnaires used to evaluate food intake of older adults is scarce, particularly in the East Mediterranean region. In neighboring countries and in Lebanon, other questionnaires were used to estimate food and nutrient intake in adult population, but so far, no FFQ was validated exclusively among Lebanese older adults , 19–24.The aim of our study is therefore to validate a quantitative FFQ, specifically tailored to evaluate nutrient consumption of community dwelling Lebanese, 60 years of age and older, using the 24HDR as a reference method. This questionnaire could serve for possible use in Lebanon and other Arabic speaking Mediterranean countries as well.To study the validity of the FFQ, the mean of two 24HDR was used as a reference method, and to test the reproducibility of the FFQ, the same questionnaire was administered twice within a one-month period.From January 2017 till June 2017, recruitment of participants and data collection were carried out in collaboration with the Ministry of Social Affairs (MOSA) through the medico-social centers that serve low to middle class income families. As illustrated in Fig. To avoid inter-rater and reporting biases, participants were administered twice the FFQ and 24HDR by the same interviewer, and all filled questionnaires were reviewed by the principal investigator and field interviewers before data entry.As illustrated by Fig. n = 7 declining to do the second interview, and n = 1 for incomplete FFQ). A total of 13 out of 42 had their questionnaire answered by accompanying person, because of low cognitive test results. From an initial sample of 100 individuals included to test for the validity of the FFQ, and after a preliminary analysis, 24 participants were not included in the study: subjects with previously undeclared active cancer (n = 2), unaccompanied individuals with altered mental status and who did not comply with the study protocol (n = 9), those refusing to undergo the second interview (n = 7), those having extreme daily dietary intake  or > 4000 Kcal/day (n = 2)), as well as those having extreme restrictions and may not be representing a regular feeding pattern (interview was performed during fasting for more than 14 hours period) (n = 3). A total of 34 out of 76 had their questionnaires answered by accompanying person, because of low cognitive test results. Retention rates within a one-month interval for repeated questionnaires, for FFQ and 24HDR, were respectively 84 and 76%.For both analyses, the size of samples was set for an expected intra-class correlation (ICC) value of 0.75 and a minimum acceptable value of 0.5, with 95% confidence and a desired power of 80%. The number of participants for the reproducibility study was 42 out of 50 initially selected for this analysis , coffee, tea and other drinks, methods of preparation were also detailed, and specific ingredients were added to the report.Daily food consumption, reported by the FFQ was analyzed by the Nutrilog software  to extract daily nutrient intake. The United States Department of Agriculture/ Standard reference 28 (USDA/SR28) and the Canadian Nutrient File (CNF 2015) databases were used to analyze nutrient composition of simple foods, and branded foods. For composite traditional dishes and some locally consumed desserts, not found in the database, recipes from known local cookbooks and pastry chefs were entered to the database and used in the nutrient analysis. Data on food consumed were reported simultaneously in (g) and as portions. The nutrient intakes of the two 24HDR were also extracted using the same methods.To analyze the concordance between FFQ and mean 24HDR in classifying individuals in terms of nutrient adequacy, we calculated The Nutrient Adequacy ratio (NAR) of 17 selected nutrients, including vitamins A, D, E, K, C, B1, B2, B3, B6, B9, B12, and minerals such as calcium, phosphorus, magnesium, iron, selenium and zinc. The NAR was calculated by dividing the estimated nutrient intake of individuals by the age and sex-specific recommended dietary allowance (RDA) for these nutrients, according to the established dietary reference intake (DRI) recommendations  For all p < 0.05 was considered significant. To assess the internal consistency of the FFQ, after standardization of the variables, all items were computed in Cronbach’s alpha statistic. Alpha values between 0.7 and 0.8 indicate a good reliability, and a value higher than 0.9 would imply that all items on the questionnaire were very strongly related to each other and reliable for assessing the construct. To measure within-person variability, intra-class correlations (ICC) were calculated between the first and the second FFQ administration, using one-way random effects model, where people’s effects are random whereas interviewers performed under the same conditions. Values of r < 0.5 indicate a poor reliability, between 0.5 and 0.75 a moderate reliability, between 0.75 - 0.9 a good reliability, and greater than 0.9 excellent reliability [IBM-SPSS 20.0 was used for statistical analysis. All macronutrients, 10 vitamins and 5 minerals estimated intakes were reported and compared for the validity and the reproducibility studies respectively. For numerical variables, data was expressed as mean ± standard deviation and median (interquartile ranges), and as numbers and percentages, for categorical variables. Student t-test and chi-square test were used to compare numeric data and categorical data respectively. Normal distribution of calories and nutrients was assessed using Kolmogorov-Smirnov tests, and data was processed accordingly. Characteristics of the reproducibility and the validation study samples were compared using Mann-Whitney U test, for continuous data and Chi-square test for categorical data. Wilcoxon signed-ranks tests were used for comparisons of both related samples of food frequency questionnaires, and for the difference between FFQ and mean 24HDR. For normally distributed data, Pearson’s correlation coefficient was used to test the reproducibility (test-retest) of the FFQ and the relation between estimated macro and micronutrients between the FFQ and 24HRs. For data that was not normally distributed, Spearman’s correlation coefficient was used. Correlation coefficients and their respective 95% confidence intervals (CI) were calculated and iability . Energy To measure agreement of NAR estimation between the FFQ and the mean 24HDR methods, we calculated the percentage agreement between NAR estimated by FFQ and mean 24HDR by comparing classification in the same or adjacent quartile to distant quartiles, and through Kappa statistics. Concordance between the 2 NAR classifications was also measured using Spearman’s correlation coefficient.N = 76), had a mean age of 74.4 ± 6.9 years and a mean BMI of 30.3 ± 6.2 kg/m2. In this sample, 63.2% were women, 60.5% being married, and 84.2% living with their partners or relatives. The group of individuals included in the reproducibility/reliability study (N = 42) resembles the validity study sample, and characteristics of both did not differ statistically.Characteristics of the sample population are presented in Table The reproducibility study included 42 individuals, by repeating the FFQ questionnaire within a one-month interval. Median and mean intakes ± standard deviation (SD) of calories, macronutrients, and micronutrients of both FFQ1 and FFQ2 are reported in Table r = 0.3-0.5) was noted for MUFA, PUFA, omega-3 fatty acids (ω3 FA), omega 6 to omega 3 fatty acids ratio (ω6/ω3 ratio) and cholesterol intakes.To measure within- person variability, ICC and correlation coefficients  were calculated comparing the two administrations of the FFQ. Based on ICC, a good and excellent reliability was noted for caloric intake and most macronutrients. Remaining nutrients had a moderate to good reliability except for PUFA, vitamins A, B12 and fibers. Correlation coefficients for fibers, vitamin A and B12 showed a strong association between both administrations, as found for caloric intake, all macronutrients and micronutrients. Fair correlation (r = 0.82 (95% CI = 0.792-0.846) and r = 0.845 (95% CI = 0.77-0.905).Internal consistency, measured by Cronbach Alpha based on standardized items, was respectively 0.947 and 0.962 for the first and second administrations of the questionnaire, and their mean ICC, was respectively Caloric intake and intake of macronutrients and some relevant micronutrients estimated by the FFQ, were compared to the mean of both 24HDRs, allowing to evaluate the accuracy of the FFQ in our study sample and estimate its agreement with this chosen standard. Details of mean and median daily intake of nutrients and their correlations are shown in Table Interrater reliability between FFQ and 24HDR was evaluated thru percentage agreement and Kappa statistics for all listed nutrient intakes. Percentage agreement between the two tools, calculated by the ratio of classification in the same or adjacent quartile, to distant quartiles, placed older individuals in the same or adjacent quartiles, on average, in 80% of the sample population, with agreements ranging from 90.8% for sugars to 61.8% for vitamin B12. Based on Kappa coefficient, agreement between FFQ and 24HDR was, strong (> 0.61), for total calories, total fat, sugars, vitamins E, C, B1, B3 and calcium. Agreement was moderate (0.41-0.6) for all remaining nutrients except for vitamins A, B12 and fibers with poor agreement. Mean Kappa value was 0.56.Energy adjusted correlation between FFQ and mean 24HDR was performed by pairwise linear regression models and shown in Table We selected a group of nutrients to evaluate the bias between the nutrient mean differences and determine the agreement interval of the differences, between the FFQ and reference method we used. This agreement, performed thru Bland-Altman analyses, is represented in Fig. Table The present study showed that the proposed 90 food-items FFQ gives a good estimation of nutrient intake of older individuals in an Arabic-speaking Mediterranean country. The analysis showed that the FFQ has a good reproducibility and reasonable validity in relation to the mean of two 24HDR.The results obtained in our repeatability study showed that all pairs of intakes estimated by the 2 FFQ administrations showed no significant difference for most macronutrients and micronutrients. FFQ administrations showed excellent internal consistency. The ICC analysis of the FFQ showed good reliability. For most nutrients, these findings align well with those reported in the meta-analysis on the reproducibility of FFQ by Cui et al., where the median of ICCs for the elderly (> 50 years) was good, with ICC coefficients ranging from 0.482 to 0.866, lower than those for younger age-groups for 22 of 40 nutrients analyzed. . When coIn our validity study, the percentage difference between the two tools showed a mean percentage difference between the FFQ and the reference method of 19.45%, with FFQ overreporting nutrient intake. As often reported, when comparing the FFQ to the mean of 24HDR, FFQ tends to overestimate nutrient intake for almost all nutrients , 42.Agreement between FFQ and 24HDR was measured by first ranking participants’ dietary intake in quartiles, then assessing the percentage agreement between the same or adjacent quartiles and calculating Cohen’s Kappa coefficient and correlation coefficients. We were able to show that the used instruments are highly concordant. For most of nutrients, our FFQ ranked correctly around 80 and 78% of individuals, in the same or adjacent quartile as the mean 24HDR, whether in classifying nutrient intake or nutrient adequacy respectively. Moreover, Kappa coefficients detected moderate to good agreement between nutrient estimation and adequacy classification of participants between FFQ and mean 24HDR. Our results are concordant with previous reports where it was established that 50% of subjects should be correctly classified, and weighted kappa values should be above 0.4 for nutrients of interest , 44.Our findings showed that FFQ overestimated intakes when compared to 24HDR, and correlation improved when adjustment for calories is made. Energy-adjusted nutrient intake estimated by FFQ compared to mean 24HDR, indicated correlation coefficients higher than 0.5 for all macronutrients and most micronutrients. A fair correlation only occurred with vitamins A, and B12. As reported by other studies, vitamin A intake is usually difficult to assess by FFQ, probably because of double counting of items, uneven distribution of vitamin A across food items, seasonal changes in dietary habits, social desirability bias, and overreporting of healthy nutrient-dense foods , 45, 46.The main strength of our study was the consideration of age-related cognitive decline while assessing the validity and reproducibility of the FFQ, performed in a homogeneous older population. Previous reports in elderly population found errors in evaluating total energy intake related to cognitive ability, with an inverse association between reported calories and cognitive functions , 48. In To our knowledge, no previous validated questionnaire was developed to specifically describe and evaluate dietary habits of older individuals in the region.This study has few limitations. To assess intake over 1 year, 4 days recall (1 day per season) are recommended to rule out seasonality bias . Our choSpecial consideration should be given for seasonality that might affect older adults reporting on their food intake. We suggest repeating the FFQ bi-annually to represent more specifically habitual intake. A self-reported version of the FFQ questionnaire could be tested for validation to allow a wider use of the questionnaire among literate elderly participants in the future.In this study we were able to test the validity and the reliability of a culture specific FFQ adapted to the elderly population in the Middle East Mediterranean region as compared to mean of two 24HDR and confirm that the FFQ remains one of the most suitable and inexpensive choices to describe food intake of elderly people of low economic status .Evaluation of food intake in elderly individuals is quiet challenging, and few countries in the Middle East developed and validated questionnaires in people aged 60 years and more. Our questionnaire showed satisfactory reliability and validity and could be suggested for a broader application as a suitable tool to estimate and evaluate dietary habits and nutrient intakes of older individuals living in Lebanon and in the East Mediterranean Arabic speaking countries.Additional file 1.


The results showed that denitrification rates were significantly decreased by 23.83–50.08%, 20.64–57.31% and 6.12–54.61% with salt gradient increasing from 1 to 3‰, 8‰, and 15‰ under 0.05‰, 0.10‰ and 0.15‰ urea addition conditions, respectively. Similarly, denitrification rates were significantly decreased by 44.57–63.24% with an increase of the salt-alkali gradient from 0.5 to 8‰. The abundance of nosZ decreased sharply in the saline condition, while a high salt level significantly decreased the abundance of nirK and nirS. In addition, the increase of nitrogen concentration attenuated the reduction of nirK, nirS and nosZ gene abundance. Partial least squares regression (PLSR) models demonstrated that salinity, dissolved oxygen (DO) in the overlying water, N concentration, and denitrifying gene abundance were key determinants of the denitrification rate in the saline environment, while pH was an additional determinant in the saline-alkali environment. Taken together, our results suggest that salinity and high pH levels decreased the denitrification rates by significantly inhibiting the abundance of the denitrifying genes nirK, nirS, and nosZ, whereas increasing nitrogen concentration could alleviate this effect. Our study provides helpful information on better understanding of reactive N removal and fertilizer application in the coastal areas.Denitrification, as the main nitrogen (N) removal process in farmland drainage ditches in coastal areas, is significantly affected by saline-alkali conditions. To elucidate the effects of saline-alkali conditions on denitrification, incubation experiments with five salt and salt-alkali gradients and three nitrogen addition levels were conducted in a saline-alkali soil followed by determination of denitrification rates and the associated functional genes (i.e.,  Excess N fertilizer application in farmlands leads to substantial Nr releasing into rivers and lakes through runoff and drainage, which results in a series of environmental problems including agricultural non-point source pollution4. The drainage ditch system is a type of wetland in the agricultural ecosystem between the output of farmland field and the receiving water bodies . The migration and transformation of pollutants in these drainage ditches plays a vital role in controlling their final output load5. The urgency of eliminating Nr from farmland drainage ditches and reducing the environmental pollution from exogenous N in rivers and lakes has drawn substantial scientific and public attention. Denitrification which is the microbial reduction of nitrate/nitrite (NO3−/NO2−) to the gaseous products nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2) under hypoxic conditions6, occurs essentially in all water-flooded systems and is considered the major N removal process in ditch systems10.Nitrogen (N) is an essential nutrient for crop growth, thus synthetic N fertilizers are commonly applied in the farmland to achieve high yield. However, yield increases that rely on increased N inputs likely result in a high risk of reactive N (Nr) losses14. However, due to its special physicochemical properties, the N fertilizer use efficiency of crops grown therein is generally very low, even to 5.63%15 and below the national average of 34.3%16 increasing the risk of Nr loss. Meanwhile, the salt and alkali in farmland soils can flow into the farmland drainage ditches with runoff and drainage, resulting in an increase of salt and alkali contents in these ditches. Salinization and alkalization can alter the fundamental physicochemical nature of the soil–water environment, not only in ionic concentrations but also in chemical equilibria and microbiological composition18. The increased ionic concentrations change physicochemical processes and chemical equilibria, facilitate aggregation and sedimentation of suspended solids, and induce the fast displacement of cations  that are bound to the cation adsorption complex in the sediment18. Moreover, the microbial community structure and diversity of denitrifying bacteria in the soils are greatly affected by salinity19. Soil salinity adversely affects the activity of soil enzymes14 and the abundances of N cycling associated genes21. The altered physicochemical and biological properties induced by salinization and alkalization will inevitably influence N transformation processes therein. For instance, salinization can directly affect denitrification rate by increasing or decreasing N availability in the soil23. Some studies have also shown that the denitrifying community structure changes greatly under high salinity conditions and sometimes even loses its function25. Moreover, the high pH value of saline-alkali soil is another crucial factor affecting the composition of the denitrifying bacterial community26, enzyme activity27, and the abundance of associated functional genes6.Mitigating agricultural non-point source N pollution is quite important in agricultural production areas since N fertilizer is easily lost therein. Saline-alkali soil in the coastal region of eastern China has become an important land resource for agriculture with increasing population growth and socioeconomic development28; however, previous studies on denitrification and denitrifying bacteria in saline-alkali soil were mostly restricted to low salinity soils20 and rarely involved alkaline environments27. It has been reported that high saline-alkali environments may induce physiological stress in denitrifying bacteria and can eventually result in large shifts in the composition of denitrifying bacteria and their associated ecosystem functions17. Furthermore, saline-alkali soil with a high salinity and pH often results in large losses of farmland fertilizer into the ditches, increasing the input of exogenous N into the ditches. A comprehensive understanding of how salt and alkali contents regulate denitrification by affecting the composition of denitrifying bacteria is critical to improve the removal of Nr in farmland drainage ditches and to reduce non-point source pollution in coastal areas. However, until recently, relatively little was known about the interaction effects of high saline-alkali conditions and exogenous N on the denitrification rate and the composition of denitrifying bacteria in farmland drainage ditches of coastal areas. To analyze the effect of saline-alkali condition on denitrifying bacteria, we focused on process-specific functional genes, mainly nirK, nirS, and nosZ, which encode pivotal reductases associated with each nitrate reduction pathway21 and are frequently used as functional markers for analyzing denitrifying microbe communities29. In this study, we examined the variation in denitrification rates of three urea levels under five salt and salt-alkali gradients (from low to high) through a flooded incubation experiment and investigated how the abundance of three denitrifying functional genes changed in saline and saline-alkali environments. Concurrently, we determined the controlling factors of the denitrification process using a partial least squares regression (PLSR) model and explored the key determinants of Nr removal in the drainage ditches of farmlands with saline-alkali soils. Our study addressed the question: what are the key determinants of denitrification in ditch systems under saline-alkali conditions and the underlying microbial mechanisms?Saline-alkali soil has a high salinity and pH1:5) of 2.24 mS cm−1, a cation exchange capacity (CEC) of 4.99 cmol·kg-1, and an organic carbon content (OC) of 3.1 g kg−1. Additional details on the general chemistry of this soil can be found in Pan et al.30.The experimental soils were collected from a ditch in Liuzong Village  in the town of Juegang, Rudong Country, Jiangsu Province. This area is a typical coastal reclamation area and is mainly used for agriculture. The sampled soils were transported to the laboratory, salt-leached, air-dried, ground and sieved to 5 mm. The soil had a silt loam texture with a pH of 8.0, a soil electrical conductivity 31 gradients were set, including a control treatment , four salt addition treatments  and four salt-alkali addition treatments . The exogenous N treatments included three analytically pure urea addition levels . Each treatment was replicated three times.The incubation experiment was conducted in the Water-Saving Park of Hohai University  from October 2020 to November 2020 to investigate the effects of different salt and salt-alkali gradients on denitrification rates under three N addition levels. Different salt 3 solutions were gently sprayed onto the sieved soil samples to prevent soil agglomeration, and sprayed and mixed several times to prevent an influence of uneven salt distribution on the results. Soil samples were placed into incubators  after being naturally dried, and each incubator was filled with 8.0 kg soil. Details on procedure of the incubation are provided in previous studies30. The incubation was maintained in a shallow water layer of approximately 5 cm for two weeks to stabilize the soil properties and restore microbial communities. Three urea concentration solutions were subsequently applied to the corresponding incubator. An undisturbed sediment sampler was used to collect the overlying water–sediment samples after 24 h of fertilization.Different concentrations of NaCl and NaHCO32, which quantifies changes in dissolved N2:Ar ratios within the water overlying the sediments. Details on this procedure are provided in previous studies33 and Supplementary Fig. The denitrification (DNF) rates were measured using membrane inlet mass spectrometry  according to the protocol by Kana et al.+) and inorganic nitrogen concentration of the overlying water–sediment system. The NH4+-N and NO3–N of the filtered water and soil extracts were analyzed using a flow injection analyzer . More details on these environmental factor measurements are provided in a previous study30 and the summary of the results showed in Supplementary Table After 24 h of fertilization, before measuring the denitrification rates, overlying water samples (100 mL each) and sediment samples  were collected to measure environmental parameters, including dissolved oxygen (DO) content, EC, pH, sodium ion  samples were extracted from frozen sediment subsamples using an Ultra Clean Soil DNA Isolation kit  according to the manufacturer’s instructions. For PCR amplification of all functional genes, a microfluidics Fluidigm Gene Expression chip was used to quantify all genes simultaneously38. All the above statistical analyses were performed using SPSS 22.0 . All figures were created using Origin 9.0 .One-way analysis of variance (ANOVA) was carried out and examined with Duncan’s test to investigate the statistical significance of the impact of different salt or salt-alkali gradients on denitrification rates and gene abundances under the same urea level. Variations in the data for denitrification rates and gene abundance affected by the interactions of salt or salt-alkali and N were evaluated by two-way ANOVA. The normality of all data was checked and met before the ANOVA was performed. Pearson’s correlation analysis was conducted to determine the relationship between the denitrification rate, environmental parameters and denitrifying gene abundance. Statistical significance was determined at a probability level of 0.05. Redundancy analysis (RDA) was performed with salinity, pH, DO and N concentration as variables to identify the primary environmental factors affecting the abundance of denitrifying genes. A classical regression method was not ideal for this dataset because some variables may be highly correlated, which could lead to reduced statistical power of the classical regression model and even cause a misinterpretation of regression coefficients. A partial least squares regression (PLSR) model which allows for a strong collinearity between the variables was used to examine the effect of the variables on the denitrification rate2 m−2 h−1 and 146.51–241.13 μmol N2 m−2 h−1, respectively  affected the denitrification rates  was performed to further evaluate the relationship among environmental factors and the abundance of denitrifying genes , DO, N concentration  and denitrifying gene abundance (nirK and nosZ) were identified as important predictors with VIP values > 1  were important predictors in the PLSR model, and salinity, DO, pH and N concentration (NO3–N and NH4+-N in the sediment) were the second most important predictors  generated by PLSR analysis reflects the contribution of the independent variables to the denitrification rates; that is, the greater the VIP value is, the greater the explanatory ability of the environmental factors on the denitrification rates Fig. . It is g> 1 Fig. a. The VI> 1 Fig. a. In theors Fig. b. In conors Fig. b. The geely Fig. .Figure 621, especially for removal of Nr from farmland drainage ditches through denitrification process5. Salinity plays a critical role in the denitrification process of drainage ditch ecosystem. Elevated salinity has been reported to alter N concentration21, the substrate for denitrification, and decrease denitrification significantly , in the saline-alkali environment, which hydrolyze to produce hydroxide ions (OH−), causing an increase in system pH. The NO3–N concentration in the overlying water in the saline-alkali environment with high pH was further reduced, which decreasing the availability of denitrification substrate  in saline environments further inhibits nitrification23 and decreases the substrate for denitrification.The identification of salinity as a strong driver of denitrifying gene abundance is consistent with several studies57; values beyond this range may hinder the denitrification process and lead to the accumulation of intermediates: NO2-, NO2 and N2O57. However, the pH of the sediment in the saline-alkali environment exceeding the suitable range of denitrifiers. High pH in the saline-alkali soils further decreased denitrifying gene abundance and denitrification rates  slightly decreased under a low salt gradient but decreased significantly under high salt conditions, and the abundance of the nitrous oxide reductase gene (nosZ) sharply decreased in the saline environment. Compared with the saline environment, the saline-alkali environment decreased the abundance of denitrifying genes more significantly. In addition, the increase in N concentration could limit the reduction in denitrifying gene abundance. The salinity, DO in the overlying water, N concentration and denitrifying gene abundance were key determinants of the denitrification rate. In the saline environment, the salinity and DO were the main limiting factors of the denitrification rate, and in the saline-alkali environment, high pH was also a determinant. Salinity and high pH decreased the denitrification rates by both inhibiting the abundance of denitrifying genes nirK, nirS, and nosZ and reducing the substrate availability for denitrification process. In addition, the decrease of substrate availability would also affect the abundance of denitrifying genes.Based on flooded incubation experiments of farmland ditches under various salt and salt-alkali gradients, we examined the changes in denitrification rates and denitrifying gene abundance in saline and saline-alkali environments. With an increase in the salt gradient from 1‰ to 3‰, 8‰, and 15‰, the denitrification rates significantly decreased by 23.83–50.08%, 20.64–57.31% and 6.12–54.61% under the addition of 0.05‰, 0.10‰ and 0.15‰ urea conditions, respectively. The inhibition of denitrification rates in a saline-alkali environment with a high pH was stronger than that in a saline environment. With the increase in the salt-alkali gradient from 0.5‰ to 1‰, 3‰, and 8‰, the denitrification rates significantly decreased by 44.57–59.44%, 54.88–65.19% and 57.84–63.24% under the addition of 0.05‰, 0.10‰ and 0.15‰ urea conditions, respectively. The abundance of nitrite reductase genes (Supplementary Information.


Otogenic brain abscess is a severe infection that must be treated as early as possible. Rare cases with a high mortality rate can be reduced by recognizing the red flags of a brain abscess, such as headaches, mental status changes, fever, and focal neurological deficits. Those could be supported by modern diagnostic management and adequate antibiotic therapy that was able to penetrate the central nervous system and abscesses. We report a case of a cerebellar abscess of the 49-year-old man with the chief complaint of vertigo. It was accompanied by chronic progressive headache, fever, bidirectional nystagmus, abnormal Romberg test, and abnormal cerebellar signs. Magnetic resonance imaging (MRI) of the head with contrast showed a right cerebellar abscess with an infectious source of otitis media and mastoiditis. The MRI evaluation showed improvement after admistered metronidazole 500 mg every 6 hours (week 22) and cefixime 200 mg every 12 hours (week 13). Long-term antibiotic treatment can be an alternative if surgery cannot be performed. However, surgery is still considered if there is no good clinical response during medical therapy. Central vertigo is divided into vascular and nonvascular causes, the most common being vertebrobasilar ischemic stroke. Among nonvascular reasons, cerebellopontine tumors such as acoustic neuroma are the most frequent and rare cases. In cases where central vertigo is chronic, headache accompanied by an ataxic gait may suggest a cerebellar abscess. Most abscesses that occur in the cerebellum are otogenic brain abscesses. Common symptoms of cerebellar abscess are gait ataxia and abnormal manifestations shown on cerebellar function tests. A cerebellar abscess is a rare case. The incidence of cerebellar abscess is estimated to be 0.3-0.9 per 100,000 population per year in developed countries, with a male-to-female ratio of 2:1 to 3:1 and an age range of 30-40 years. The frequency of cerebellar disturbances and vertigo is often underestimated due to inaccurate examination of the motor and vestibular systems. In acute vertigo, cerebellar lesions account for about two-thirds of central lesions, or about 2.5-6.5% of all critical cases. Morbidity and mortality rates are very high, but with advances in imaging and good antibiotic management, mortality rates can be reduced by 30%-50%. Inappropriate use of antibiotics can alter or hide the disease's characteristics, making diagnosis difficult. Morbidity and mortality rates are very high, but with advances in imaging and good antibiotic management, mortality rates can be reduced by 30%-50%. Inappropriate use of antibiotics can alter or hide the characteristics of the disease which can make diagnosis difficult Therefore, it is essential to fully understand the early symptoms and clinical manifestations and make proper diagnoses and treatments. Appropriate antibiotics and surgical intervention are crucial in treating otogenic brain abscesses. However, the extent of the surgical procedure and the optimal timing for this intervention is still a matter of debate. We report a patient with an otogenic brain abscess that developed central vertigo while treated with appropriate systemic antibiotic management.A man, 49 years old, came to the Emergency Unit (ER) RSUD Dr. Soetomo Surabaya with complaints of headaches for 5 months. The pain comes and goes and gets worse. A throbbing headache felt all over the head. For the past 4 months, the patient felt unbalanced walking, spinning, and never getting better. Complaints were accompanied by fever and vomiting. In the past year, the patient complained of ear pain accompanied by decreased hearing and a thick discharge from the right ear that he felt, but the patient did not go to the doctor. Physical examination revealed normal and conscious vital signs, with focal neurological deficits in the form of bidirectional nystagmus, positive open and closed Romberg test to the right, right past pointing dysmetria test, and right dysdiadokinesia. Complete blood count showed leukocytosis 15.980/uL, neutrophils 83.4%, C-reactive protein (CRP) 2.7 mL/dL, erythrocyte sedimentation rate (ESR) 30 mm/hour. The patient underwent a blood culture, but no bacterial culture was found. MRI examination showed multiple lesions, intra-axial, infratentorial, well-defined, regular edges, measuring about ±2.2 × 2 × 2 cm on the right cerebellum with perifocal edema, which was hypointense on T1-weighted, hyperintense T2-weighted on diffusion-weighted imaging (DWI). Contrast administration showed rim enhancement. The lesion was seen pressing the right posterior aspect of the pons and the fourth ventricle medially, accompanied by leptomeningeal enhancement and right mastoiditis a. MR speThe patient was referred to the otolaryngology department and the dental and oral departments to find the source of the infection. The results of the examination revealed bilateral chronic suppurative otitis media. Then the patient underwent an audiometric examination and found that the sensorineural hearing loss was very severe, with pure tone audiometry (PTA) >120 dB. Dental and oral analysis revealed chronic apical periodontitis. The patient was given antibiotic therapy, namely ceftriaxone 2 g every 12 hours intravenously (IV) for 4 weeks and metronidazole 500 mg every 6 hours intravenously (IV) for 4 weeks. The patient had an MRI of the head with contrast 5 weeks after antibiotics administration. MRI of the head with contrast evaluation showed improvement. The size of the lesion appeared to be smaller, with a size of ±1.8 × 1.7 × 2.2 cm in the right cerebellar hemisphere b.The patient went to the outpatient clinic for clinical and radiological evaluation. Patient underwent an MRI of the head with contrast evaluation after administering metronidazole 500 mg every 6 hours (week 22) and cefixime 200 mg every 12 hours (week 13). MRI showed no restricted diffusion area lesions on DWI. The lesion showing contrast enhancement appeared to be shrinking with a size of ±0.6 × 0.6 × 0.9 cm in the right cerebellar hemisphere c. Then tOtogenic brain abscess is an accumulation of pus in the cerebrum or cerebellum that develops into encephalitis. Otogenic brain abscess is caused by pyogenic microorganisms originating from the inflammatory process in the middle ear cavity. Acute and chronic otitis media both cause autogenic brain abscesses. Possible pathways of propagation of brain abscess consist of direct extension pathway and hematogenous pathway. Among these, the most common cause is a direct extension of infection through the osteitic bone. Autogenic brain abscess usually presents as a solitary abscess rather than in multiple forms. The infection then develops in the temporal lobe 2-4 times more often than in the cerebellum. But there are many different reports about the location of the abscess. After the development of antibiotics, the incidence of autogenic brain abscesses decreased statistically. Although rare, the mortality rate is high enough to consider the possibility of intracranial complications in the treatment of otitis media. Symptoms of an otogenic brain abscess are generally mild in the early stages. Nonspecific symptoms such as headache, nausea, vomiting, and fever may develop in the early stages of a brain abscess. Danger signs/red flags that need attention in cerebellar abscess cases are headache, mental status changes, fever, and focal neurological deficits. Headache is the most common symptom of otogenic brain abscess in about 70%. Alterations in mental status (65%) and seizures (25%-35%) may occur due to meningeal irritation and increased intracranial pressure. In rare cases, lateralization may occur on motor examination. Nausea and vomiting occur in about 40%. Other neurological deficits are the stiff neck (40%) and papilledema (25%). Vertigo symptoms will be more dominant in the vestibulocerebellum structures . In addition, gaze-evoked nystagmus and saccadic movements are often found on physical examination, symptoms of head-shaking nystagmus appear (2%-15%), and central-positional nystagmus (12%-28%). The diagnosis of a cerebellar abscess in this patient was made based on anamnesis with complaints of chronic headache, fever, history of ear infection and focal neurological findings in the form of bidirectional nystagmus, positive open and closed Romberg test for right eye, right past pointing dysmetria test, right rebound phenomenon and right dysdiadokinesia Investigations in patients with brain abscesses can provide a general picture of infection, such as erythrocyte sedimentation rate, blood leukocytosis or elevated CRP. However, the infection blood parameters were in the normal range at 30%-40% Arlotti et al. stated that surgery should still be considered if antibiotic therapy fails after 1-2 weeks Before mass expansion occurs, early antibiotic therapy can prevent progression from cerebritis to abscess. Patients with symptoms for less than one week respond better to medical treatment than those who persist for more than one week. In certain circumstances, brain abscesses can be treated without surgery. Small abscesses (<2.5 cm) and cerebritis may respond to antibiotics alone. Patients treated with medical therapy alone improved clinically before the CT scan showed improvement. CT scan and MRI will show a decrease in lesion size and edema and a reduction in the number of lesions. Improvements on CT scans generally show complete resolution in 1-11 months, although radiological abnormalities may persist for months after successful therapy The early stages of cerebritis can be managed in a shorter time, about 4-6 weeks. However, patients with encapsulated abscesses, tissue necrosis, uncontrolled abscess growth, multiple abscesses, lesions at vital sites, and immunocompromise require 6-8 weeks. It takes a long time for brain tissue to repair and close the abscess space. Initial antibiotics are administered intravenously, followed by 2-6 months of oral therapy. Penetration of antibiotics is poor across the blood-brain barrier, so the choice of antibiotics is limited and maximum doses are often required Empirical antibiotic therapy should be based on the most likely underlying etiologic agent, source of primary infection and pathogenesis. Parenteral antibiotics are active against pathogens, penetrate the abscess fluid and the site of the underlying disease in adequate concentrations and are bactericidal. The combination of penicillin or a third-generation cephalosporin (cefotaxime or ceftriaxone) plus metronidazole is effective empiric therapy in most cases Abscess evacuation surgery without proper antibiotic coverage is less meaningful in handling. Brain abscesses are generally treated with antimicrobial therapy for a minimum of 4-8 weeks but longer for abscesses treated with medical therapy without surgical excision Long-term antibiotic treatment can be an alternative if surgery cannot be performed. It should be accompanied by close monitoring of the side effects of antibiotics. Initial empiric antibiotic therapy should be based on the expected etiologic agent, possible predisposing conditions, source of primary infection and suspected pathogenesis. The antibiotic selected must be able to penetrate the abscess fluid and the site of primary infection in adequate concentrations and be bactericidal. Surgery is still considered if there is no good clinical response during medical therapy.


Background. Enamel microabrasion can eliminate enamel irregularities and discoloration. This study evaluated the staining susceptibility of enamel after microabrasion using different clinical protocols.Methods. A total of 210 extracted bovine incisors were used in this study. The teeth were randomly divided into five groups of 42 teeth each (n=42), including group 1: control (no treatment), group 2: microabrasion, group 3: microabrasion + fluoride polishing, group 4: macroabrasion (fine-grit diamond bur) + microabrasion, and group 5: macroabrasion (finegrit diamond bur) + microabrasion + fluoride polishing. The groups were then randomly divided into two subgroups for discoloration procedures: coffee and distilled water (n=21). A spectrophotometric evaluation was carried out at baseline and on the 1st, 7th, 14th, and 28th days of the staining period. Statistical analyses were performed using repeated-measures ANOVA and the post hoc Bonferroni test at a significance level of 0.05.Results. The greatest color change was observed in group 2 specimens, which were immersed in coffee solutions. The color change values for groups 3, 4, and 5 specimens, which were immersed in distilled water, were higher than those in group 1 specimens (P<0.05). The fluoride polishing + enamel microabrasion treatment groups (groups 3 and 5) exhibited greater resistance to color changes than the matched non-fluoride-polished groups (groups 2 and 4) (P<0.05).Conclusions. The teeth that underwent enamel microabrasion treatment and were polished with fluoride gel became more resistant to color changes. Our findings confirm that enamel microabrasion treatment is a conservative method for localized discoloration. According to Croll et al, an ideal microabrasion system has a low acid content and includes abrasive particles in a water-soluble mixture applied with a slowly rotating handpiece to minimize the spread of the compounds and ensure that the procedure is safe. Furthermore, to shorten the application time, Croll et al also recommended using an extra-fine diamond bur before applying microabrasion agents.5 Microabrasion is a treatment option for color differences and surface texture inconsistencies on teeth, especially in cases of superficial tooth stains, enamel decalcification, and minor defects.6 Acid erosion and the polishing impact of abrasive particles eliminate surface discoloration and produce a smoother surface by removing micropores, yielding a prism-free outer enamel surface following microabrasion treatment. This increases the esthetics of the enamel by brightening it, making it more reflective.7 Many studies have found enamel microabrasion to be an effective and conservative treatment.10 According to Sundfeld et al,6 5‒10 applications of a microabrasion system can result in enamel erosion, which is tolerable under clinical conditions. According to a recent study,11 120 seconds of microabrasion therapy reduces enamel thickness by approximately 10%, indicating that it is a safe and conservative approach. Depending on the severity of the enamel staining, the number of applications or techniques of application may vary. The degree of enamel staining also determines the quantity and technique of applying the microabrasion product. To minimize clinical hours, enamel macro-reduction can be performed by first using a tapered fine-diamond bur, followed by polishing the micro-abraded surface using felt disks and polishing paste or fluoride paste. Enamel microabrasion can also be used in combination with bleaching treatments.4 Contemporary microabrasion treatment is based on Croll’s concepts, a procedure involving the removal of a layer of enamel tissue. In previous studies, various microabrasion products have been used, and the depth abraded has been reported to vary from 20 to 200 µm, depending on the acid content and the application time.7 Individuals with tooth enamel staining and discoloration typically visit dental clinics. The etiology of superficial enamel discoloration, which frequently results in esthetic concerns, tends to vary. It can be caused by stains from extrinsic sources, including coffee, tea, and cigarettes, or it may have an intrinsic etiology, e.g., amelogenesis imperfecta or tetracycline staining. Color abnormalities are frequently restored using direct or indirect restorative materials, which yield satisfactory esthetic outcomes. However, these restorations require long-term repair and recurring replacements, causing the tooth to enter the restorative cycle.12 including the studies mentioned earlier, focused on enamel loss and surface roughness attributed to the microabrasion technique. This technique increases the roughness of the enamel surface and is also related to reduced enamel microhardness. However, both effects can be reversed by the polishing procedure or saliva exposure.4Despite the benefits and resources available for this procedure, there is still a lack of knowledge about the outcomes of this strategy. Furthermore, there is little evidence that these treated surfaces will resist extrinsic discoloration if they are later exposed to extrinsic discoloring agents. Previous research, Therefore, this in vitro study evaluated the staining susceptibility of enamel after different microabrasion application protocols after 28 days of immersion in distilled water and coffee. The null hypothesis tested was that microabrasion application protocols did not affect the staining susceptibility of enamel. In this study, 210 sound bovine incisors without cracks or erosion were used as specimens that were cleaned and disinfected using 0.1% thymol. The required minimum sample size was calculated using G*Power 3.1 statistical software . An alpha-type error of 0.05, an effect size of 0.25, and a beta power of 0.80 were specified, and the minimal estimated total sample size was calculated at n = 200. The roots of the incisors were removed, and the crowns were cut into 5-mm blocks with a diamond disk on a low-speed cutting machine . With their labial surfaces exposed, the specimens were embedded in transparent autopolymerizing acrylic resin . Silicon carbide paper was not applied to the labial surfaces of the enamel. The specimens were randomly divided into five groups, and each group was randomly assigned to a microabrasion technique.Group 1: Control: No treatment was applied to the enamel surface.Group 2: Microabrasion: A water-soluble gel  containing mildly concentrated hydrochloric acid (6%) and a fine-grit silicon carbide abrasive paper was applied to the enamel surface using a rubber cup specially designed for a 500-rpm handpiece. The abrasive compound was applied in three applications9 of one minute each, with irrigation between each application.Group 3: Microabrasion + fluoride polishing: The enamel microabrasion procedure was similar to group 2. After the abrasion compound treatment, a 2% neutral-pH sodium fluoride gel  was applied to the enamel surfaces for four minutes.Group 4:Macroabrasion (fine-grit diamond bur) + microabrasion: Before administering the enamel microabrasion therapy, the superficial layer of the enamel surface was removed for 5–10 seconds with a high-speed fine-grit water-cooled tapered diamond bur . The enamel microabrasion procedure was similar to group 2.Group 5: Macroabrasion (fine-grit diamond bur) + microabrasion + fluoride polishing: The enamel microabrasion procedure was similar to group 4. After the abrasion compound treatment, a 2% neutral-pH sodium fluoride gel  was applied to the enamel surfaces for four minutes. All the specimens were kept in distilled water for 24 hours. Following the application of the various enamel microabrasion procedures, the specimens in the five groups were randomly divided into two subgroups (n = 21) to undergo staining procedures.Coffee was used as the staining solution, and distilled water was used on the control subgroup. The coffee was made with two grams of Nescafe powder  and 200 mL of hot water. The solutions were refreshed every day.13 The color change in the specimens was formulated with the ΔE parameter calculated using the values L, a, and b14: A spectrophotometer was used to determine the baseline color readings of all the specimens after the enamel microabrasion procedure . To remove background influences, each measurement was taken against a standard white background, and all the measurements were made at the same time of the day. While taking measurements, the instrument’s fiber-optic tip was set perpendicular to the specimens and parallel to the ground, and calibration was performed before each measurement. For each specimen, three measurements were taken. After determining the baseline color values, half of each experimental group’s specimens were immersed in distilled water and the other half in a coffee solution. Color measurements were taken on the 1st, 7th, 14th, and 28th days.2 + (Δa0 − Δa1)2 + (Δb0 − Δb1)2]1/2 ΔE = [(ΔL0 − ΔL1) All the restorative procedures and color measurements were performed by an operator on the research team. The acceptable limit of ΔE was established at 3.5 in this investigation.P < 0.05. The data were analyzed using SPSS 22.0 . The mean and standard deviation were used in evaluating the color measurements using the descriptive statistical method. The data distribution was controlled with the Shapiro–Wilk test, and a normal distribution was observed. The repeated-measures analysis of variance (ANOVA) test was used to determine the differences between intragroup repeated measurements. Differences between more than two groups were analyzed using the post hoc Bonferroni test at a significance level of  The greatest color change was observed in group 2 specimens  (ΔE = 31.9 ± 0.8) immersed in a coffee solution. The group least affected by the discoloration treatment was group 1, i.e., the control group (ΔE = 3.4 ± 0.7), with specimens immersed in distilled water .P = 0.000;  The color change values recorded for group 1 specimens (control), immersed in coffee, were higher than the values recorded in group 1 specimens (control), immersed in distilled water after 14 days and 28 days  and group 4 specimens (diamond bur + enamel microabrasion treatment without fluoride polishing), immersed in coffee, were higher than the values recorded for group 2 specimens (microabrasion without fluoride polishing) and group 4 specimens (diamond bur + enamel microabrasion treatment without fluoride polishing), which were immersed in distilled water for all periods  and group 5 specimens (diamond bur + enamel microabrasion treatment with fluoride polishing), immersed in coffee, were higher than the values recorded for group 3 specimens (enamel microabrasion treatment with fluoride polishing) and group 5 specimens (diamond bur + enamel microabrasion treatment with fluoride polishing), immersed in distilled water, after 7, 14, and 28 days , immersed in coffee, were higher than the values recorded for groups 3 and 5 (microabrasion with fluoride polishing), immersed in coffee, after 7, 14, and 28 days  . Figure  The present study evaluated different clinical application protocols of microabrasion (enamel microabrasion/with or without fluoride polishing and macroabrasion + microabrasion/with or without fluoride polishing) and the subsequent staining susceptibility of the treated enamel in distilled water and coffee. Based on the results, microabrasion and macroabrasion + microabrasion with fluoride polishing resulted in better staining outcomes than the microabrasion treatment without fluoride polishing. Therefore, the null hypothesis was rejected.16 For homogeneous specimen allocation, bovine enamel was chosen as the test substrate in this study. Although using human enamel in dental material testing is preferable, it has been established that bovine enamel could be safely used as a substitute for human enamel, especially since a large crown size is required for preparing samples with similar crown sizes.abrasion effect.8 Loguercio et al17 examined two commercially available microabrasion treatments to eliminate fluorosis stains and reported that Opalustre was more effective than Prema. They attributed this finding to the potentially large size of the silica granules in Opalustre. Therefore, Opalustre was used in our study because it was the most readily available and most widely used microabrasion product. In addition, it contains less HCl and has larger silica granules than similar dental materials. Microabrasion involves removing tooth surface discoloration by applying a material containing acid and abrasive particles to the enamel surface. This procedure causes minimal enamel loss and yields an enamel surface with significant homogeneity, smoothness, and gloss over time—clinically known as the 11 Based on years of clinical use, in some severe cases, it has also been established that macroabrasion with a tapered, fine-diamond bur as the first step reduces the clinical hours required for the procedure.8 In this study, different clinical application protocols for enamel microabrasion treatment, including the macroabrasion step, were investigated, and the technical factors were standardized, including the number of applications, intervals, and pressure applied. Many factors affect the enamel surface after microabrasion treatment, including manual or mechanical processes used, the interval between applications, handpiece speed, the pressure applied, the type/concentration of acid, and abrasive particle type.16 In this study, a spectrophotometer (VITA Easyshade) was used for standard color change quantification. In terms of sensitivity, reliability, reproducibility, and accuracy, the VITA Easyshade spectrophotometer has been validated in numerous studies.19 The CIEL*a*b* color system is used in the VITA Easyshade and is especially useful because it allows the user to specify how the color parameters change in the system. Changes in the color components  were used to assess the resistance of the enamel to discoloration after undergoing microabrasion treatments.9 Furthermore, in this study, different protocols of microabrasion followed by fluoride polishing (groups 3 and 5) exhibited greater resistance to staining than the unpolished groups (groups 2 and 4). After microabrasion treatment, polishing with fluoride reduced surface roughness and increased the resistance of the enamel surface to discolorations, affecting esthetic properties and clinical longevity. According to Fragoso et al,20 microabrasion followed by polishing with diamond paste or prophylactic fluoride paste increased hardness and enhanced surface smoothness of the enamel. In this study, the specimens undergoing enamel microabrasion treatment without fluoride polishing (group 2) showed the most color changes by the 28th day after being immersed in coffee. Furthermore, specimens that underwent enamel microabrasion treatment without fluoride polishing, immersed in distilled water (group 2), exhibited greater color changes than the control group (group 1). This finding could be explained since the remaining enamel surface was slightly thinner and translucent, and the dentin seemed more evident following treatment on micro-abraded teeth; in addition, they might have a darker shade or yellowish coloration.21 The acceptable limit of ΔE was established at 3.5 in this investigation. Color change values (ΔE) > 3.5 units could be recognized with the naked eye in all stages of the assessment across all the experimental groups. In this study, the color change values (ΔE) in all the groups, including the control group, were above the acceptable threshold of 3.5. These findings can be explained because the color values of the teeth were high before the microabrasion treatment, and the teeth undergoing microabrasion exhibited reduced lightness.9 A visible color change of 3.5 units is the standard in most clinical trials.3 investigated the color changes of human enamel surfaces after using two microabrasion compounds  with different treatment periods of 10, 20, 30, and 40 seconds, respectively. According to Paic et al, there were no significant changes in the color measurements between the pastes during the treatment period. Jahanbin et al22 investigated the color change values of an artificially white spot lesion treated with two microabrasion techniques with five minutes of daily immersion in a tea-coffee solution for one week and found color change values (ΔE) above the acceptable threshold of 3.5 after enamel microabrasion treatment. Baĝlar et al23 evaluated the color change values for prototype microabrasion paste and Opalustre on fluorosis-stained enamel. After enamel microabrasion treatment, the color change values (ΔE) were higher than the acceptable threshold of 3.5. These findings are consistent with the results of the present study. In the dental literature, there was no study on color changes after enamel microabrasion treatment. Paic et al13 For individuals who drink three glasses of coffee per day, on average, the 28-day holding duration used in this study is approximately equivalent to over two years of coffee consumption in real life.13 As a staining solution, coffee was chosen for the present study due to its routine use in daily life and its potential to produce distinct color changes in esthetic restorative materials. Several studies have established that the average time spent drinking a cup of coffee is 15 minutes in a single day. One important limitation of the present study was that the specimens used were intact enamel surfaces without any superficial defect and discoloration . However, it is difficult to determine whether the enamel removal via the microabrasion techniques performed would be sufficient to eliminate any enamel anomaly. Furthermore, the reduction in enamel thickness and surface roughness following the microabrasion procedures was not measured in this study. These parameters are useful for investigating the correlation with color changes. Another important limitation of the study was that the specimens were stored in distilled water instead of artificial saliva after the enamel microabrasion treatment. Enamel remineralization in artificial saliva would reduce any potential bias in the methodology. Another limitation of the study was that only two staining solutions were used, which is one of several factors that could influence the intensity of enamel staining. Finally, additional in vitro and in vivo research to address these shortcomings may be needed to corroborate the conclusions of this study. Within the limitations of this study, the following conclusions can be drawn:The greatest color change was observed in specimens that underwent enamel microabrasion treatment without fluoride polishing and were then immersed in a coffee solution. Specimens that underwent enamel microabrasion treatment in combination with polishing with fluoride became more resistant to color changes. In comparison to direct and indirect restorations, enamel microabrasion therapy appears to be a conservative approach, despite the limitations of this in vitro study.  BHS, HM, and ÇC contributed to the study concept and design. BHS, HM, and ÇC contributed to material preparation and data collection and analysis. All the authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript. None. Based on Kirikkale university animal experiments local ethics committee directive, we did not treat or extract teeth on any animals for this study. We only received samples to be thrown away at the slaughterhouse. The authors do not have any financial interest in the companies whose materials are included in this article.


Drug treatment centres provide the highest level of rehab services for patients diagnosed with drug addictions. Most inpatient drug rehab programs focus on medical detox and mental health interventions. However, how to optimize the later remains a challenge. The aim of this study was to examine the psychophysiological and psychosocial profile of patients attending drug addiction centres in comparation with the general population. A total of 105 inpatient drug rehab patients and 50 participants from the general population were compared based on standardized psychophysiological and psychosocial measures. Results of this study suggest that patients attending drug addiction centers differ from general population in several different psychophysiological and psychosocial factors. Patients reported significantly lower levels of physical activity and increased sympathetic responsiveness, and significantly higher levels in loneliness, psychologically inflexibility and neuroticism. The results of this study highlight the importance of address healthy lifestyle behaviors such as sport practice and psychological variables such as loneliness, psychological (in)flexibility and neuroticism to improve current programs aim to prevent or reduce problematic drug consumptions. Drug abuse is a disruptive behavior, being the principal approximation to this public health problem the as a disease or a learned, maladaptive behavioral pattern  under drug addiction treatment in an addiction treatment center and a non-clinical control group of 50 participants  were analyzed using a number of sociodemographic, psychophysiological and psychosocial measures. The study was conducted according to the principles expressed in the Declaration of Helsinki and approved by the direction of the addiction treatment center. All the participants signed a written informed consent prior to start the study and they were free to leave the study at any moment.A descriptive cross-sectional study was conducted. We analyzed the autonomic modulation of participants by the Heart Rate (HR) and Heart Rate Variability (HRV). For this aim, participants were in a quiet and temperature-controlled room , sited in a chair in a comfortable position and without speaking. The participants were 15 min in this position, but only the 10 last minutes were used for analysis. The HR and HRV was recorded with the Polar V800 HR monitor . with a sampling frequency of 1000 Hz, that allow to monitor the RR intervals  for the analysis of the HRV and the number of beats per minute for the HR analysis. Subsequently, HRV parameters analyzed using the Kubios HRV software program v.3.0 . Three HRV domains were included as indicators of autonomic stress reactivity:Time-Domain  Analysis. This analysis was based on the assessment of the intervals between normal beats on ECG recordings. During the statistical analysis, generally all the QRS complexes, the duration between consecutive QRS complexes , or the instantaneous heart rates during continuous ECG recordings are determined. We recorded the following time-domain indices: pNN50 which is the percentage of successive normal sinus RR intervals exceeding 50 ms (%) and RMSSD (ms): Is the square root of the mean value of the sum of squared differences of all successive R-R intervals.Frequency-Domain  Analysis. Frequency-domain measures give information about how the power is distributed as a function of frequency. This analysis give us smoother spectral components that can be distinguished as independent from preselected frequency bands and easy post-processing of the spectrum with an automatic calculation of low (LF)- and high-frequency (HF) power components and an easy identification of the central frequency of each component, and accurate estimation even on a small number of samples .HF and LF (n.u) were measured in order to measure the peaks of parasympathetic, high-frequency component, frequency range: 0.15–0.40 Hz (HF) and sympathetic low-frequency component frequency range: 0.04–0.15 Hz (LF) values. In addition, LF/HF ratio was evaluated.Nonlinear domain. SD1 and SD2 were measured to reflect the fluctuations of the HRV throw a Poincaré chart, physiologically, the transverse axis. SD1 reflects parasympathetic activity while SD2 reflect the long-term changes of RR intervals and is considered as an inverse indicator of sympathetic activity. In addition, the Approximate Entropy (ApEn) and Sample Entropy (SampEn) of the Heart Rate Variability were measured.After the HR and HRV analysis participants went to a different room to fill a battery of psychological questionnaires with the presence of a professional to solve any questions they may have. The questionnaires were as follows.Avoidance and Action Questionnaire (AAQ-7) , weekly physical training (h), movement per day (min), sleeping time (h) and the relationship with their parents .Sociodemographic data. We asked for the participants age (years), body mass index . The descriptive analysis of the sample included the means and standard deviations (M + SD) for the quantitative variables, while frequencies and percentages were used for the nominal variables. Normality and homoscedasticity of variance were examined using Shapiro–Wilk and Levene´s test respectively. Students´ t tests were conducted to explore differences in psychosocial  and physiological variables (stress reactivity) between patient attending addiction centers vs control group.The group of patients attending addiction centers reported their first consumption at the age of Md = 16 years old (IQR = 4.75) and have been involved in the program for the last 8.33 years under rehabilitation program (IQR = 1.19). A total of 67,47% were unemployed. 44,76% had basic education, 53.3% secondary education. A total of 86.67 were smokers and 71% were policonsumers. The first drug of abuse was the following: cocaine (27.62%), heroin (20%), cannabis (9.52%), alcohol , hashish , tobacco (1.90%), opioids  and amphetamines . A total of 38.09% reported doing exercise regularly and 78.09% having a healthy diet. The control group was characterized by workers (100%) with university academic training (100%) and no history of drug consumption, although a 6% reported that smoked cigarettes. A total of 62.00% reported doing exercise regularly and 96% having a healthy diet.The results are reported as mean ± SD. Only differences between control and patients’ group were seen regarding physical activity behaviors. Control group presented greater significant hours of weekly physical training and minutes of movement per day than patients’ group Table .The group of patients attending addiction centers reported all greater significant values of Extraversion (BFI- E); Agreeableness (BFI-A); Neuroticism (BFI-N); Psychological inflexibility (AAQ II) and Loneliness (UCLA), than control group. Factors that make drug use prone, characterizing the psychological profile of patients Table .According to the HRV analysis, patient group present significant lower values of ApEN, SampEn and Pnn50, while higher HRmin than control group Table .The diagram of the 5 BFI personality traits suggest patient group has a higher score of extraversion, agreeableness, and neuroticism while similar openness to experience and conscientiousness than patients’ group, which is in accordance with the results of the Table This study provides a complete psychophysiological and psychosocial profile of patients undergoing treatments in an addiction center. To our knowledge, this is one of the first studies to approach from a holistic perspective the complete profile of the patients. The initial hypothesis was compiled since differences were found in the autonomous sympathetic modulation, and psychological and psychosocial profile between drug addiction patients and control group.Patients attending drug addiction centers presented a significantly less physical activity levels and formal education background than controls, and significantly higher scores in extraversion, agreeableness, and neuroticism. Higher scores in neuroticism are particularly important, because suggest a tendency for stress reactivity, in line with previous literature Lahey, . PatientPhysical activity levels were higher in control group, indicated by the significant higher values of weekly physical training hours and minutes of movement. This result is consequent with previous studies comparing physical activity levels of inmate’s acute drug users, and chronic drug users , which is in line with the significantly higher levels of physical activity, either daily minutes and hours of weekly training, denoting a higher cardiovascular capacity , increase perceived social support and formal education of patients. Overall, these results are consistent with the importance of moving beyond classical health care treatment towards interventions based on social determinants of health flexibility and neuroticism to improve current programs aim to prevent or reduce problematic drug consumptions.


The muscle fiber ultrastructure in Idiopathic Inflammatory Myopathies (IIM) has been scarcely explored, especially in Inclusion Body Myositis. The aim of this study was to implement the Scanning Electron Microscopy (SEM) in a small cohort of IIM patients, together with the characterization of immunological profile for a better understanding of the pathophysiology. For immunological profile characterization, we identified the presence of autoantibodies  and quantified cytokines  and chemokines . The histological analysis was made by hematoxylin–eosin staining while the muscle fiber ultrastructure was characterized by SEM. We observed changes in the morphology and structure of the muscle fiber according to muscle strength and muscle enzymes. We were able to find and describe muscle fiber ultrastructure with marked irregularities, porosities, disruption in the linearity and integrity of the fascicle, more evident in patients with increased serum levels of muscle enzymes and diminished muscle strength. Despite the scarce reports about the use of SEM as a tool in all clinical phenotypes of IIM, our work provides an excellent opportunity to discuss and reframe the clinical usefulness of SEM in the diagnostic approach of IIM. The diagnostic tools used for IIM include electromyography, magnetic resonance imaging, serum levels of muscle enzymes, presence of autoantibodies and muscle histological characterization; however, they may not be specific enough for some IIM phenotypes according to classification criteria6. The Scanning Electron Microscopy (SEM) has not been fully explored, since the lack of availability in clinical centers and the expertise needed for its interpretation represent major limitations.There are still many fields to explore deeply in IIM research, for example, the immunological profile of IIM patients, including the clinical relevance of the myositis specific antibodies (MSA) beyond cancer risk development or anti-synthetase syndrome (ASS) and myositis associated antibodies (MAA), as well as the participation of cytokines and chemokines. Notwithstanding, the histological analysis of the muscle is commonly performed by hematoxylin–eosin (HE) staining, however, the information about the description of muscle fiber ultrastructure is scarce and limited to IBM. There are only a few descriptive reports regarding muscle fiber in other pathologies or conditions7; likewise, a few reports have included muscle analysis of DM and PM patients8. However, neither of these studies described rigorously the muscle fiber ultrastructure of IIM patients using SEM, therefore our study represents the first report in this context.Some reports have implemented transmission electron microscopy (TEM), which is useful particularly for IBM patients due to their classic inclusion bodies structuresThe use of SEM will allow us, with the highest resolution, to appropriately characterize the muscle fiber ultrastructure, and along with cytokines, chemokines and the presence of autoantibodies, might provide us a better understanding of the pathological features of IIM. Therefore, our study aim was to characterize the muscle fiber ultrastructure and to identify the possible association with the immunological profile and clinical features of the IIM clinical phenotypes.Our study group was conformed of eight women (66.7%) and four men (33.3%) classified with IIM according to EULAR/ACR 2017 criteria. Regarding extra muscle manifestations, we found that arthralgias and interstitial lung disease (ILD) were the most prevalent. Other demographical, clinical, and serological features are shown on Table s = − 0.660, P = 0.020), ALT  and AST , meaning that patients with higher levels of muscle enzymes had lower muscle strength  and MMT8 score 0–150). We found an inverse correlation between the MMT8 score and CPK   and IFN-γ (12.1 ± 10.80 vs. 1.7 ± 2.23 pg/mL P = 0.048)  to compare the serum levels of these molecules in IIM patients. We found higher serum levels of IL-6 in IIM patients  .Since we observed marked alterations in muscle fiber ultrastructure in patients with higher muscle enzymes and lower muscle strength, we analyzed if there are any associations between these findings with the immunological profile. We initially looked for a possible correlation between muscle enzymes or MMT8 score with serum levels of cytokines or chemokines. We did not find any correlation regarding cytokines or chemokines with CPK, AST, ALT nor MMT8 score, however, we observed a correlation between serum levels of IFN-γ and LDH (r27) Fig.  panel b P = 0.018)  in seropositive patients . On the other hand, we did not find difference in the frequency distribution of MSA/MAA seropositivity according to normal or elevated levels of muscle enzymes.Regarding the muscle enzyme serum levels according to MSA/MAA presence, we found higher serum levels of LDH , perifascicular atrophy (41.7%), endomysial inflammatory infiltrate (33.3%) and the presence of basophilic fibers (25%).P = 0.015). Regarding patients with endomysial inflammatory infiltrate, we found higher levels of IL-18  and lower score of MMT8 . Finally, we observed higher serum levels of CPK , AST  and ALT  as well as lower score of MMT8  in patients with basophilic fibers.We observed that patients with perivascular inflammatory infiltrate had higher erythrocyte sedimentation rate (ESR) , however, since this feature is present in almost all of our patients, it is considered characteristic of muscle fiber ultrastructure independently of the clinical phenotype.P = 0.005) as well as lower levels of CXCL8 .Other findings included altered muscle morphology (60%), non-linear muscle fibers (60%), cellular infiltrate (50%), and myofiber surface porosities (30%). In this regard, in patients with altered muscle morphology, we observed non detectable levels of IL-1β , as well as a tendency of diminished serum levels of IL-23 in mycophenolate mofetil-treated patients ; moreover, although no significative, serum levels of IL-1β and IL-17A were not detectable in patients under mycophenolate treatment. We did not observe differences concerning hydroxychloroquine. Regarding MSA/MAA presence, we did not observe a difference in the frequency distribution of autoantibody seropositivity according to immunosuppressive treatment.The patients included in this study are under conventional synthetic disease-modifying drugs including methotrexate, prednisone, hydroxychloroquine, mycophenolate, and azathioprine. We looked for differences in cytokine and chemokine serum levels as well as the frequency of seropositivity. We could not compare methotrexate because almost all our patients were under this treatment  and overlap myositis (OM) by SEM. Since the unexplored clinical use of SEM in these patients and the lack of classification criteria including SEM findings, we decided to categorize and describe the muscle fiber ultrastructure according to muscle damage and muscle weakness markers represented by muscle enzymes and MMT8 score, respectively. To complement the obtained information on muscle fiber ultrastructure, we also characterized the immunological profile of IIM patients aiming to get a better understanding of their pathophysiology.9, meanwhile the MMT8 score is a validated tool for muscle weakness evaluation in myositis patients10, therefore both parameters are considered adequate markers for muscle damage and muscle weakness.Serum levels of muscle enzymes are considered a biomarker for myositis classification considered in EULAR/ACR 2017 criteria. Is important to remind that serum levels of muscle enzymes might reflect the muscle damage or the lack of physical training in some IIM patientsAccording to muscle fiber ultrastructure description, we presented the SEM images in decreasing order of the MMT8 score Fig. . Only twAn important aspect to consider regarding the muscle fiber description of our patients is the fact of the observed discordances between optical and electronic microscopy; according to HE staining, we did not observe any finding suggestive of muscle pathology in three of our patients, meanwhile, we found alterations in the ultrastructure of muscle fiber of the same patients. This situation denotes the usefulness of SEM as a tool with higher resolution to get a better opportunity to observe histological changes and a better comprehension of the muscle tissue alterations of IIM patients.11. In our study, the clinical phenotype was not concordant in 30% with muscle biopsy results; this is one of the reasons why the decision of muscle biopsy is debatable nowadays, due to the lack of expertise in its interpretation. These findings highlight the importance of muscle biopsy for an accurate diagnosis and classification of IIM patients.Another important fact regarding muscle histological examination of IIM patients is the discordance between clinical and histopathological diagnosis that has been previous reported, indicating up to 55% of change in diagnosis after muscle biopsy in IIM patients. Muscle biopsy examination has also a great utility in deep characterization of IIM patients13.In addition to muscle fiber description, we also aimed to characterize the immunological profile of our study group by serum detection of MSA and MAA as well as cytokines and chemokines quantification. We found a seropositivity of 41.7% of MSA or MAA in concordance with the phenotypes previously described as the case of the presence of anti-MDA5 in ADM phenotype or ILD development in anti-EJ seropositive patientsRegarding cytokines, it is important to denote that not all cytokines or chemokines were detected because their serum levels were lower than the minimum considered in the detection range, we considered them as zero for comparisons and reported the detection rate in Table 14; IL-6 promotes vascular endothelial activation by an increase of E-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cellular adhesion molecule 1 (VCAM-1) expression which is important for leukocyte migration to muscle tissue15, otherwise, IL-6 elevated levels have been reported in other rheumatic diseases including rheumatoid arthritis, Sjögren’s syndrome, and Crohn’s disease18. The role of IL-6 in IIM has been previously reported as the case of the model of myosin-induced experimental myositis where mice developed myopathy, but IL-6 deficient mice did not show clinical nor histological signs of muscle damage19. Additionally, elevated serum levels of IL-6 in IIM patients have been also reported and proposed as an activity disease biomarker in DMJ20. Considering this background as well as our results, we could consider IL-6 as a one of the main mediators of tissue destruction in IIM immunopathology.We found higher levels of IL-6 in IIM patients respecting to control group. This cytokine is one of the main mediators of pro-inflammatory responses, mainly secreted by macrophages, fibroblasts and endothelial cells22, did not show difference. This behavior might be explained by the clinical remission of the IIM, the immunosuppressive treatment or the low concentrations of cytokines and chemokines in peripheral blood, however, it would be important to determine the expression of these molecules directly in muscle tissue.Otherwise, IFNγ and IL-17A, which are related cytokines to IIM pathogenesis because of their participation in MHC-I overexpression and autoimmunity developmentAfterwards, according to MSA/MAA seropositivity, we observed only a tendency of IL-6 higher levels in seropositive patients. On the other hand, we found higher levels of IFN-γ and CCL2 in seropositive patients. To our knowledge, this is the first report comparing cytokine and chemokine serum levels according MSA/MAA seropositivity, however, due to small sample size, we could not carry out the analysis for each autoantibody. We propose the comparative of cytokine and chemokine serum levels according to each MSA and MAA as a perspective for a better understanding of the immunological profile of IIM patients.23. This cytokine is mainly produced by monocytes, macrophages, and natural killer24 cells after pattern recognition receptors (PRR) activation, but IFN-γ is also synthesized by TCD4+ lymphocytes (Th1 profile) and TCD8+ lymphocytes25. It has been demonstrated that the IFN-γ gene is overexpressed in myofibers surrounded by TCD8+ lymphocytes26. Otherwise, it is highly recognized that this cytokine induces the MHC-I and MHC-II overexpression in human myoblasts28, which is associated with IIM immunopathology because it facilitates myofibers recognition by TCD8+ lymphocytes15, and triggers endoplasmic reticulum stress29.The implication of IFNs in IIM is well known and has been widely reported since the first time in 198630. In addition, the inflammatory infiltrates which surround the blood vessels of muscle tissue are mainly composed of lymphocytes and macrophages, which are target cells of CCL215. It has been shown distinct evidence of CCL2 implication in myositis including its expression at mRNA level in patient muscle tissue, CCL2 protein expression in endothelial cells, as well as higher levels of CCL2 in muscle tissue of IIM patients32. Moreover, we could infer that CCL2 synthesis could be induced by membrane attack complex24 formation in endothelial cells surface of DM patients, the main phenotype in our study. Otherwise, there are few reports which suggest the consideration of this cytokine as an early biomarker for ILD development, since CCL2 elevated levels have been observed in DM/PM patients with ILD complication34.On the other hand, it has been reported that the chemokine CCL2, mainly produced by monocytes and macrophages, participates in IIM pathogenesis by the stimulation of leukocyte migration to the muscle tissueOnce we characterized the damage and muscle strength as well as immunological profile, we looked for correlations between these parameters. We did not find any association according to the MMT8 score, however, one of the most remarkable findings is the association of LDH enzyme with immunological profile, reflected in: the correlation between LDH and IFN-γ serum levels, the difference of IFN-γ serum levels according to normal or elevated LDH levels, higher LDH levels in seropositive patients, as well as a tendency to the higher frequency of seropositive patients with LDH elevated levels.Concerning a specific histopathological finding and its association with immunological profile, we observed that the most common finding according to HE results, was perivascular inflammatory infiltrate. We also observed a decreased MMT8 score and increased muscle enzymes when there was presence of regeneration markers (basophilic fibers), as well as lower muscle strength and IL-18 elevated serum levels when the patients had endomysial inflammatory infiltrate.+ T cells, which could have an association with this cytokine since it has been proved that IL-18 receptor (IL-18R) has higher expression in functional phenotypes of CD8+ T cells35, meanwhile other studies have demonstrated that exhausted CD8+ T cells downregulates its IL-18R expression36. In addition, IL-18 has been proposed as a biomarker in IIM due to its correlation with disease activity37, and its increment in serum as consequence of muscle damage38. Despite the low number of patients in our study, we were able to find an association of higher IL-18 levels in patients with endomysial infiltrate probably due to an active state of immune response mainly orchestrated by CD8+ T cells.This infiltrate in muscle tissue is commonly composed by CD8Moreover, the most common findings by SEM included myofiber surface irregularities, altered muscle morphology and non-linear muscle fibers. We observed lower levels of IL-1β and CXCL8 in patients with altered muscle morphology, probably due to a chronic damage.In search of a possible association between HE and SEM findings . Concerning the association between immunological parameters, the serum levels of IFN-γ are higher in MSA/MAA seropositive patients. Regarding to muscle and immunological parameters association, the LDH enzyme even unspecific of muscle damage showed association to immunological profile.In conclusion, despite the scarce reports about the use of SEM as a tool in all clinical phenotypes of IIM, our work provides an excellent opportunity to discuss and reframe its clinical usefulness in the diagnostic approach of IIM.Twelve patients classified with IIM according to EULAR/ACR 2017 criteria, attending Hospital Civil de Guadalajara “Dr. Juan I. Menchaca” were enrolled in this cross-sectional study. We also included twelve clinically healthy subjects as control group for cytokines and chemokines serum levels comparison. Informed written consent was obtained from every patient and subject before enrollment in the study. This protocol was approved by Ethical and Research committees of Hospital Civil de Guadalajara “Dr. Juan I. Menchaca” .We obtained peripheral blood and muscle samples of each patient and all our experiments were performed in accordance with relevant guidelines and regulations. Muscle tissue was obtained from the quadriceps by a muscle biopsy procedure performed by a surgeon. Immediately the muscle tissue was stored as corresponding: buffered formaldehyde 10% for HE staining and paraformaldehyde 4% for SEM.10.The muscle weakness examination was performed by a rheumatologist using the MMT8 score. This tool included seven proximal muscle groups tested bilaterally  and neck flexors which complete the eight muscle groups tested. Each muscle subgroup receives a score of ten points obtaining a maximum score of 150 when the patient did not present muscle weakness45.Disease activity is defined as potentially reversible clinically evident pathology or physiology resulting from the myositis disease process according to the International Myositis Assessment and Clinical Studies Group (IMACS), we evaluated the disease activity in each of our patients using the Disease Activity Core Set Measure, the MYOACT45.Damage is defined as persistent changes in anatomy, physiology, pathology or function, which are present for at least 6 months according to the IMACS, we evaluated the damage in each of our patients employing the recommended Disease Damage Core Set Measure, the MDIOnce the muscle tissues were obtained, they were processed by a pathologist using a validated protocol for hematoxylin–eosin staining. The muscle sample was fixed from 24 to 48 h in 10% neutral buffered formalin. The samples were dehydrated in increasing ethanol solutions, cleared in xylol, and embedded in paraffin on an automatic tissue processor . Subsequently, 3 μm sections were obtained and stained with the conventional hematoxylin and eosin technique, and then evaluated by light microscopy (Microscope Carl Zeiss Axio Lab.A1).Patients were grouped according to histological features in connective tissue increased, diffuse and multifocal atrophy, perifascicular atrophy, perivascular inflammatory infiltrate, endomysial inflammatory infiltrate, degenerated fibers, necrotic fibers, basophilic fibers, edema and bleeding, muscle fiber size variation, endothelial hyperplasia, and tumefaction.Samples were processed according to validation guidelines by an expert in electronic microscopy. Muscle was fixed with glutaraldehyde 2.5% and phosphate-buffered saline (PBS) 0.1 M, pH 7.2 for twelve hours, subsequently tissue was washed with PBS 0.1 M, pH 7.2 three times. Samples were dehydrated with increasing concentrations of ethanol solutions , each step had a duration of fifteen minutes. Posteriorly they were collocated in hexamethyldisilazane with absolute alcohol (1:1) for fifteen minutes, then tissues were reposed in hexamethyldisilazane for fifteen minutes and subsequently were dried with the air flux of the extraction hood . The dehydrated tissues were prepared in slides with carbon tape and were then coated with a layer of gold by a sputter coater . Finally, the samples were observed in the scanning electronic microscope .Patients were grouped according to ultrastructural findings in non-linear muscle fibers, myofiber surface irregularities, altered muscle morphology, muscle fibers perforation, cellular infiltrates, size fiber variation, and myofiber surface porosities.Autoantibodies were detected in serum samples of our patients using the line-blot assay kit Euroline: Autoimmune Inflammatory Myopathies from EUROIMMUN Medizinische Labordiagnostika AG  according to test instructions. This kit allows the detection of twelve MSA  and four MAA . This kit includes test strips coated with highly purified and biochemically characterized autoantigens which were incubated with diluted serum samples and, in the case of positivity, the autoantibodies bounded to the corresponding site and catalyzed a color reaction. For the correct evaluation of the test strips, they were read by EUROLineScan software and the results were interpreted according to the signal intensity of the bands.https://legendplex.qognit.com/).Serum levels of cytokines  and chemokines  were quantified using the kit LEGENDplex Multi-Analyte Flow Assay Kit from BioLegend  following test instructions as corresponding. This multiple bead-based immunoassay is based on the basic principle of ELISA type sandwich assay. Each bead is coated with a specific antibody for the cytokine or chemokine of interest, subsequently, these beads were differentiated by size and internal fluorescence intensity using a flow cytometer . Measures were analyzed in the online software of LEGENDplex  were considered as muscle damage marker, they were recorded at the time of diagnosis and recruitment.The microscope employed to performed Light Microscopy  features 10x  and 40 × objectives  as well as an ocular HC PLAN s 10x/20. For image acquisition we utilized the microscope camera  and the software Leica Acquire for iMAC, version 3.4.7. Blank adjustment to the pictures was made and pictures were edited in Figma, online version, only for fitting in the panel.Variables were assessed for normality using the Kolmogorov–Smirnov test; values are presented as mean ± SD, or percentages (%), as appropriate. Comparisons were made using Mann–Whitney U test with Fisher’s exact test for quantitative variables, due the low number of patients, meanwhile qualitative variables were analyzed using χ2 or Fisher’s exact test as appropriate.P < 0.05 to be significant for analysis.Our data were analyzed using SPSS 24.0 software  and GraphPad Prism version 6.00 for Windows , considering a two-tailed level of Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.