Age/sex  and playing position  also had significant effects on external load. The present data comprehensively describe and analyze important external load measures in a sample of high-performing beach handball players, providing valuable information to practitioners and coaches aiming at improving athletic performance in this new sport.Beach handball is a young discipline that is characterized by numerous high-intensity actions. By following up on previous work, the objective was to perform in-depth analyses evaluating external load  in beach handball players. In cross-sectional analyses, data of 69 players belonging to the German national or prospective team were analyzed during official tournaments using a local positioning system (10 Hz) and inertial measurement units (100 Hz). Statistical analyses comprised the comparison of the first and second set and the effects of age and sex  and playing position  on external load measures. We found evidence for reduced external workload during the second set of the matches ( Beach handball has its origins in 1992 and has been growing ever since then. Derived from classic indoor team handball, beach handball is played predominantly during the summer break of team handball schedules. Although both sports share many characteristics, there are also substantial differences, placing new requirements on athletes and coaches in terms of physical demands, tactics, and designing appropriate training programs. In contrast to indoor team handball with seven players, beach handball is played on the sand by only four players, including the goalkeeper. In the offensive phase, player positions are specialist, wing, and pivot. During defensive phases, usually, other players take over the tactical tasks of the goalkeeper and defenders. Moreover, beach handball is played on a field measuring 27 × 12 m, and the game is divided by a five-minute break into two halves with a duration of 10 min each, followed by a shoot-out in the case where both teams win one half. Two more examples of the modified rules in beach handball are the possibility of repeatedly substituting players at any time, as well as variable scoring depending on the type of goal throws, e.g., trick shots such as spin shots or goalkeeper’s goals count double. These new requirements for athletes and their coaches have prompted further research efforts in various sports science disciplines, encompassing sports medicine .The increasing professionalization of the discipline and its public appeal makes beach handball an interesting candidate for the Olympic tournament. This potential is currently not reflected in the scientific literature, with only a small number of studies on requirements and conditional analyses in this sport. However, recent technological advances in terms of improved portability when using inertial measurement units (IMU) compared with the more established motion-analysis equipment allows for enhanced ecologically valid data collection outside the lab ,5, whichThe physical requirements in sports can be categorized as internal and external workload. External load refers to the physical work performed in training or competition . Among oIn recent years, multi-camera video systems have been replaced by Global Positioning Systems (GPS) and Local Positioning Systems (LPS) for location tracking. Both tools avoid the drawbacks of camera-based technology such as the overlapping of players in team sports or the tracking of individuals under unfavorable light and shadow conditions ,16,17. A−1 and 70 m·min−1, respectively ), as well as maximum velocity . IMA metrics, captured at a sampling rate of 100 Hz, include total player load and player load per minute, acceleration and deceleration, explosive efforts, changes in direction (CoD), and jump counts. Player load is a commonly used variable to describe external load in arbitrary units (au) that can be calculated using 3D-acceleration data based on the formula, where a equals acceleration [The parameters presented here can be categorized as positional data, captured at a sampling rate of 10 Hz, and encompassing the distance traveled  in meter (m) and distance per minute , which is in line with previous reports, e.g., . Shoot-oout) if they fulfilled one of the following conditions:Statistical analyses were performed using IBM SPSS v.28 . Data sets were examined for outliers by initial standardization using z-transformation and subsequently identifying outliers using the interquartile range (IQR) method [N = 16), male adolescents , and male adults , comprising subgroup 1: “age/sex” and between the five different playing positions . Multivariate analysis of variance (MANOVA) with Bonferroni adjustments for multiple comparisons was performed to test for anthropometric differences between the subgroups. Repeated-measures MANOVA with Bonferroni adjustments for multiple comparisons were conducted to test for differences in external load measures between the first and second sets of matches. Interaction effects (set × subgroup 1 and set × subgroup 2) were analyzed to evaluate the effects of the match set on external load measures as a function of age/sex and playing position. Multivariate analyses of covariance (MANCOVA) with Bonferroni adjustments for multiple comparisons were carried out to examine differences in all variables analyzed, with subgroup 1 adjusted for playing position and subgroup 2 adjusted for age/sex. Results are presented as means ± standard deviation and effect sizes . Statistical significance was set at p < 0.05.IQR represents the range between the first and third quartile (25th to 75th percentile). The constant k, used to scale the limits of the outlier tolerance field, was set at 1.5 . SubgrouN = 23) and invalid data (N = 2). The overall sample had a mean age of 20.1 ± 4.9 years (range 15.3–34.4 years). The mean age among the subgroups was 16.6 ± 0.5 years for female adolescent athletes, 17.1 ± 0.4 years for male adolescent athletes, and 24.3 ± 4.7 years for male adult players. The distribution among the subgroups and corresponding anthropometric data are presented in Following the data cleaning, 285 data sets were valid and included in the analyses. Reasons for missing data were limited time on the pitch . Likewise, no differences among the subgroups were found in the mean play time during the first and second sets. A repeated-measures MANOVA showed a statistically significant difference between the first and the second sets of the matches played (F(9250) = 2.70, p = 0.005, ηp2 = 0.09, Wilk’s λ = 0.91). Post hoc analyses with Bonferroni adjustments indicated significant differences in player load per minute and changes in direction per minute (F(2256) = 0.95, p = 0.388, ηp2 = 0.01, Wilk’s λ = 0.99, and set × subgroup 2 (“playing position”): F(4254) = 0.91, p = 0.457, ηp2 = 0.01, Wilk’s λ = 0.99.Mean play time during the first and second set was 6.7 ± 2.7 min and 6.6 ± 2.6 min, respectively  = 7.10, p < 0.001, ηp2 = 0.22, Wilk’s λ = 0.61. Likewise, external player load was also affected by playing position: F  = 10.38, p < 0.001, ηp2 = 0.29, Wilk’s λ = 0.25.−1, with a mean decrease of 11 m and −1.2 m·min−1 during the second set. Male adolescents covered the greatest distances (870 ± 217 m), followed by male adults (790 ± 205 m) and female adolescents (760 ± 217 m). In terms of playing position, the rank order was specialists (889 ± 239 m), wings (823 ± 245 m), pivots (804 ± 158 m), followed by defenders (785 ± 172 m), and goalkeepers (737 ± 251 m). The distance traveled per minute was calculated to account for active playing time on the pitch, with similar trends among the subgroups  covered significantly more distance compared with male adults (62.2 ± 13.0 m·min−1) but not compared to female adolescents (63.3 ± 12.0 m·min−1). Specialists traveled significantly larger distances (73.9 ± 15.0 m·min−1) compared to the other playing positions . Results from pairwise comparisons indicated that male adolescents (17.3 ± 2.0 km·h−1) attained higher maximum velocities compared with female adolescents  and male adults , whose values did not differ significantly (p = 0.056). Regarding playing positions, the highest maximum velocity was measured in wings (17.3 ± 1.9 km·h−1), the lowest in goalkeepers . The remaining pairwise comparisons revealed no further significant differences (The average maximum velocity in this sample was 16.5 ± 2.0 km·hferences .ηp2= 0.01). In order to account for playing time, player load per minute was calculated (p = 0.039). The analysis of interaction effects (set × subgroups) was not significant, indicating that this reduction was neither affected by age/sex nor by playing position. Male adolescent players attained a significantly higher player load compared with female adolescents and male adults (each p < 0.001), whose values did not differ significantly (p = 0.051). In terms of playing position, player load per minute was considerably lower in goalkeepers compared with the other positions, but pairwise comparisons did not reveal any significant differences  and deceleration values (−3.34 ± 0.67 m·s−2) of the sample did not show significant differences between the first and second set of the matches, as was the case for absolute numbers of explosive efforts (9.7 ± 5.9 counts) and efforts per minute (0.78 ± 0.48 counts). Male adolescents attained the highest accelerations and decelerations and performed the highest number of explosive efforts per minute, followed by male adults and female adolescents. Pairwise comparisons indicated significantly higher maximum accelerations and decelerations in male adolescents compared to female adolescents and significantly higher maximum decelerations between male adolescents and male adults  and IMA decelerations (21.1 ± 9.6 counts), the average number of changes in directions (87.8 ± 36.4 counts), and the number of jumps (11.3 ± 7.2 counts) per match played. p < 0.001). No interaction effect (set × subgroups) was found, indicating that these reductions were neither affected by age/sex nor by playing position. The results further reveal a similar trend across subgroup 1 “age/sex” for the various variables analyzed, according to which male adolescents reveal the highest external load, followed by male adults and female adolescents, with the variable DECIMA/min being the only exception  and also compared to pivots (p = 0.044) (p < 0.001), while defenders performed significantly less compared to pivots (p = 0.008). Contrary, IMA deceleration counts per minute were significantly higher in defenders compared to goalkeepers (p = 0.003), specialists (p < 0.001), and wings (p = 0.004) (p = 0.003), pivots, and specialists (both p < 0.001). The remaining comparisons were not statistically significant  . Special= 0.004) . Besides= 0.004) b, while nificant .The objectives of this study were to comprehensively describe important external load parameters using valid assessment methods and considering analyses presented in previous publications. In summary, only two variables (player load per minute and changes in direction per minute) revealed a significant reduction from the first to second set without interaction effects of subgroups, indicating limited evidence for reductions in external load measures during the second set of analyzed matches. Several variables point to differences between female and male players, as well as between adolescent and adult players, while the most significant differences in external load were found between playing positions. Additionally, this comparison revealed the largest effect size, indicating that among all factors analyzed, playing position had the greatest impact on external load measures.One of the most common variables examined to describe external load in beach handball is the distance traveled. Our data show that the mean distance covered during matches equals 806 m, ranging from 760 m in female adolescents to 870 m in male adolescents. This is comparable with previous studies reporting a mean distance of 898 m per match in nine elite female athletes . However−1 in males and 59.8 m·min−1 in females, compared to 69.2 m·min−1 in male adolescents and 63.3 m·min−1 in female players in the present study. Remarkably, relative distances traveled in beach handball are much lower compared to small-sided team handball, which is played 4 × 4 on a court of comparable size (24 × 12 m). Corvino et al. reported a mean distance traveled of 118.5 m·min−1 [In this regard, our results align well with the findings of Pueo et al. , who dem m·min−1 , distinc m·min−1 , thus li−1 in female elite beach handball players, compared to 15.6 km·h−1 in female adolescent athletes in the present study. Zapardiel et al. [TM IMU devices [Beach handball is further characterized by numerous high-intensity actions throughout the game. The game dynamics in beach handball emerge from the rapidly occurring transition from attack to defense and counterattack, requiring high-intensity game actions such as sprints, jumps, changes in direction, as well as high accelerations and decelerations. In order to design reasonable training programs aiming at sustainable performance optimization, valid assessments and detailed analyses of these high-intensity bouts of play are required. In this respect, our data largely support previous studies in beach handball but also expand them by conducting in-depth analyses on workload in subgroups. In summary, our data on maximum velocity well align with Sánchez-Sáez et al. , who usel et al.  reported devices . They rePrevious studies demonstrated a reduction in external load measures from the first to the second set, likely indicating fatigue effects over the course of a match. For instance, Sánchez-Sáez et al. reported significantly reduced total distance and average velocities during the second set . Pueo etWith regard to the effects of sex and age on external load measures in elite beach handball players, previous studies consistently demonstrated differences between female and male players ,20, but −2. These results indicate that physical demands in beach handball vary considerably according to the different playing positions. Hence, the availability of highly differentiated, valid, and reliable technical solutions to analyze external workload in sports using IMUs and LPS/ GPS should be complemented by differentiating more meaningfully within analyses according to playing positions. This will help in laying the basis for individualization when designing sound position-specific exercise programs and thus provides the foundation for optimal performance enhancement in already high-performing athletes.Zapardiel et al. were the first to perform conditional analyses in elite beach handball considering specific playing positions , and ourThe results of this study must be interpreted in light of some limitations. First, all measurements took place during tournaments in which we had to adhere to fixed schedules based on pre-determined match plans. Hence, no standardization was feasible regarding weather conditions such as temperature and/or humidity, which might have affected player performance and the condition of the sandy surface. We examined the effects of age and sex on external player load. To obtain a complete picture, the inclusion of a female adult group would have been desirable but, unfortunately, was not feasible to carry out within the funding period. Furthermore, the teams played up to four games on a given tournament day and up to seven games on a tournament weekend, which might have affected the outcomes as well. However, in sum, active playing time during seven games still falls below the mean active play time in male and female elite team handball in a given match . The motOur results underline that beach handball can be characterized by numerous high-intensity actions such as sprints, jumps, changes in direction, as well as high-intensity accelerations and decelerations. In line with previous studies, we found moderate evidence of reduced external load during the second half of the matches, but contrary to previous reports, no interaction effects, which is indicative of overall fatigue instead of workload reductions affecting solely specific age/sex groups or playing positions. Our results further confirm studies demonstrating differences in external workload between female and male beach handball players but contradicted findings of higher workloads in adult compared to adolescent players. Particularly important information is derived from analyzing workload considering specific playing positions, which helps in designing individualized training programs for optimal performance enhancement in beach handball.


We aimed to identify eldercare wards with different types of resident handling characteristics (‘phenotypes’) and determine the prospective association between these characteristics and musculoskeletal pain and sickness absence among workers during a one-year follow-up.Our study was based on the DOSES cohort, including 467 workers at 103 eldercare wards. At baseline, resident handlings were assessed using onsite observations. Workers’ self-reported musculoskeletal pain and sickness absence were assessed during the following year using text messages. Observations of the frequency of handlings per shift, use of assistive devices, assistance from others, and barriers (interruptions and impediments) were estimated for each worker, aggregated at ward level, and entered into a latent profile analysis, identifying ward phenotypes. We then used generalized estimating equations to determine associations between ward phenotypes, musculoskeletal pain and sickness absence.We identified four ward phenotypes: ‘turbulent’ , ‘strained’ , ‘unpressured’  and ‘balanced’ . Compared to workers in balanced wards, workers in turbulent wards had more days with neck-shoulder and low-back pain (LBP); and those working in strained wards had more days with LBP and higher pain intensities.We found that ward phenotypes based on resident handling characteristics were predictive of musculoskeletal pain and sickness absence over one year. This shows that organizational factors related to resident handling are important determinants of musculoskeletal health among eldercare workers. Eldercare workers often report low-back pain (LBP), neck-shoulder pain (NSP) and musculoskeletal sickness absence, likely due to high physical workload and poor psychosocial working conditions –3. This As proposed in previous exposure–effect models , 7, muscTo a major extent, previous studies in eldercare have also been based on self-reported data on physical workload, which are prone to bias . As an aa set of observable characteristics of an organism , which are known to have limited effectiveness . In the organism .This study aims to identify phenotypes of wards sharing similar physical and psychosocial conditions for resident handlings and then determine the extent to which they are associated with musculoskeletal pain and sickness absence among workers at the wards over the course of one year.This prospective study uses data from the Danish Observational Study of Eldercare work and musculoskeletal disorders (DOSES), collected between September 2013 and January 2016 and previously described . BrieflyAt baseline, ward managers were asked to complete a questionnaire addressing conditions at their ward. These ward managers were, in general, responsible for communicating with higher-level management, taking decisions at their own ward, and managing the staff there. The questionnaire asked about type of ward , resident information , staff ratio, and how often assistive devices were not in place . Physical function level was categorized based on the resident’s need for physical assistance . Psychosocial function level was categorized based on the resident’s usual behavior .2).The eldercare workers answered a baseline questionnaire asking about age, smoking habits (smoker or non-smoker) and musculoskeletal pain and sickness absence due to NSP and/or LBP, as described in detail below. Body mass index (BMI) was calculated based on measured height and weight . Reasons for not observing a resident could be that (i) the resident was not in need of care or was hospitalized, (ii) the eldercare worker taking care of that resident declined to be followed, and (iii) there was a shortage of research assistants at that point in time. Estimates of handling characteristics for non-observed residents were obtained by imputation. Next, handlings at the level of individual eldercare workers were estimated based on work schedules, which were obtained from the ward managers for all workers, irrespective of whether they participated in observations or not. Schedules were then combined with the data based on handlings at the resident level to give a task-based estimate of the number of handlings per shift for each worker, as well as the associated handling characteristics. In our eventual analysis of phenotypes, we considered (i) the frequency of handlings per shift, and calculated (ii), the proportion of those handlings done without any assistive device and (iii), the proportion of handlings done without any assistance from others. We also considered the number of (iv) interruptions per shift and (v) impediments per shift. We aggregated observations of these five variables at the ward level by averaging the handling exposure estimates of all eldercare workers in each specific ward. In this study, observations during day and evening shifts were summarized in terms of values per shift, disregarding time of day. Then, we used these ward-level scores in a latent profile analysis of handling characteristics.www.sms-track.com)  , 26. At At baseline, and every 12 weeks in follow-up, the workers were also asked to report the number of days on sickness absence due to NSP and/or LBP in the past three months (0–84 days). Answers to pain-related work interference were dichotomized into 0 or ≥1 days, while the other two variables describing number of days were kept continuous.Based on the handling characteristics of each of the 103 wards , we performed a latent profile analysis, using the Latent Gold software . This technique allows multiple variables to be combined in an analysis identifying latent profiles, ie, subpopulations within a population sharing similar properties with respect to the set of observed variables –29. In oThe latent profile analysis requires the user to decide the number of profiles that suits data the best. In this process, we considered model fit indices as well as the interpretability of identified profiles. Model fit statistics included the Akaike information criterion (AIC) and the Bayesian information criterion (BIC), showing the trade-off between the simplicity of the model and the goodness of fit, and entropy, describing the degree of certainty of the classification. We also required each profile to contain at least five wards so that even the smallest profile would be generalizable. To better understand the distinct handling characteristics of the eventually identified profiles, we compared the characteristics of wards, workers and residents in each profile, using analysis of variance (ANOVA) for descriptive continuous variables and Chi-squared tests for descriptive categorical variables. At this point, and after further examining the specific ward characteristics expressed in each profile, we labeled the ward profiles as phenotypes, in the sense that they reflect a set of observable and interpretable characteristics pertaining to the ward.To estimate the effects of working at a certain ward phenotype (ward-level predictor) on reported musculoskeletal pain and sickness absence during the one-year follow-up  we used generalized estimating equations (GEE) . In thesWe adjusted each of the six models for their corresponding outcome values obtained at baseline. We further adjusted the models for age, BMI and smoking habits. We also did a sensitivity analysis by re-running each of the six primary models solely with participants reporting ‘little’ pain at baseline, defined as pain ≤5 days/month, pain intensity ≤2 , 0 days with pain-related work interference, and 0 days with sickness absence. We performed all tests in SPSS  and set the significance level at 0.05.https://www.sjweh.fi/article/3979, table S1), models with four and five profiles were the more certain , while still being sufficiently parsimonious according to BIC and AIC values. These two models also fulfilled the criterion of the smallest profile containing at least five wards. Taking into account that the model with four profiles showed better parsimony and had more workers in the smallest profile than the model with five profiles, and that the differences in the handling characteristics between the four ward profiles were more intuitive than those in the five-profiles model, we settled on the four-profile solution.According to the model fit statistics  7.1 per shift], some of which are done without assistive devices [mean 41 (SD 18) %] or assistance from others [mean 20 (SD 9) %], and with many barriers .‘Strained’ wards: many handling operations [mean 11.4 (SD 4.3) per shift], many of which are done without assistive devices [mean 52 (SD 18) %] or assistance from others [mean 67 (SD 10) %], with some barriers .‘Unpressured’ wards: few handling operations [mean 4.9 (SD 1.8) per shift], even if many of which are done without assistive devices [mean 53 (SD 21) %] or assistance from others [mean 61 (SD 24) %], and few barriers .‘Balanced’ wards: some handling operations [mean 9.2 (SD 1.6) per shift], some of which without assistive devices [mean 47 (SD 12) %] or assistance from others [mean 34 (SD 11) %], and with some barriers .Based on the latent profile analysis, mean 7 (6.8%), 21 (20.4%), 52 (50.5%) and 23 (22.3%) of the 103 wards were categorized as turbulent, strained, unpressured, and balanced, respectively. At turbulent wards, the staff ratio was smaller than at the other ward phenotypes and a higher proportion of residents were classified as needing complete physical assistance. At strained wards, devices were reported to be misplaced daily more often than in the other ward phenotypes, and the workers had higher BMI. Unpressured and balanced wards were not extreme in any respect. Descriptive characteristics of the eldercare workers’ musculoskeletal pain and sickness absence at baseline and during the one-year follow-up, stratified by ward phenotype, are shown in During the one-year follow-up, turbulent wards were significantly associated with more days per month with NSP  and LBP  among the workers than balanced wards . SimilarStrained wards were associated with higher intensity of NSP pain , more days with LBP , and higher intensity of LBP  and were more likely to report pain-related work interference  compared with balanced wards. Strained wards were also associated with more days with musculoskeletal sickness absence  than balanced wards. After adjustments these results remained significant and with slightly higher effect estimates . UnpressThe sensitivity analysis of workers with low pain and no sickness absence at baseline showed, in general, stronger and more significant associations at follow-up in this sub-population than in the total population (supplementary table S2).To our knowledge, our study is the first to collect onsite observations of resident handlings in eldercare, interpret those in terms of ward phenotypes, and address the prospective association of these phenotypes with musculoskeletal pain and sickness absence among eldercare workers. We identified four distinct ward phenotypes, labeled as turbulent, strained, unpressured, and balanced, representing distinctly different combinations of various handling characteristics. After adjusting for age, BMI and smoking habits, we found that workers at turbulent and strained wards reported a larger increase in pain during the one-year follow up than workers at balanced wards.Among the four ward phenotypes, two (turbulent and strained) appeared to have a less favorable combination of handling characteristics, while the two others  had more beneficial profiles. To our knowledge, this is the first study to evaluate different aspects of resident handlings concomitantly.At turbulent wards, workers had high physical demands reflected by a high frequency of handlings, but they also had resources to perform these handlings, as shown by the high proportion of handlings done with assistive devices and assistance from others. Handling assistance has been shown to reduce physical load during handling tasks , 31. On The strained wards also appear to have less favorable handlings characteristics; handlings were both frequent and, to a considerable extent, performed without assistive devices or assistance from others, likely have increasing the physical load on workers , 16, 31.Compared with balanced wards, working in a turbulent ward was associated with more frequent NSP and LBP, while working in a strained ward was associated with more days of LBP, higher intensity of NSP and LBP and a higher likelihood of pain interfering with work. This suggests that the risk of musculoskeletal pain increases with the frequency of handlings, particularly when handlings are more often performed without the use of assistive devices or assistance from others. This is consistent with previous studies reporting that a higher frequency of handlings is associated with persistent LBP in eldercare workers  and a hiPerforming resident handlings without assistive devices, as observed to a larger extent in strained wards, have been associated with LBP among nurses , but eviWe found a large variability in reported pain and sickness absence at baseline, and so we also performed a sensitivity analysis including only those workers with little pain and no sickness absence. Ward phenotype predicted musculoskeletal pain and sickness absence even stronger in this sub-population, suggesting that the handling characteristics at turbulent and strained wards could, indeed, increase the risk of workers developing musculoskeletal disorders.The use of a systematic and reliable onsite observation tool for assessing resident handlings characteristics , 24 is aStill, the study also suffers some limitations. Even though the use of an observational tool can be considered an advantage compared to self-reports, it should ideally be combined with direct measurements of exposures , which wThe assessment of pain and sickness absence during the one-year follow-up relied solely on self-reports, which could introduce bias. However, we trust this risk to be reduced by the frequent assessment of outcomes, reducing recall bias, and we emphasize that common method variance of exposures and outcomes is likely small, due to the use of observational-based, aggregated exposures. Finally, while we believe each of the six selected outcomes to be important in reflecting different aspects of pain  and its consequences , we acknowledge the risk of obtaining significant results by chance in multiple tests.Our results, in illustrating the importance of ward-level characteristics to health can be used in future studies as a basis for designing ward-level tools for preventing musculoskeletal symptoms in eldercare workers, as an alternative to, or complementing, individualized interventions, such as having workers attend courses in lifting techniques. While we believe that intervening on organizational and structural determinants of handlings would lead to more effective and sustainable results than interventions directed towards the behaviors of individual workers, future studies should verify the effectiveness of ward-level organizational tools, for example in randomized control trials, before they are generally recommended for practice .Based on onsite observations of resident handlings in eldercare, we were able to identify four handling phenotypes at the ward level. Our results show that turbulent and strained ward phenotypes were associated with some indicators of musculoskeletal pain during the one-year follow-up. Thus, our study suggests that ward phenotypes with frequent handlings, either combined with limited use of assistive devices or with many barriers, may impose an increased risk of musculoskeletal pain among the workers. Our findings encourage future studies devoted to the development of ward-level, organizational interventions in eldercare homes.


Acute malnutrition is one of the main causes of morbidity and mortality among children under 5 years worldwide, and Action Contre la Faim (ACF) aims to address its causes and consequences. To better tailor humanitarian programs, ACF conducts standardized contextual studies called Link NCAs , to identify factors associated with severe acute malnutrition (SAM) and moderate acute malnutrition (MAM). Data from three Link NCAs performed in 2018 and 2019 in Haiti, Burkina Faso and Madagascar were used to explore the prevalence of malnutrition by different indicators and associated risk factors among children aged 6–59 months.Cross-sectional data, collected via household surveys applying two-stage cluster sampling, were pooled to build a sample of 1,356 children. Recommended anthropometric thresholds were used to define SAM (Weight-for-Height Z-score (WHZ) <-3 or Mid-upper Arm Circumference (MUAC) <115 mm and/or presence oedema), MAM (-3≤WHZ<-2 or 115≤MUAC<125 mm) and global acute malnutrition GAM (SAM or MAM) among children. Multivariate analyses for each anthropometric indicator were performed using logistic mixed models and adjusting for potential confounders.The prevalence of acute malnutrition was the highest in Madagascar. The risk of having GAM and MAM varied across countries, while the risk of having SAM varied across clusters. Being male, suffering from diarrhea, and having unwashed face and hands, were significantly associated with GAM by WHZ with adjusted odds ratio of 1.9 [95%Confidence interval (CI):1.1–3.2], 1.7 (95%CI: 1.0–3.1) and 1.9 (95%CI: 1.0–3.6) respectively. These factors were also associated with MAM by WHZ. None of the studied factors was significantly associated with SAM, which could be due to a small sample size.These results obtained from a large sample contribute to the evidence of the factors associated with undernutrition in children aged 6–59 months. Further research with larger sample sizes is needed to confirm these results. Acute malnutrition is one of the main causes of morbidity and mortality among children under 5 years of age worldwide. In 2018, over 49 million children under 5 year of age were wasted and nearly 17 million were severely wasted . A sample Link NCA questionnaire can be found in the Across all surveys, data on retrospective mortality, food security, livelihoods, and WASH were collected at household level. Among women 15–49 years of age, health data were collected. Among children 6–59 months of age, data on health, infant and young child feeding practices, and anthropometric measurements  were collected.Anthropometric measurements were taken from all eligible children in order to assess acute malnutrition by three main indicators: Weight-for-Height Z-score (WHZ), MUAC, and bilateral pitting oedema. The outcomes of interest were acute malnutrition per WHZ, acute malnutrition per MUAC, and acute malnutrition per combined WHZ and/or MUAC .Recommended thresholds for anthropometric measurements were used to define SAM (WHZ <-3 or MUAC <115 mm and/or presence oedema), moderate acute malnutrition (MAM) (-3≤WHZ<-2 or 115≤MUAC<125 mm without oedema) and GAM (SAM or MAM) among children –21 Tabl.For the combined variables, combined GAM (cGAM) is defined as all cases of GAM by WHZ and/or MUAC and/or oedema , 20. ComRisk factors that were likely to be associated with acute malnutrition based on either published or grey literature, were validated by technical experts during an initial workshop were collected as part of the quantitative survey. Only comparable variables collected across all three surveys were retained for this analysis and they were grouped into three categories as per the UNICEF conceptual framework of the determinants of child undernutrition:Child characteristics: sex of the child , vitaminHousehold characteristic: sex of the head of household  , large fEnvironmental characteristic: place of birth of the youngest child (home / outside of home) , number versus non-protected. In order to harmonize the variable for all countries, this reclassification of the variable as a protected and unprotected main drinking water source was done with the help of a WASH specialist , MUAC  and combined categories  were structured as binary outcomes (positive or negative) for the analysis. The prevalence of each anthropometric variable was calculated for each survey area. A univariate descriptive statistical analysis of anthropometric variables and risk factor variables for each of the three studies was conducted.Several logistic regression models, classical models (with fixed effect only) and random effect models were used for each anthropometric variable. As a level of heterogeneity was observed between clusters and by country, these last two variables were introduced into the models as a random effect . Despitecluster as random effect, a model with country as random effect, and a model with cluster and country as random effect. The variance components of each model were tested with the likelihood ratio test. The models were tested in pairs and the best model was the one with cluster and country as random effect. Furthermore, the result of the Akaike information criterion (AIC) also identify the model with cluster and country in random effect as the best suited model.Four types of models were tested: a model without random effect, a model with cluster as random effect. For models with SAM by MUAC as outcome, the best models are the models without random effects. For the other models, it remains with cluster and country in random effects , the explicative variables introduced in the models were all variables with a p-value < 0.20. For models with SAM by WHZ or SAM by WHZ & MUAC combined as outcome, the best models are those with A calculation of the percentage of missing values was made. The number of missing data was reduced, and the fact of having missing data was considered to be random. For that reason, the complete case analysis was used to treat the missing data . All anaOffice National de Nutrition (ONN) and the Regional Department of Health. In Burkina Faso, permission was obtained from the Direction Nationale de la Nutrition. A new approval was not requested to conduct these analyses, which were secondary in nature. Further, a new authorization was not necessary because the data are anonymous and did not include any information that would enable to identify participants [The Link NCAs were each reviewed and approved by an institutional review committee prior to the beginning of each study. In Haiti, permission to conduct the survey was obtained from the Nutrition Unit of the Ministry of Public Health and Population ). In Madagascar, permission was obtained from the regional office of the icipants .After describing the survey, its objectives and duration in the local language, informed consent was obtained verbally  from the head of household and entered in the tablets before data collection began. Heads of households and mothers of children under five years of age provided oral consent to participate in the survey as well as permission for the participation of all children under five years of age in the household. All the answers were recorded in a way that ensured and maintained anonymity and confidentiality. Participants were allowed to ask questions before giving their consent or refusal to participate.The Link NCA quantitative surveys collected data on 395 children in Haiti, 481 children in Madagascar and 441 children in Burkina Faso between the ages of 6 and 59 months. All children were retained for analysis. According to the WHO thresholds, among the 1,317 children with anthropometric measurement across the three countries, 87 (6.6%) children had GAM by WHZ including 16 (1.2%) with SAM by WHZ. Furthermore, 43 (3.2%) children had GAM by MUAC and 11 (0.8%) had SAM by MUAC. The prevalence of acute malnutrition by all anthropometric indicators was found to be higher in Madagascar than in the other two countries . MUAC inThe univariate model results are presented in The results of the multivariate analyses can be found in Analyses using the two different models (univariate and multivariate) demonstrated that the risk of having GAM  or MAM  varied between countries see . This meThis study examined cross-sectional data collected from Burkina Faso, Madagascar and Haiti on a pooled sample of 1,317 children aged 6–59 months allowing to study the WHZ and MUAC specific prevalence of and characteristics associated with acute malnutrition across the three contexts; however, the number of SAM cases was low. Results indicated a non-significant difference in the prevalence of GAM, MAM and SAM between WHZ and MUAC based indicators whereby WHZ based classification resulted in higher prevalence of malnutrition compared to MUAC. Furthermore, different risk factors were associated with acute malnutrition by WHZ and by MUAC.et al. (2005), clinical features of malnutrition or malaria parasitemia were significantly more common among children with MUAC ≤ 115 mm than among those with WHZ ≤ −3 [et al. (2021) on children with SAM aged 6 to 59 months in Bangladesh, Burkina Faso, and Liberia showed that children with WHZ < - 3 and MUAC ≥ 115 mm had equally or more severe deficits in nutritional status, hydration, and iron balance than children with WHZ ≥ -3 and MUAC < 115 mm, and presented lower levels of leptin, a marker of mortality risk among children with SAM [Previous studies have demonstrated that WHZ and MUAC indicators do not necessarily capture the same populations –36, thatWHZ ≤ −3 . Howeverwith SAM .versus 11.9% in the current study, 7.8% in 2017 in Kénédougou in Burkina Faso [versus 4.8% in the current study, and 5% in 2018 in the Grande Anse region in Haiti [versus 2.3% in the current study. Although all three areas are parts of low-income countries, they do not have the same socio-demographic, economic and environmental specificities. Child characteristics were found to be worse in Madagascar than in the other two countries: low vitamin A supplementation coverage, lower personal hygiene  scores and higher prevalence of illness such as fever and diarrhea. This is coherent with studies documenting that wasting is often associated with a deteriorated health status [The results from this study demonstrated that the prevalence of malnutrition was highest in Madagascar, followed by Burkina Faso and finally Haiti. The prevalences of GAM by WHZ in each country was slightly lower but close those of previous studies in the same areas: 12.9% in Amboasary in Madagascar in 2018  versus 1in Haiti  versus 2h status . Diseaseh status .The factors found to be significantly associated with GAM by WHZ in all three pooled countries were being male, suffering from diarrhea, having unwashed face and hands, and using a protected water source. Only diarrhea was found to be significantly associated with GAM by MUAC. For GAM from combined indicators, risk factors were diarrhea and a protected water source. By distinguishing MAM and SAM, we found no significant association between potential risk factors and SAM according to the 3 indicators and MAM by MUAC. On the other hand, being male, suffering from diarrhea, having unwashed face and hands and using a protected water source were significantly associated with MAM by WHZ and MAM by combined indicators. Furthermore, suffering from fever was associated with MAM by WHZ.These findings on the significant predictors of malnutrition are aligned with results from Link NCAs’ qualitative surveys on the communities’ perceptions of risk factors and underlying mechanisms of malnutrition. In Madagascar and Haiti , 41, parMoreover, the significant association between sex of the child  and GAM/MAM by WHZ demonstrated by this analysis is consistent with several studies that have shown that males have a higher risk than females to be affected by GAM or/and MAM by WHZ . A systeA significant association was found between the sex of the child and SAM by MUAC with a higher risk of malnutrition in females than males in the univariate model only. However, our sample contained a small number of SAM cases, therefore results should be interpreted cautiously.et al. (2017), 80% of the studies showed an association between a recent episode of diarrhea and malnutrition [Results of this study suggested that suffering from diarrhea (in the 14 past days before the survey) was significantly associated with GAM and MAM for the three indicators. This is consistent with the literature, where the association between recent episode of diarrhea and malnutrition is found in several studies , 43, 44.utrition . Diarrheutrition  with a butrition . Lack ofutrition –49. It iutrition , 51. ThiUnexpectedly, a significant negative association was found between water source and GAM/MAM by WHZ and by combined indicators indicating that unprotected source water decreases the risk of GAM and MAM by WHZ and by combined indicators. This protective effect opposes several studies that have demonstrated that an unprotected water source increases the risk of malnutrition , 53. AccMost risk factors found to be associated with malnutrition in this study are related to the Water, Sanitation and Hygiene (WASH). It has been shown that exposure to inadequate WASH may result in a range of poor health outcomes including malnutrition . SeveralWith reference to the UNICEF framework, we were able to identify a factor considered to be an immediate cause of malnutrition, namely diarrhea (disease), and another one considered to be an underlying cause of malnutrition namely unwashed face and hands (inadequate care practice). We have also identified a basic cause of malnutrition, namely sex .The inclusion of data obtained via two-stage cluster sampling from three countries with different socio-demographic, geographic, climatic and cultural characteristics increases the robustness and external validity of findings from this study. To our knowledge, only a few studies have evaluated the risk factors of GAM, MAM and SAM with different anthropometric indicators in the same population and none across various countries. One strength of this study was the ability to compare risk factors for malnutrition by different indicators in both univariate and multivariate models. This study demonstrated, for example, that in a univariate model, the intake of vitamin A and deworming were significantly associated with GAM by MUAC and not by WHZ.Limitations of this study include the limitations inherent to cross-sectional studies. First, the lack of longitudinal follow-up of individuals can reduce the reliability of the results. Thus, it was not possible to identify a causal relationship between the tested factors and undernutrition. Second, potential misclassification may have occurred during the collection of retrospective information, namely regarding the occurrence of diarrhea and fever episodes that are measures of disease prone to recall biais . Third, This study has identified some risk factors for malnutrition generally shown in the literature. The results taking into account three samples from different regions of the world suggests that whatever the context, these variables contribute to malnutrition. However, several risk factors were not considered because the selection of potential risk factors was based on contextual and country-specific assumptions.This study aims to support decision making in humanitarian situations where anthropometric measurements are based on the WHO thresholds that define malnutrition as a medical condition. Therefore, anthropometric variables were studied as categorized variables. Additional analyses exploring both WHZ and MUAC as continuous variables through linear mixed models did not change our main findings. Indeed, the choice of the best model (the mixed model with cluster and country as random effect) remained the same. Regarding the risk factors, diarrhea remained the only one significantly associated with a decrease in both WHZ and MUAC. For the model with MUAC as outcome, the absence of deworming was significantly associated with a decrease of MUAC—an association that was not significant when considering MUAC as a categorized variable.This study should be considered as a pilot, and further research is needed to consolidate the results for a better orientation for field interventions against malnutrition. For future research, it will be interesting to include data from other cross-sectional surveys from different contexts and performed in the same area but at different time of the year. Furthermore, it will be important to consider the collection of a larger number of variables in each country and an expected prevalence of SAM in the calculation of sample size in order to be able to conclude on the risk factors associated with SAM. Those pooled results can be completed and improved by Link NCA data from other countries of intervention. These findings could help to better orient humanitarian interventions in the field.The comparison of various anthropometric indicators enabled to draw attention to differences in malnutrition prevalence by different indicators (WHZ and/or MUAC) and associated risk factors. Based on these results, we can assume that improved and accessible WASH services preventing infectious diseases related to diarrhea might help reduce the risk of malnutrition regardless of the context of intervention. Additionally, interventions to promote and facilitate optimal and gender-neutral childcare practices to maintain protective hygiene levels might also help reduce the risk of malnutrition. Furthermore, comprehensive and multisectoral programming in areas with high levels of malnutrition including sensitisation to appropriate water management and hygiene practices could contribute to addressing the multicausal pathways leading to poor child health in general and undernutrition in particular. It is important to continue to collect and analyse the different indicators of malnutrition in other settings to obtain an overall and more accurate overview of the nutritional situation and the related factors, and to propose targeted and comprehensive actions.This study included a large sample of children aged 6–59 months from three different countries in order to identify the factors associated with acute malnutrition by three indicators . The factors associated with GAM and MAM found in this study have a high level of evidence allowing for a better representation of universal factors associated with acute malnutrition in this segment of the population . This study also provided an understanding of the variation in the prevalence of GAM, MAM and SAM and the associated factors involved.These findings deepen the knowledge of the factors associated with undernutrition in children to better guide the humanitarian interventions. This may ultimately improve the relevance of the upcoming Link NCAs and the effectiveness of multi-sector humanitarian assistance delivered by ACF and its partners.S1 Appendix(PDF)Click here for additional data file.S1 FigWHZ: Weight for Height Z-score; age in months.(TIF)Click here for additional data file.S2 FigMUAC: Mid-upper Arm Circumference; age in months.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table* Recoding as “1” corresponds to unprotected main drinking water source and “0” corresponds to protected main drinking water source.(DOCX)Click here for additional data file.S4 Table***p-value < 0.001; **p-value < 0.01; *p-value < 0.05; ●p-value < 0.20; Ref: the reference class for a qualitative variable; OR: Odds ratio; CI 95%: confidence interval at 5% alpha risk; WHZ: Weight for Height Z-score; MUAC: Mid-upper Arm Circumference; GAM: Global Acute Malnutrition; MAM: Moderate Acute Malnutrition; SAM: Severe Acute Malnutrition; cGAM: combined Global Acute Malnutrition; cMAM: combined Moderate Acute Malnutrition; cSAM: combined Severe Acute Malnutrition.(DOCX)Click here for additional data file.S5 TableFor each indicator, several random effect mixed models were tested and compared to find the best model according to the AIC criterion. Different random-effect combinations were tested: country alone, cluster alone, cluster and country, and cluster nested in country. For Global Acute Malnutrition and Moderate Acute Malnutrition , the best model was the one with cluster nested in country. For Severe Acute Malnutrition, the best model was the one with cluster alone. Cluster: the smallest geographic units in each country. WHZ: Weight for Height Z-score; MUAC: Mid-upper Arm Circumference. cGAM: combined Global Acute Malnutrition; cMAM: combined Moderate Acute Malnutrition; cSAM: combined Severe Acute Malnutrition. Std. Dev.: Standard deviation. * Conditional modes of the random effects.(DOCX)Click here for additional data file.


Visual representation learning is ubiquitous in various real-world applications, including visual comprehension, video understanding, multi-modal analysis, human-computer interaction, and urban computing. Due to the emergence of huge amounts of multimodal heterogeneous spatial/temporal/spatial-temporal data in the big data era, the lack of interpretability, robustness, and out-of-distribution generalization are becoming the challenges of the existing visual models. The majority of the existing methods tend to fit the original data/variable distributions and ignore the essential causal relations behind the multi-modal knowledge, which lacks unified guidance and analysis about why modern visual representation learning methods easily collapse into data bias and have limited generalization and cognitive abilities. Inspired by the strong inference ability of human-level agents, recent years have therefore witnessed great effort in developing causal reasoning paradigms to realize robust representation and model learning with good cognitive ability. In this paper, we conduct a comprehensive review of existing causal reasoning methods for visual representation learning, covering fundamental theories, models, and datasets. The limitations of current methods and datasets are also discussed. Moreover, we propose some prospective challenges, opportunities, and future research directions for benchmarking causal reasoning algorithms in visual representation learning. This paper aims to provide a comprehensive overview of this emerging field, attract attention, encourage discussions, bring to the forefront the urgency of developing novel causal reasoning methods, publicly available benchmarks, and consensus-building standards for reliable visual representation learning and related real-world applications more efficiently.


The primitive trigeminal artery (PTA), an abnormal carotid-basilar anastomosis, forms the vascular anomaly connection between the internal carotid artery and vertebrobasilar system. Rarely, PTA can be complicated by several other cerebrovascular disease, including arteriovenous malformations (AVMs), intracranial aneurysms, moyamoya disease, and carotid-cavernous malformations. Herein, we reported a rare case of PTA combined with an AVM in a male patient. The patient was a 28-year-old male with epileptic seizures at the onset of symptoms. Magnetic resonance imaging showed abnormal signal foci and localized softening foci formation with gliosis in the right parietal temporal lobe. Furthermore, using a digital subtraction angiogram (DSA), it was found that an abnormal carotid-basilar anastomosis had developed through a PTA originating from the cavernous portion of the right internal carotid artery (ICA) and a large AVM on the surface of the right carotid artery. The lesion of AVM tightly developed and draining into superior sagittal sinus. A hybrid operating room was used for the surgery. The main feeding arteries of the AVM originating from three major arteries, including the right middle cerebral artery, the right anterior cerebral artery, and the right posterior cerebral artery, were clipped and subsequently, then the AVM was thoroughly removed. The intraoperative DSA showed that the AVM had been resected completely. Postoperative pathological examination of the resected specimen indicated the presence of an AVM. The patient recovered well after surgery and has been symptom-free for more than 3 months. In summary, the pathogenesis of the coexistence of PTA and AVM remains unknown. As highlighted in this case report, hybrid surgery can be used to remove AVMs and can improve the patients' prognosis. To our best knowledge, this is the first case in the literature of successful AVM treatment using hybrid surgery. The primitive trigeminal artery (PTA), also known as the persistent trigeminal artery is a relatively rare vascular anomaly characterized by the embryonic arteries that connect the internal carotid artery (ICA) to the vertebrobasilar system . To datePTA is associated with a higher incidence of AVMs (approximately 4.5%) . GeneralA 28-year-old male presented with sudden cerebral hemorrhage 5 years ago and underwent decompressive hemicraniectomy with hematoma evacuation. The patient recovered into a good condition after surgery. The patient experienced grand mal epileptic seizures at 5 years after surgery. The patient was treated with antiseizure medication and showed poor response. Following the onset of symptoms, the patient was admitted to our hospital. The right limbs moved freely, while the left limbs were reflexive (muscle strength grade IV). The positive Babinski sign was presented in the left lower limb. Magnetic resonance imaging showed abnormal signal foci and localized softening foci formation with gliosis in the right parietotemporal lobe . Magnetivia endovascular and intravascular embolization using a biplane flat-panel angiographic suite  and 3D reconstruction under general anesthesia. After the incision of the skin, a 6-F guiding catheter  was placed in the right femoral artery by a percutaneous puncture. The presurgical embolization procedure involved identifying the main supplying artery of the AVM, guiding superselective cannulation into the feeding artery with a microcatheter system, embolizing the vessels with an ethylene-vinyl copolymer , and confirming continued injection until the feeding artery stasis (Headaches and dizziness restricted the patient's daily activities; therefore, he underwent hybrid surgery to completely resect the AVM. AVM resection was scheduled to be performed in a hybrid operating room. AVM resection and preoperative embolization were performed in a single stage. Emboli were performed y stasis . Intraopy stasis . Subsequy stasis . The dury stasis . The lesy stasis . The finy stasis . The paty stasis . The paty stasis .The PTA ranging from 0.1% to 0.6%, is an embryological anastomosis between the cavernous sinus segment of the ICA and the basilar artery and is the most common anomalous traffic between the ICA system and the vertebrobasilar system . Other aPTA was first proposed by Saltzman in 1958 and is divided into two main types . In SaltAssociations between PTA and cerebrovascular diseases, especially with vertebrobasilar embolic ischemia, and with vascular nerve compression syndrome, have been reviewed –25. ThesTo our knowledge, only eight reported cases of AVM have occurred in association with PTA  12–14, , 26–29. Recently, multimodality treatment, especially hybrid surgery, has received increasing attention as an effective treatment for intracranial AVMs , 31. PreIn summary, coexistence of AVM and PTA was successfully treated with hybrid surgery. A one-stop hybrid operation combining embolization and microsurgical resection could be performed as a safe and effective intervention strategy for AVMs. More follow-up data is required to determine the long-term effects of surgery.


Springbank Ward, in the CPFT NHS trust, is a specialist unit for patients with a diagnosis of emotionally unstable personality disorder (EUPD). Psychiatric wards often use restrictive practices to try and minimise suicide risk. Using risk assessment checklists to decide whether to grant leave is one example. Research shows that it is not possible to predict suicide or self-harm risk at an individual level, regardless of the assessment method used, so we questioned the utility of such an approach. A previous evaluation of our leave protocol showed that patients and staff would favour a less restrictive and more personalised approach. We introduced a new protocol that eliminated use of checklists, replacing them with an optional 1:1 conversation with staff before leaving the ward. Our aim in this service evaluation was to determine whether there was any significant change in rates of incidents on the ward and during leave as a result of this new, less restrictive leave protocol.Data were obtained from the records of incidents on Springbank ward from March 2019 to March 2021. These incidents were recorded by members of staff on the ward, and ranked according to the severity of harm that resulted from these incidents. The rankings from least severe to most severe recorded during the study were ‘No harm’, ‘Low ’, and ‘Moderate (Short term harm)’. The number of incidents which occurred for the year before and the year after the policy change were compared. The comparison compared both the total amount of incidents and the sub-types of incidents.In the 365 days following the change in protocol, there was a 15.5% decrease in total incidents and a 51.0% decrease in incidents occurring off the ward compared to the 365 days before the change in protocol. Notably there was a 61% decrease in total (both on and off the ward) Moderate (Short term harm) incidents, the most harmful type of incident recorded, following the change in protocol.The decrease in incidents following the change in protocol suggests that replacing the use of a formal risk assessment checklist with a holistic alternative improves patient safety.


Brugia malayi muscle cells producing spastic paralysis. DEC also has stimulatory effects on ascarid nematode parasites. Using PCR techniques, we detected, in Ascaris suum intestine, message for: Asu-trp-2, Asu-gon-2, Asu-ced-11, Asu-ocr-1, Asu-osm-9 and Asu-trpa-1. Comparison of amino-acid sequences of the TRP channels of B. malayi, and A. suum revealed noteworthy similarity, suggesting that the intestine of Ascaris will also be sensitive to DEC. We used Fluo-3AM as a Ca2+ indicator and observed characteristic unsteady time-dependent increases in the Ca2+ signal in the intestine in response to DEC. Application of La3+ and the TRP channel inhibitors, 2-APB or SKF 96365, inhibited DEC mediated increases in intracellular Ca2+. These observations are important because they emphasize that the nematode intestine, in addition to muscle, is a site of action of DEC as well as other anthelmintics. DEC may also enhance the Ca2+ toxicity effects of other anthelmintics acting on the intestine or, increase the effects of other anthelmintics that are metabolized and excreted by the nematode intestine.The nematode parasite intestine absorbs nutrients, is involved in innate immunity, can metabolize xenobiotics and as we show here, is also a site of action of the anthelmintic, diethylcarbamazine. Diethylcarbamazine (DEC) is used to treat lymphatic filariasis and activates TRP-2, GON-2 & CED-11 TRP channels in  Ascaris, Trichuris, and hookworm, are a primary medical and public health concern in many developing countries. It is estimated that approximately 807 million to 1.2 billion people are infected with Ascaris lumbricoides1. Although not usually fatal, parasitic infections have a detrimental effect on morbidity, reducing worker productivity by 6.3 million Disability Adjusted Life Years (DALYs) per year. Parasitic infections affect worker health, output, and the performance of children in school. In livestock, infestations lead to reduced food yields, impacting economic returns and exacerbating poverty2.Soil-transmitted helminths (STHs), including 3. With the limited number of anthelmintic drugs and the risk of resistance development, identifying the modes of action of other existing anthelmintics and predicting rational anthelmintic combinations, which are more potent and effective, is anticipated to counter the resistance.Currently, there are no effective vaccines against STH infections, and treatment relies on the use of chemotherapeutics, usually using one of the three major classes of anthelmintics: benzimidazoles , macrocyclic lactones (ivermectin), or nicotinic cholinergics (levamisole and pyrantel). There is a risk of resistance developing in humans associated with regular use of these compounds, as seen in animal parasitesBrugia malayi and Wucheria bancrofti5, but DEC has also been used to treat STH infections, including ascariasis6. Although DEC has been used for over seventy years, its mode of action is poorly understood. DEC's recently discovered actions are to open nematode muscle Transient Receptor Potential (TRP) channels, including the TRPMs, GON-2 & CED-11, and the TRPC, TRP-27. The opening of muscle cell TRPs allows entry of Ca2+ and transient spastic paralysis. TRP channels in nematodes are not only found in muscle cells, but they are also found distributed throughout the different tissues of nematodes including the intestine9. This suggests that DEC will also affect the cells of the intestine and impair its vital functions.The classic drug diethylcarbamazine (DEC) is used to treat lymphatic filariasis caused by filarial parasites including 14. It is involved in defense against environmental toxins and microbial infection by mediating innate immunity responses and the release of antimicrobial peptides16. The intestine is also a major site of metabolism of xenobiotic drugs by cytochrome P450 oxidative enzymes17: it excretes xenobiotic compounds and their metabolites (including anthelmintic drugs) via its numerous ABC transporters and organic anion transporters18. Toxic effects on the nematode intestine will produce a cascade of damage on other tissues including the neuromuscular and reproductive tissues of the parasite.The nematode intestine is a long tube of single-layer columnar epithelial cells that connects the base of the pharynx to the anus. The intestine is vital for nematode survival. It is essential for digestion and the transport of ion solutes and nutrientsAscaris suum19. Levamisole, a cholinergic anthelmintic, targets nicotinic ion-channel receptors that are present in muscle20 and intestinal cells of A. suum21. Another potential anthelmintic acting on the intestine is Cry5B, a Cry protein recovered from Bacillus thuringiensis, which forms pores in the intestine membranes26. Cry5B has potent toxic effects on a range of parasitic nematodes, including hookworm, Strongyloides and A. suum28.The intestine of parasitic nematodes is also one of the sites of action of anthelmintic drugs. The benzimidazoles, like mebendazole, disrupt the microtubules and block the transport of secretory granules and movement of subcellular organelles in the intestine of Ascaris suum, which is genetically the same species as the human worm, Ascaris lumbricoides30. We used Ca2+ imaging to record simultaneously from groups of intestine cells in isolated open intestine flap preparations. This allowed us to separate and characterize the effects of anthelmintics on the intestine from the underlying muscle and hypodermis of the worm. We identified the message of different types of TRP channels in the intestine to compare it with the underlying muscle bags of the parasite and found different levels of tissue expression. The Ca2+ imaging also showed us that DEC produces an increase in intracellular Ca2+ in the intestine by opening TRP channels that allow entry of extracellular Ca2+ but DEC did not mediate an intracellular release of Ca2+. The rise in the cytoplasmic Ca2+ will be toxic and limit normal functions, including metabolism and excretion of xenobiotic drugs. By this mechanism, DEC is predicted to synergize with and enhance the effects of other anthelmintics.Here we report the effects of DEC on the intestine of the pig worm, Brugia malayi, DEC stimulates spastic muscle paralysis by opening muscle TRP channels of the TRPM group (Bma-GON-2 and Bma-CED-11) and the TRPC group (Bma-TRP-2)7. We blasted these genes against the A. suum (ASU) database (Wormbase) and found Asu-gon-2, Asu-ced-11, Asu-trp-2. We also found the TRPV genes, Asu-ocr-1 & Asu-osm-9, and the TRPA channel gene, Asu-trpa-1. We compared the sequences of these six TRP A. suum genes: Asu-gon-2, Asu-ced-11, Asu-trp-2, Asu-ocr-1, Asu-osm-9, and Asu-trpa-1 with the sequences of the same genes in: (1) the Clade III nematode parasites, Parascaris equorum (PEQ) and Brugia malayi (BMA); (2) the Clade V nematode, C. elegans (CEL); (3) the Clade I nematode parasite, Trichuris muris (TMU); (4) the schistosome, Schistosoma mansoni (SMA) and; (5) the fly, Drosophila melanogaster (DME). Accession number links to all sequences used can be found in Supplementary TableS3. The dendrogram, Fig. A. suum and B. malayi are more closely related than to the TRP genes of C. elegans, S. mansoni, and D. melanogaster. The close relationship of the TRP channels of A. suum and B. malayi encouraged the hypothesis that TRP channels of A. suum and B. malayi have a similar pharmacology and sensitivity to DEC.In the filarial nematode, Ascaris, we generated screening primers targeting the coding region of these six genes  that are associated with mediating DEC effects on the muscle of the filarial parasite, Brugia malayi, are similar in sequence and present in the intestine of A. suum.To determine if these TRP channel genes are expressed in the intestine and muscle bags of Asu-gapdh. We compared the TRP channel expression levels to paired muscle bags from the same Ascaris parasite.Variations in band intensity for the different TRP channels between the intestine and muscle bag regions suggested differences in the relative mRNA expression levels for each channel. We used qPCR to measure the mRNA expression levels for each of the six TRP channels in the intestine relative to the reference gene, Asu-gon-2 had a higher expression level in the intestine compared to the muscle bags . Increasing the Ca2+ concentration in the APF bathing solution from 1 to 10 mM provides a valuable control test of the physiological condition of the intestine preparation. In fresh preparations, it produced a rapid and smooth increase in the Fluo-3 fluorescence starting within 20 s of application. This Ca2+ induced rise in cytosolic calcium declined immediately after the bath CaCl2 concentration was returned to 1 mM. The time for the CaCl2 signal to reach the peak was relatively fast, taking a mean of 3 min, Fig. 2+ signal when the sample was constantly perfused long term with 1 mM CaCl2 APF; there was only small spontaneous fluctuations that had an average amplitude of 0.6% , causing spastic paralysis7. We anticipated that DEC would also open TRP channels in the intestines of Ascaris. We bathed the intestines in 10 µM DEC for 5 min and observed that DEC produced a slow but uneven ‘sometimes step-like’ Ca2+ signal in the intestine as opposed to the smoother rise caused by the CaCl2 and was initiated within 30 s of the DEC application , Fig. Previous work on the muscle cells of the filarial nematode, ion Fig. B. The inAscaris body flaps (intestine and underlying muscle cells) from the same worms to 10 µM DEC for 5 min and then washed them in 1 mM CaCl2 APF for 10 min (average time to peak) or 60 min (average length of a recording). We then compared the enterocytes of the sections taken: before DEC treatment; 10 min after 5 min exposure to 10 µM DEC and; after the 60 min wash. We observed no evidence of DEC causing death or damage to the cell body of the enterocyte or their brush borders  preventing movement of calcium through the plasma membrane into the enterocytes36. We tested the effects of 100 µM La3+ on our DEC responses. Figure 3+ blocks the DEC increase in Ca2+ in normal extracellular Ca2+ APF. Interestingly, in low extracellular calcium (no La3+), DEC produces a reduction in the cytoplasmic calcium rather than an increase, due to leaching of intracellular calcium from the cell, Fig. 3+, Fig. 2+ permeable plasma membrane channels in the enterocytes that are blocked by La3+ without producing release of intracellular Ca2+. Figure 2+ signal produced in the different experiments, n = 5 preparations for each experiment. The entry of Ca2+ to, and loss of Ca2+ from, the cytosol through the plasma membrane channels that is inhibited by a low-concentrations of La3+, is consistent with DEC opening TRP channels. The effects of La3+ were readily washed off and control responses to our test 10 mM CaCl2 then returned (not shown).The TRP channels, GON-2/GTL-1 in the 3+ is recognized for blocking TRP channels of the nematode intestine, it is possible that other plasma membrane channels permeable to calcium could be a target of DEC. The plasma membrane channels may include the voltage-activated calcium Cav channels, ICRAC and other calcium entry pathways that are also blocked by La3+. We used 2-APB and SKF 96365 to explore the pharmacology of the DEC calcium signaling response and the role of TRP channels in the response.Although LaCRAC channels40. We tested the effects of 10 µM 2-APB on DEC responses, Fig. 2+ signal  which at low concentrations (10 µM) also has stimulatory effects of Store Operated Calcium entry (SOC) mediated by I2 after 2-APB and DEC applications to ensure that the preparation was still responding normally and saw the usual ~ 30% increase in the fluorescent signal  concentrations can stimulate SOC extracellular Ca2+ entry pathways mediated by ICRAC (Orai) channels40. The inhibitory effects of La3+ and 2-APB together can be explained by the effects of DEC opening TRP channels in the plasma membrane to promote Ca2+ entry into the intestinal cells via TRP channels.We checked the response to 10 mM CaClCRAC SOCs40 and that La3+ blocks calcium entry pathways including: TRPs and voltage-activated calcium Cav channels37. We wanted to determine if our observed DEC signal included a role for a more specific TRP channel subtype. We exposed intestinal tissues to the TRPC selective antagonist SKF 9636541. We have previously demonstrated that 10 µM SKF 96365 is able to inhibit DEC mediated inward currents in Brugia muscles7. We exposed intestinal tissues to 10 µM SKF 96365 for 5 min and observed no changes in the Ca2+ amplitude  and, K+ channels42. However, of all antagonists we tested, there is one target in common, the TRP channels. Our results here indicate that DEC gives rise to the entry of calcium into the intestine by opening plasma membrane TRP channels as we have observed in Brugia muscle. The continued increase in Ca2+ fluorescence after removal of DEC may relate to a slow wash-off effect of DEC and downstream secondary Ca2+ induced Ca2+ release from intracellular stores35.We have pointed out that the broad-spectrum TRP inhibitor, 2-APB, may have off target effects including opening of IB. malayi produces a temporary spastic paralysis that lasts for less than 4 h that is followed by desensitization and recovery7. We were interested in determining if there was accommodation to DEC in A. suum mediated by reducing TRP channel message during a 4-h exposure. We treated isolated intestine flaps and muscle flaps with 10 and 100 µM DEC for 4 h. Again, we used qPCR to measure relative mRNA levels of the six TRP channels in the treated flaps and compared them to untreated controls that were bathed in RPMI. We observed no significant changes in message levels for any TRP channels in either the intestines or muscle cells samples treated with either 10 µM or 100 µM DEC when using a one-way ANOVA with a post-hoc Tukey test . These observations show that the level of TRP channel transcript expression in the intestines and muscle cells of A. suum is not sensitive to DEC over this timescale.Maintained applications of diethylcarbamazine to adult 12; (2) pH regulation via apical membranes V-type ATPases18; (3) storage of lipids43; (4) innate immunity44; (5) secretion of bactericidal peptides15; (6) drug metabolism with P450 cytochromes and UDP transferases17 and; (7) excretion by organic-anion-transporter (OAT-1), P-glycoprotein- and MDR-transporters into the lumen of the intestine45. Damage to the cells of the intestine will adversely affect these vital functions.The nematode intestine carries out essential functions for survival, including: (1) digestion and nutrient absorption46. The processes of absorption of nutrients and secretion of proteases also require significant endocytosis and exocytosis utilizing different types of vacuoles and vesicles that are present in the intestinal cells; these vesicles are moved within the cell on an elaborate network of microtubules that includes a terminal web under the apical microvilli11. Absorption of Ca2+ and Mg2+ is mediated by TRPM-like ion-channels (GON-2)36.The intestine cells are columnar cells, electrically coupled to their neighbors, and polarized with the apical region bearing microvilli and the basolateral region facing the somatic body muscle cells. The digestion of food involves the secretion of proteases and lipases and the absorption of nutrients including glucose and lipids. It is facilitated by a range of selective transporters47. These anthelmintics now include:Diethylcarbamazine (DEC): The disturbance in the Ca2+ homeostasis of the intestine by an action of DEC on TRP channels can disrupt vital functions of the enterocytes and will contribute to the anthelmintic action of DEC against ascariasis. DEC activates TRP-2, GON-2 & CED-11 channels that produce an inward sarcolemma current in Brugia malayi muscle cells where 2-APB and SKF 96365 are also effective as DEC antagonists7. The presence of TRP channel message in Ascaris intestine that is similar to that of B. malayi, Fig. A. suum intestine. The actions of La3+ and 2-APB taken together on the intestine inhibiting the actions of DEC are explained by DEC also opening TRP channels in the intestine of A. suum. The effect of SKF 96365 also supports a role for TRP-2 channels.Ascaris intestine mediated by activating TRP-2, and GON-2 TRP channels leading to the entry of Ca2+ into the intestinal cells. We have included here STIM-1 and ORAI-1  binding selectively to the CDH-8 cadherin present on the surface of the cell membrane of C. elegans intestines, leading to oligomerization of the toxin and possible internalization25 and; (2) interaction with glycolipids produced by BRE-5, a glycosyltransferase that catalyzes the addition of monosaccharides onto glycolipids, leading to Cry5B insertion into the membrane forming the pore23. Interestingly, C. elegans that are resistant to the anthelmintic levamisole, show increased hyper-susceptibility to Cry5B and vice versa with Cry5B resistant strains showing increased sensitivity to levamisole, resulting in a synergistic relationship between two anthelmintics23. Here, we have observed that DEC has a direct effect on the intestine, in addition to the known effects of levamisole21. In addition to levamisole, the two other cholinergic agonist anthelmintics, pyrantel and tribendimidine also have strong synergistic effects with Cry5B in C. elegans23 that is also seen in the hookworm, Ancylostoma ceylanicum24. The observations that we describe here suggest a mechanism by which DEC will add to, and potentiate, the effects of albendazole, levamisole, ivermectin, and Cry5B in combinations treatments for STH infections.The The intestine of parasitic nematodes is increasingly being recognized as a major site of action of a number of anthelmintic drugs in addition to the neuromuscular system2+ homeostasis and vital functions and suggest that combinations of these anthelmintics will have additive or synergistic effects. Adverse effects on the intestine will potentiate the effects of other anthelmintics that are metabolized or excreted by the intestine. One example is the macrocyclic lactone, ivermectin, which acts as positive allosteric modulators of the Glutamate-gated Chloride channel. P-glycoproteins have been suggested as candidate genes for ivermectin resistance by acting as efflux pumps. The P-glycoprotein gene pgp-9 has been repeatedly associated with macrocyclic lactone resistance in different nematode parasites52. Although P-glycoproteins are found in other nematode tissues, they are most strongly expressed in the intestine of the parasites53. The combination of albendazole with ivermectin or diethylcarbamazine is anticipated to enhance the efficacy of the treatment of STHs compared to albendazole alone, ivermectin alone, or diethylcarbamazine alone. This has, in fact, been seen in the study of the effects of albendazole, ivermectin, and diethylcarbamazine alone and combinations against ascariasis and trichuriasis54. The parasite intestine offers a common target where anthelmintics can have synergistic effects.We have pointed out above that the nematode parasite intestine is a site of action of DEC, the benzimidazoles, cholinergic anthelmintics like levamisole, and Cry5B. These anthelmintic drugs disrupt the normal CaA. suum worms were collected from the JBS Swift and Co. pork processing plant at Marshalltown, Iowa. Worms were maintained in Ascaris Ringers Solution  at 32 °C for 24 h to allow for acclimatization before use in experiments. The solution was changed daily, and worms were used for a maximum of three days for experiments. All the worms were examined at the start of each day and excluded if they were damaged or immotile.Adult female A. suum females as previously described, McHugh et al., 2020. Subsequently, muscle bags and intestinal tissue were homogenized separately in 1 ml of Trizol reagent using a mortar and pestle, followed by total RNA extraction according to the Trizol Reagent protocol . One microgram (1 µg) of total RNA from each tissue was used to generate cDNA by reverse transcription (RT) using SuperScript IV VILO™ Master Mix  following the manufacturer's protocol. PCR was conducted to detect the presence of Asu-gon-2, Asu-trp-2, Asu-ced-11, Asu-ocr-1, Asu-osm-9, and Asu-trpa-1 using primers that were designed for targeting encoding regions of each gene . The PCR products of each gene were then separated on an individual 1% Agarose containing SYBR® Safe DNA Gel Stain (ThermoFisher Scientific), for 45 min at 100 V, followed by visualization under UV light to confirm the presence of the genes. All photographs were acquired using Visionworks™ software  with an exposure setting of 3 s per 1 frame. Original gel pictures are presented in Supplementary Fig. Dissection of muscle bags and intestinal tissue was conducted on adult A. suum as described above. Target genes with fragments ranging from 150 to 200 bp were amplified by qPCR from each cDNA sample in triplicate. The same procedure was used for the reference gene Asu-gapdh. All primers for qPCR are presented in Supplementary Table Asu-gon-2, Asu-trp-2, Asu-ced-11, Asu-ocr-1, Asu-osm-9, and Asu-trpa-1) were estimated relative to the reference gene (Asu-gapdh) using the Pfaffl Method. The qPCR experiments were repeated 3 times for each gene and for each DEC treatment .To quantify the relative mRNA transcript levels of each identified TRP channel in the intestine and muscle bags, cDNA was synthesized from 1 µg of RNA from each adult female http://mafft.cbrc.jp/alignment/software/)55. Amino-acid similarity and identity was analyzed using MAFFT alignments. Percent identity was the percent ratio of identical amino-acids and the total number of residues in the longest sequence. Similarly, percent similarity was determined by dividing the number of identical and similar amino-acids by the total number of residues in the longest sequence (× 100). Phylogenetic analysis was done using the software MEGA X software56 using the maximum likelihood method based on Le and Gascuel model57.All protein sequences used for the construction of the dendrogram were acquired either from Wormbase Parasite, The European Nucleotide Archive (ENA) or UNIPROT repositories. Accession number links to sequences used for each gene of each organism can be found in Supplementary Table Ascaris Perienteric Fluid APF  in a laminar flow chamber . Fluo-3AM loading was achieved by incubating the intestine in APF solution with no added CaCl2 (< 100 µM Ca2+) containing 5 µM Fluo-3AM and 10% Pluronic F-127 (10% v/v) for one hour with the chamber connected to a Dual Automatic Temperature Controller  maintained at 34–36 °C. After incubation, the Fluo-3AM solution was discarded, and the sample was incubated in APF containing 1 mM CaCl2 for an additional 15 min at 34–36 °C to promote Ca2+ loading. Intestinal tissues were then continuously perfused with APF containing 1 mM CaCl2 and exposed to either 10 µM diethylcarbamazine (DEC), 10 µM 2-APB, 10 µM DEC and 2-APB, 10 µM SKF96365, 10 µM SKF96365 and DEC or 10 mM CaCl2, which was used as a positive control. For no added  Ca2+ experiments, 1 mM CaCl2 APF was exchanged for no added CaCl2 APF (measured < 100 µM Ca2+) after 10 min incubation and the samples were incubated for an additional 5 min before the beginning of each experiment at 34–36 °C. During these recordings, preparations were continually washed in solutions with no added CaCl2. For recordings with La3+, intestinal preparations were incubated in 1 mM CaCl2 APF containing 100 µM LaCl3 to inhibit TRP channels for 10 min. These intestine preparations were the incubated in no added CaCl2 and 100 µM LaCl3 for additional 5 min at 34–36 °C. For all experiments, samples were exposed to solutions containing no added CaCl2 and 100 µM LaCl3. All incubations were done in the absence of light to prevent degradation of the fluorescent dye.A 2 cm section of the intestine was removed from the body piece using fine forceps and cut open. The intestinal flap was placed onto a coverslip (24 × 50 mm) and pinned using a slice anchor , immersed in 2+ signaling before application of any compound.All solutions were delivered to the chamber under gravity feed through solenoid valves controlled using a VC-6 six-channel Valve Controller  through an inline heater set at 37 °C  at a rate of 1.5 mL/min. At the start of all experiments, intestinal preparations were left under blue light for a minimum of 3 min to promote settling and equilibration of the fluorescent signal and monitor for any spontaneous CaAll recordings were performed on an Eclipse TE3000 microscope (20X/0.45 Nikon PlanFluor objective), fitted with a Photometrics Retiga R1 Camera . Light control was achieved using a Lambda 10–2 two-filter wheel system with a shutter controller . Filter wheel one was set on a green filter between the microscope and camera. Filter wheel two was set on the blue filter between a Lambda LS Xenon bulb light box, which delivered light via a fiber optic cable to the microscope . Blue light emission was controlled by using a shutter. Minimal illumination exposure was used to prevent photobleaching.2+ signal recordings were acquired and analyzed using MetaFluor 7.10.2  with exposure settings at 150 ms with 2 × binning. Maximal percent Ca2+ signal amplitudes (ΔF) were calculated using the equation F1–F0/F0 × 100, where F1 is the fluorescent value and F0 is the baseline value. All F0 values were determined as being the value when any stimulus was applied to the sample for all traces analyzed.All CaDetails of numbers of regions and numbers of preparations used for measurements of the Ca2+signals.2 that included 800–1000 individual enterocytes. The average fluorescence amplitude was calculated for each intestinal exposure of all 50 regions. For the DEC experiments, intestines were exposed to 10 µM DEC for 5 min followed by 10 mM CaCl2 as the positive control. For the 2-APB experiments intestines were exposed to 10 µM 2-APB for 5 min followed by 10 mM CaCl2. For the study of the effects of 2-APB on the DEC responses, preparations were exposed to 10 µM 2-APB and 10 µM DEC at the same time for 5 min before 2-APB was removed and DEC was left for an additional 5 min. 10 mM CaCl2 was then used as a positive control. For SKF 96,365 experiments, 10 µM SKF96365 was applied for 5 min followed by applying of 10 µM SKF96365 + 10 µM DEC for 5 min. SKF96365 was removed, and the DEC was left on for an additional 5 min. 10 mM CaCl2 was then used as a positive control. Finally, for the no added Ca2+ and La3+ experiments 10 µM DEC was applied for 5 min before subjected to 10 mM CaCl2.Calcium signals from each intestine were collected from 50 square 50 µm × 50 µm areas across the intestine covering a total area of 125,000 µMAscaris body sections were cut along the lateral line and opened, exposing the intestine. Sections were then incubated in 1 mM CaCl2 APF containing 10 µM DEC for 5 min and then transferred to 1 mM CaCl2 APF for 10 min, average time for Ca2+ signal to reach peak  or 55 min, the average length of recording . Control samples were exposed to 1 mM CaCl2 APF for 3 min.Sections of Ascaris worms were preserved in 10% buffered neutral formalin. Preserved specimens were placed in histology cassettes for paraffin embedding and sectioning. Hematoxylin and eosin staining was performed on 5 µm slides as described previously58. Histological sections were reviewed by an ACVM board-certified parasitologist (Brewer). Sections were viewed and images were captured on an Olympus BX60 microscope fitted with an Olympus DP70 camera . Image captures were achieved using Olympus DP controller software  under 20 × magnification and exposure settings of 1/350 s per frame.Following treatment, the segments of adult 3+ are provided in the legends of the figures. Analysis of Ca2+ amplitudes and time to peak were done using either unpaired or paired student t-tests P < 0.05. For qPCR experiments one-way analysis of variance (ANOVA), with a post-hoc Tukey test P < 0.05 was used. The data are presented as relative expression levels in mean ± SEM for each TRP channel subunit relative to Asu-gapdh in muscle bags and intestinal tissue. Comparisons were made using Tukey's post hoc test.Statistical analysis of all data was done using GraphPad Prism 5.0 . To ensure reproducibility, we repeated our experiments: the numbers of female worms, intestine preparations, the concentrations, and durations of applications of 2-APB, DEC, LaSource of chemicals: 2-APB and SKF96365 were procured from Tocris; Sigma Aldrich supplied all other chemicals.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.