The persistence of serum-specific anti-chikungunya IgM antibodies (CHIKV-IgM) can vary after chikungunya fever (CHIK) infection. However, the factors related to its production are not yet known. We described a case series drawn up from data collected from 57 patients between 12 and 36 months after the acute phase of CHIK infection in Northeastern Brazil. CHIKV-IgM was detectable in 7/57 (12.3%) patients after 28.3 months of infection. No frequency differences in chronic musculoskeletal manifestations and underlying conditions were detected between patients with or without CHIKV-IgM. CHIKV-IgM was detected for up to 35 months in Brazilian patients after CHIK infection. Togaviridae family, transmitted by Aedes mosquitoes and causes chikungunya fever (CHIK),Chikungunya virus (CHIKV) is an alphavirus of the -It is known that in the viremia phase, CHIKV causes B-cell activation and early production of immunoglobulins, with the detection of serum-specific anti-chikungunya IgM antibodies (CHIKV-IgM) expected from the onset of infection until approximately three months after infection,,Although this antibody does not present an established pathogenic mechanism, it has been questioned whether there is a relationship between the serum persistence of this immunoglobulin and chronic joint symptoms in CHIKFrom December 2018 to July 2019, a case series was prepared from data collected from 57 patients diagnosed with CHIK, who were previously evaluated in a cross-sectional study, between 12 and 36 months after the acute phase of infection. These patients were followed-up in a specific outpatient clinic for people with chronic symptoms after CHIK infection at the Hospital das Clínicas at the Universidade Federal de Pernambuco, Northeastern Brazil. A diagnosis of CHIK was established based on clinical and epidemiological criteria and confirmed by the detection of serum CHIKV-IgM or IgG by enzyme-linked immunosorbent assay (ELISA). The study was approved by the local ethics committee, and consent forms were obtained from all patients before inclusion in the study. At the time of the evaluation, blood samples were collected and tested for CHIKV-IgM using a commercially available qualitative ELISA, following the manufacturer's instructions . All patients underwent clinical examinations by experienced rheumatologists. We compared the frequency of chronic musculoskeletal manifestations and comorbidities in the two groups of patients: CHIKV-IgM-positive and CHIKV-IgM-negative. Data were analyzed using SPSS software and Fisher’s exact test. The significance level was set at p<0.05.Between 12 and 36 months after CHIK acute infection, seven of 57 patients (12.3%) presented detectable CHIKV-IgM with a median time of 28.3 months . Six of seven patients (86%) who presented with positive CHIKV-IgM reported persistent musculoskeletal symptoms. All these patients displayed joint pain, and two experienced persistent arthritis. The descriptive characteristics of the patients with persistent detectable CHIKV-IgM are shown in Patients positive for CHIKV-IgM were compared to those without detectable antibodies with respect to the presence of chronic musculoskeletal manifestations and underlying previous disease. The descriptive characteristics of the patients are presented in CHIK was first observed in Brazil in the Northern and Northeastern regions. Northeastern Brazil was severely affected by the epidemic, and it was necessary to establish a specific outpatient clinic for CHIK at the Hospital das Clínicas at the Universidade Federal de Pernambuco due to the high frequency of chronic manifestations. This led us to assess whether the long-term progression of CHIK in Brazilian patients was different from that in patients from other affected countries. Herein, we present the serological and clinical data of patients with long-term detectable CHIKV-IgM followed in a specific outpatient clinic for people with chronic symptoms after CHIK in Northeastern Brazil.-According to initial studies, after CHIKV infection, there is a progressive reduction in the production of CHIKV-IgM, which was detected in 76.2% of patients between two and six months, 69.8% between seven and 12 months, and 55.5% between 13 and 18 months after the acute phaseIt is plausible to accept that the difference observed in the present study may only be related to the time elapsed since the acute phase of CHIK as our assessment occurred after a mean interval of 28 months, which is longer than the intervals in previous studies. Furthermore, a commercially available ELISA kit was used in the present study; this may have reduced the sensitivity for detecting such antibodies,,,,,Whether the persistence of CHIKV-IgM in the chronic phase of CHIK is related to a possible pathogenic role has not yet been fully established. Studies that only assessed individuals with persistent joint pain in the chronic phase of CHIK have demonstrated detectable CHIKV-IgM in around 63% after six months and in 11−19% after one year,In addition, some studies have controversially reported that the severity of CHIK, as well as the chronicity of symptoms, may be related to the presence of comorbidities, such as hypertension and diabetesIn summary, in contrast to most studies that have analyzed patients up to 12−18 months post-CHIKV infection, this case series detected CHIKV-IgM for up to 35 months in 12.3% of Brazilian patients. The presence of persistent joint symptoms and underlying conditions did not seem to be associated with the long-term production of CHIKV-IgM in this sample, although further investigations are required to validate these observations. This study has indicated that a positive serological test for CHIK-IgM alone should not define the diagnosis of a recent disease.


Introduction: Patients in the pediatric intensive care unit (PICU) are at risk of developing long-term morbidities following recovery from their critical illness. One such health outcome is called post-intensive care syndrome (PICS). PICS in pediatrics may be mitigated by interventions that facilitate adjustment to the PICU setting.Methods: The PICU implemented a two-pronged Peer and Volunteer (P/V) Program to help: (a) families adjust to the PICU experience with the support of a peer mentor (PM); and (b) patients receive non-medical interaction from trained volunteers (V). We designed a mixed-methods program evaluation targeting perspectives and feedback from PICU families and healthcare professionals (HCPs).Results: All stakeholder groups agreed that the PICU P/V Program was a valuable resource for PICU patients and their families. HCPs reported that they lack both time and training to provide regular developmental care to patients. However, the P/V Program may influence both families' and HCP's confidence in their ability to offer non-medical interaction to children in the PICU.Discussion: Important initial and on-going strengths and barriers to successful implementation were identified, including the need to clarify roles and intervention scope. The program evaluation served as a change management strategy and also helped to identify both areas for improvement and strategies for on-going sustainability. HCP's exposure to the program and modeling by PMs may have helped HCPs to feel that it is within their job description and capacity to provide emotional support and guidance to families. Post-intensive care syndrome (PICS) is the new or worsening impairment in various domains of health arising after critical illness and persisting beyond acute care hospitalization . This syIn pediatrics, the following factors affect a patient's baseline health and lasting impairment in the physical, cognitive, emotional and social domains: length and type of PICU admission, the number of medical procedures performed in the PICU, cardiac arrest and/or receipt of advanced life support, invasive mechanical ventilation, receipt of sedation and opioids, and family function and structure . The effDevelopmental care is a popular concept in the field of neonatal intensive care that is known to affect neonate's physical, cognitive, emotional and social domains of health. Developmental care encompasses an approach to individualize infant's care to maximize neurological development and reduce long-term cognitive and behavioral problems . There iPatient and Family Centered Care (PFCC) describes both a philosophy and practice in pediatric healthcare . The phivia volunteers (V), and (b) offer families guidance through their hospital experience via peer mentors (PM).The aim of this program evaluation was to facilitate PFCC at the Stollery Children's Hospital by implementing and evaluating a two-pronged service to assist with patient and family experience following admission to the PICU; a Peer Mentor and Volunteer (P/V) Program for patients and their families. Specifically, our goals were to: (a) offer patients age-appropriate, non-medical interaction We started this initiative by establishing a multi-disciplinary team which consisted of Child Life Specialists, the Stollery Volunteer Coordinator, PFCC Advisors, Social Workers, a PICU Clinical Nurse Educator, a Clinical Nurse Specialist, and a Physician to lead this initiative and to recruit and train Vs and PMs. Child Life Specialists are trained professionals in Canada who assist children and adolescents to cope with the impact of hospitalization, by assessing, planning, implementing, and evaluating mutually agreed upon goals. They are well positioned to engage with both patients and their families about social, psychological, and developmental-related needs while in hospital.We defined Vs as those trained to provide age-appropriate, positive and non-medical interaction to PICU patients, including cuddling, rocking, reading, playing, coloring, singing, crafting, or hand holding. To be eligible, Vs needed to be >18 years of age and have had inpatient Stollery V experience. This service was offered by Vs during the day to patients in the PICU. The V portion of the program was intended to support patients especially when their families were taking self-care breaks or were busy with other life demands such as work or family.via the Stollery Children Hospital's normal volunteer intake program. If they met the criteria as defined above, they were connected to the PICU P/V project team for further information and training. Training for both positions included on-line and in-person modules about the organization and hospital, patient, and family centered care goals; effective communication; confidentiality; professional boundaries; self-care; and how to facilitate appropriate referrals. The team also partnered with Stollery Social Workers to develop a new 2-h cultural sensitivity course, and Vs and PMs were encouraged to attend. PMs and Vs were required to have passed a security clearance check prior to training. Following training, Vs were supported by Child Life Specialists who helped to coordinate appropriate patients to engage with and direct the activity of that engagement. PFCC Advisors took an active role in supporting PMs, and helped to direct them to appropriate families. Families could also request Vs and PMs via PICU HCPs or directly via phone or email to the PFCC department.We defined PMs as those with a shared understanding of the PICU family experience from having previously had their child admitted into the PICU. PMs were positioned to provide peer support, normalization, validation, and guidance throughout current PICU families' hospital stays. PMs were available during the day and in the evenings. PMs and Vs were recruited While the primary outcome measures for this evaluation were related to patient and family experience and satisfaction, we recognized that change management with the PICU multidisciplinary team was an important factor for this program's success and sustainability. Thus, we designed a mixed-methods program evaluation with the help of our hospital's Department for Health Systems Evaluation and Evidence. We received ethics approval for this program evaluation from the University Of Alberta Research Ethics Board (Study ID Pro00089760). Data were collected through the course of the program's two phases of implementation in September to November, 2019 (pre-implementation) to January to March, 2020 (post-implementation). The evaluation commenced with a HCP focus group. Surveys were then distributed at pre- and post-program implementation. Surveys were developed for PICU HCPs and Family/Caregivers (henceforth “family/caregiver” is referred to as just “family”). All surveys and the focus group guide were developed collaboratively by the PICU and evaluation teams. All family data were collected prospectively while families were present in the PICU and their child was an admitted patient.A 1-hour focus group discussion took place prior to program implementation as a change management strategy to gather HCPs feedback and buy-in on: (a) what would enable the successful launch of the program, (b) their expectations of the program, and (c) the perceived benefits and challenges to implementing such a program. The evaluation team conducted the in-person focus group at the hospital with voluntary participants. The PICU team supported focus group recruitment by email until there was HCPs representation from each of the major stakeholder groups.The PICU HCPs Survey targeted PICU Nurses, Physicians, Child Life Specialists, Unit Clerks, Health Care Aids, Allied Health , and Patient and Family Centered Care. The evaluation team was responsible for both pre- and post-implementation survey distribution. All HCPs were invited to complete the pre-survey. The post-survey was also sent to all HCPs, though the questions were directed to those who had exposure to the PM or V Program.The V Survey targeted PICU families for their perspective on receiving support from a PICU V. The PM Survey targeted PICU families for their perspective on receiving support from a PICU PM. Both surveys were offered to a convenience sample of families in the PICU at program pre-implementation. Families were asked to participate in the pre-survey if their child was in the unit for at least 3 days so that they were familiar with the team, environment, and routine. Families were only offered surveys if their child was “stable,” meaning they were not actively decompensating or deteriorating or experiencing increased acuity. We also avoided families whose child was at end of life, or if it was a critical decision-making day. Surveys were offered when the PICU team was available to offer surveys . Families were asked to participate in the post-survey at the same time of day as pre-implementation, and only if they had exposure to the PM and/or V Program.SelectSurvey platform. The PICU team handed out paper surveys and iPads on the unit. Completed paper surveys were dropped in a locked survey box on the unit. Only the evaluation team had access to the locked survey box to retrieve and enter data from the paper-based surveys. The V and PM Surveys were distributed both by paper and online through the Analysis was conducted using Microsoft Excel to theme qualitative survey comments and to generate descriptive statistics and visualizations of quantitative data.Five HCPs voluntarily attended the scheduled focus group. Participants included nurses, occupational therapy, and Volunteer Coordinator who provided their perspective on expected program benefits, program communication, and program operations. Key themes that emerged about expected program benefits centered on facilitating the families' transition into the PICU environment and providing non-medical support, which was expected to help the families as well as HCPs who may see a decrease in non-medical or developmental care responsibilities.Participants suggested key communication requirements, like how to disseminate information and clarify roles and responsibilities. Participants identified poor communication as a potential barrier to program rollout, and shared suggestions for program operations including sharing communications through the management team. They also recommended resources for Vs, and opportunities for HCPs to build rapport with and provide feedback to Vs. Participants identified that it would be important for HCPs to keep an open mind about the Vs, and remember that they would be there to help ease HCPs workload. The focus group themes are summarized in n = 78), followed by allied health , and physicians . Of those respondents who identified their discipline as allied health, the majority were respiratory therapists . The disciplines of the remaining respondents included: unit clerks, social workers, health care aides, a PICU administrator, occupational therapists, and a pharmacist.A total of 120 PICU HCPs responded to the Volunteer and Peer Mentor pre-implementation survey  and that they were comfortable interacting with PICU patients in non-medical ways . However, HCPs responded that there were not enough resources available to provide developmental care to PICU patients, and only 38% (n = 45) reported providing routine developmental care . HCPs further elaborated that their ability to provide developmental care was dependent on their daily duties and how busy it was on the unit, and most frequently identified limited human resources , insufficient training , and no clear processes or clinical pathways for offering developmental care in PICU  as barriers  and guidance to families in the PICU  and were comfortable doing so , but far fewer HCPs agreed that they routinely shared their skills, experience, and knowledge of developmental care with PICU families . Moreover, few HCPs agreed that they had enough time or adequate training to provide emotional support to PICU families , while 34% (n = 41) reported no barriers. The barriers HCPs most frequently identified were limited human resources , insufficient training , and a lack of clear processes for offering emotional supports , and 84% (n = 21) of respondents further agreed that access to a trained V would be helpful for them and their family (n = 15). Families also agreed that play and interaction from a PICU V would make a difference in their child's recovery .Overall, families felt comfortable in the position of having a trained PICU V spend time with their child . Families reported exhaustion (n = 2) and fear of touching/dislodging tubes (n = 1) as barriers to interacting with their child. Four families did not reply to the survey item.Most families felt positive about their experience in the PICU; however, families felt least positive about the level of playing and developmental care their child received in the PICU (see n = 11). On average, seven families reported spending an average of 88 h per week with their child in PICU . Fourteen families skipped this question. Most families felt comfortable with their understanding of how to interact with their child and HCPs in the PICU but reported commonly experiencing negative feelings and emotions ; however, some families reported that their interest would depend on the mentor's personality. Families reported they would seek advice from a PM about the treatment experience, coping strategies, advocating for their involvement in the care team, and balancing life in the PICU (see n = 12). They perceived that health professionals would provide clinical expertise and a neutral understanding of clinical circumstances in the PICU, and that PMs would provide personal, open, and emotional support that would make them feel as if they were not alone. Families expressed that PMs may potentially have biases and different values, and they identified that they may lack familiarity with each case.When questioned about their perspectives on a potential PICU PM program, most families indicated they would welcome support from families who had similar PICU experiences and felt such support would be helpful . Just ovPICU see . Most faThe post-program evaluation surveys were distributed starting in January 2020. PICU team HCPs approached families who were known to have received either PM and/or V support in the PICU. As families are not present in the PICU all of the time, it was difficult to connect with families to obtain their feedback on the services they and their child received. Unfortunately, in March 2020, this program evaluations was forced to suspend due to the COVID-19 pandemic hospital restrictions on families and Vs allowed in the hospital. Additionally, at this time PICU HCPs were re-deployed to train in other areas of the hospital. Thus, the response rate of the HCPs and family P/V post-program evaluations were affected, and we received significantly smaller response rates. The following results should be interpreted with caution.Fifty-four HCPs responded to the post-implementation survey (response rate of 15% or 54/351 HCPs). Overall respondents were somewhat more experienced in health care but had similar PICU experience compared to pre-implementation survey respondents (see n = 53) and that they were comfortable interacting with PICU patients in non-medical ways ; however, HCPs generally did not agree that the PICU had enough physical, human, or time resources available to provide developmental care to PICU patients, and 26% (n = 14) reported routinely providing developmental care (n = 43) of HCPs agreed that there are barriers to providing developmental care in the PICU. As in the pre-implementation survey, HCPs most frequently identified limited human resources , no clear processes or clinical pathways for offering developmental care in PICU , and insufficient training , as barriers to providing developmental care  and felt that those interactions were valuable . When asked how likely they were to recommend the V Program to families on a scale of 1–10, where 10 is “extremely likely,” HCPs indicated a mean rating of 8.10, an increase from the pre-survey. HCPs commented that the V's value was in providing increased access to developmental care, especially from someone who does not also provide medical care. Further, HCPs noted that that the V Program provided value in supporting families and decreasing clinicians' workloads.HCPs were asked about interactions between Vs and patients and their families. A large majority of HCPs reported witnessing Vs interact with patients and their families on the unit  and guidance to families in the PICU  and were comfortable doing so , but few HCPs agreed that they routinely shared their knowledge of developmental care with PICU families .Most HCPs reported that they frequently provided emotional support  (n = 43); while 19% (n = 10) of HCPs felt there were no barriers. One individual did not respond to this survey item. The perceived barriers to providing families with emotional support and guidance decreased across most dimensions, except capacity. Post implementation, 21% of HCPs felt limited capacity was a barrier compared to 15% pre-implementation , limited capacity , and insufficient training  as barriers to providing emotional support and guidance  . Most HCguidance .n = 19) of HCPs reported witnessing PMs interact with PICU patients and their families. This trend may be influenced by the post-implementation program duration being cut short due to the COVID-19 pandemic. However, the majority of HCPs reported that the interactions between PMs and families were valuable . When asked how likely they were to recommend the PM Program to families on a scale of 1–10, where 10 is “extremely likely,” HCPs indicated a mean rating of 7.94. HCPs felt that PMs provided peer-to-peer supports, acted as non-medical confidants, addressed family health , and empowered families to be more proactive in their child's care.Only 35% .All families agreed that their child's comfort was important to the PICU team. As in pre-implementation, families felt least positive about the level of playing and developmental care their children received in the PICU; however, families generally agreed that the PICU team uses age-appropriate activities with their children and are attentive to their need for play; an increase compared to the pre-implementation results . One respondent cited challenges with coping, exhaustion, fear of tubing, and limited knowledge of how to interact with their child.Families reported PICU Vs engaged with their children through play with toys, hand holding, reading, singing, cuddling, and talking—including asking age-appropriate questions. The majority of families did not feel there were barriers preventing them from interacting with their children in the PICU  reported the average number of hours they spend in the PICU per week, which ranged from 14 to 50 h.One respondent's child had been in the PICU for <2 weeks, two had been admitted for more than 3 months, and the remaining had been admitted between 2 weeks and 3 months. All families reported they had been visited by a PM one to three times in the past seven days. A third of the families , massage , or play , but a few reported never engaging in these activities . Overall, engaging in cuddling was reported least frequently . 83% (n = 5) of families did not feel there were any barriers that prevented them from interacting with their child in the PICU; however, those barriers that were highlighted included difficulty coping, exhaustion, fear of touching or dislodging tubes, and physical disability .Most families felt comfortable with their understanding of how to interact with their child and HCPs in the PICU, but more often reported experiencing negative feelings and emotions. All families reported they or their family members interacted with their child in the PICU in some manner, including hand-holding. Most reported engaging in story-telling  agreed that a PM provided them support that helped them through their critical care experience, but were mostly neutral about if PMs provided tips and advice . Families reported talking about day-to-day life and their children (including those not in the PICU) with the PMs. Overall, families reported that speaking with a PM was helpful compared to a health professional, because they felt less alone and as if someone truly understood their experiences.When asked about their interactions with the PM program, most families .An important theme that emerged from the HCP data is that in both pre- and post-data, PICU HCPs reported that they lack both time and training to provide regular developmental care to patients. This finding may be corroborated by answers in the V survey that indicated families did not feel that the PICU regularly offers age-appropriate activities to patients. However, as per Another interesting finding was that families reported two extremes of their availability to be physically present at the bedside with their child. We hypothesize that those families spending 8 h weekly in the PICU would benefit from this program in having trained Vs at their child's bedside to support non-medical interaction. Those families spending 168 h weekly (24/7), could benefit from the program in potentially taking self-care breaks away from bedside or receiving support from a PM. Fourteen families did not respond to this question, and we can only speculate if the lack of response was due to either their inconsistent presence in the PICU or guilt over not being as present as they would have liked. Further evaluation with these groups would be helpful to better understand eithers' experience. Overall, this program evaluation is an important step to identifying both areas for improvement and encouraging the work toward on-going P/V Program success and sustainability.Prior to initiating our P/V Program, we conducted a snowball sampling environmental scan to determine how others had designed, developed and implemented a like program in other institutions. We found nine sites across North America that advertised a type of V program for their neonatal intensive care unit (NICU) or PICU. The majority of NICU programs employed Vs as “cuddlers” for their patient populations. PICU Vs were trained to offer developmentally appropriate non-medical interaction and play to patients, or to help families with way-finding.In general, V programs in Canada and North America are thought to contribute to significant cost savings to hospitals and are also likely to enhance quality indicators such as patient satisfaction and safety . If repon” value where appropriate. Also of note, the results of our post-implementation surveys for families may have been influenced by the amount of time they spent on the unit. If families were not present to witness V services or engage with PMs, they may have had an incomplete perspective of the programs. In the post-implementation volunteer survey, we did not ask for the time families spent on the unit, so we were unable to correlate if their presence had an influence on their perspective of the programs. We would include this demographic data in subsequent evaluation questions. Finally, as we mentioned above, in March 2020, post-implementation evaluation data collection was halted due to the COVID-19 pandemic. Frontline HCPs were deployed to other units to address pandemic needs, families were limited to one caregiver at a time at their child's bedside, and Vs were not allowed on-site. Therefore, post-implementation survey responses were understandably lower across all participant groups. The results from all post-implementation surveys are limited by the small number of surveys we were able to collect compared to pre-implementation. We look forward to continuing this robust evaluation when COVID-19 restrictions are lifted at our hospital.This program evaluation has potential limitations. Surveys are limited by sampling bias whereby the results may not accurately reflect the experiences of those who did not respond. Survey data is also limited by the completeness of survey responses as respondents may have skipped questions or offered limited explanation for their selections. This limitation was partially addressed by triangulating data and reporting the “The PICU P/V Program was designed to improve the experience of patients, families, and HCPs in the PICU. For patients, the opportunity to receive non-medical interaction has the potential to facilitate important growth and development and reduce the risk for PICS. For families, PMs may help them to more easily navigate their hospital experience as well as feel a type of comradery-support that they currently do not receive from medical professionals. For HCPs, the P/V Program may help to offset developmental care requirements so that they may focus on other quality of care activities.In summary, the PICU's P/V Program Pilot was a success, offering potential benefit to patients and families and perceived value from our HCPs. We aim to continue this program once the COVID-19 restrictions on those families and volunteers allowed in the hospital are lifted. When we are able to re-initiate the program, we will focus on fostering awareness of the program for patients, families, and HCPs, and developing more clarity between our new support roles (PMs and Vs) and our existing support roles . There was also evidence that our HCPs could benefit from more focused developmental care education and training so they can better understand the hands-on, non-medical interaction that can be offered to acutely ill patients in the PICU.This program evaluation is an important contribution to the literature in the field. There exists a paucity of empirical research dedicated to understanding the short- and long-term effects of PMs and Vs on patients, families, and HCPs in PICU settings. Further research is required to test the short-term effects of a similar P/V program on patients, families, and HCPs in in this type of environment. Longitudinal studies are required to elucidate the influence of PMs and Vs on PICS for patients and their families.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by University of Alberta Research Ethics Board (Study ID Pro00089760). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.NP, AD, CM, and AB composed and edited the manuscript. DG reviewed and edited the draft. All authors were involved in conceptualizing, developing, executing, and evaluating this project.Alberta Health Services Chief Medical Office for funding this quality improvement project.We wish to acknowledge the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The lymphovascular invasion (LVI) status facilitates the determination of the optimal therapeutic strategy for superficial esophageal squamous cell carcinoma (SESCC), but in clinical practice, LVI must be confirmed by postoperative pathology. However, studies of the risk factors for LVI in SESCC are limited. Consequently, this study aimed to identify the risk factors for LVI and use these factors to establish a prediction model. The data of 516 patients who underwent radical esophagectomy between January 2007 and September 2019 were retrospectively collected . In the training set, least absolute shrinkage and selection operator (LASSO) regression and multivariate analyses were utilized to identify predictive factors for LVI in patients with SESCC. A nomogram was then developed using these predictors. The area under the curve (AUC), calibration curve, and decision curve were used to evaluate the efficiency, accuracy, and clinical utility of the model. LASSO regression indicated that the tumor size, depth of invasion, tumor differentiation, lymph node metastasis (LNM), sex, circumferential extension, the presence of multiple lesions, and the resection margin were correlated with LVI. However, multivariate analysis revealed that only the tumor size, depth of invasion, tumor differentiation, and LNM were independent risk factors for LVI. Incorporating these four variables, model 1 achieved an AUC of 0.817 in predicting LVI. Adding circumferential extension to model 1 did not appreciably change the AUC and integrated discrimination improvement, but led to a significant increase in the net reclassification improvement (p=0.011). A final nomogram was constructed by incorporating tumor size, depth of invasion, tumor differentiation, LNM, and circumferential extension and showed good discrimination  and good calibration in the training and validation sets. Decision curve analysis demonstrated that the nomogram was clinically useful in both sets. Thus, it is possible to predict the status of LVI using this nomogram scoring system, which can aid the selection of an appropriate treatment plan. Esophageal cancer is the seventh most common malignancy in the world . For theLymphovascular invasion (LVI) means tumor cells are present in blood vessels or lymphatics and is only confirmed by histopathological methods , which pBetween January 2007 and September 2019, the data of patients with histopathologically-confirmed esophageal cancer (Tis or T1 stage) who underwent esophagus resection at Zhejiang Cancer Hospital were retrospectively analyzed. The exclusion criteria were (1) patients who received chemotherapy or radiotherapy before surgery (2); patients with a history of other malignancies or incomplete data; and (3) patients with basaloid squamous cell carcinoma, adenosquamous carcinoma, sarcomatoid carcinoma, neuroendocrine carcinoma, or spindle cell carcinoma. The final eligible patients with SESCC who were admitted between January 2007 and May 2015 were assigned to the training set and those admitted between June 2015 and September 2019 were assigned to the validation set. The flowchart of patient selection is summarized in Before the operation, the esophagus cancer diagnosis must be confirmed by biopsy and clinical stage need to be evaluated by CT or endoscopic ultrasonography (EUS). Also, the cardio-pulmonary function was evaluated by cardiac ultrasound and pulmonary function test to make sure the patients can tolerate the operation. Finally, some laboratory examinations such as blood routine examination, liver and kidney function, coagulation function, HIV infection, viral hepatitis, and syphilis were evaluated pre-operatively.Surgical specimens were fixed with formaldehyde and were then cut serially to make slices. The intervals between the tumor tissue and adjacent normal tissues in the slices were 2-5 mm. Tumors that exceed the muscularis mucosa were considered as submucosal invasion . We then2 test or Fisher’s exact test. All variables that were significantly associated with LVI in univariate analysis were candidates for stepwise multivariate logistic analysis. The least absolute shrinkage and selection operator (LASSO) method was used to select the most significant predictive features (glmnet package. The integrated discrimination improvement (IDI) is the difference in the discrimination slopes for a prediction model with and without one variable, which indicates whether the discrimination slope of a model will improve if one important parameter is added. The net reclassification improvement (NRI) is an index that attempts to quantify how well a new model correctly reclassifies subjects. Typically, this comparison is between an original model and a new model  (PredictABEL package.Continuous variables are expressed as the median (range) and were compared using the Mann-Whitney test. Categorical variables were compared using the χfeatures , 20. LASmponent) , 22. Therms package to formulate a nomogram. The nomogram can proportionally convert each regression coefficient in the logistic regression to a scale of 0 to 100 points  and R, version 4.0.3.According to the results of multivariate logistic regression analysis, we used R software (version 4.0.3) with the 0 points . The poiThe clinicopathologic characteristics of the 516 included patients are listed in Comparisons of the clinicopathological characteristics between the LVI-positive and -negative groups are summarized in In total, 11 characteristics were analyzed by LASSO regression in the training set, and seven candidate variables were determined to be associated with LVI Figure 2The base model (model 1) was then created by incorporating the four variables determined to be associated with LVI . By adding sex, circumferential extension, and multiple lesions in sequence to the model 1, we constructed three new models named model 2, model 3, and model 4 Table 4.A nomogram for LVI prediction was formed by incorporating five variables—— tumor size, tumor invasion depth, tumor differentiation, LNM, and circumferential extension  in the training sets are presented in SESCC just invade the mucosa and submucosa and lack of any subjective symptoms. Hence, early diagnosis is difficult for these patients, and most esophageal cancers are at a locally advanced stage when the diagnosis is confirmed. However, due to advancements in diagnostic techniques and the widespread use of endoscopic screening, the rate of SESCC diagnosis is increasing . In thisWe measured the largest tumor diameter under a microscope as tumor size, then used univariate and multivariate analyses to investigate the association between tumor size and LVI. It can be concluded from our study that tumor size was significantly correlated with LVI and was also identified as an important predictor of LVI. Although SESCC comprises both mucosal and submucosal cancers, the LVI status may differ between mucosal and submucosal infiltration. Taking mucosal infiltration as reference, the odds ratio of the submucosal infiltration was 4.871 for prediction of LVI in our training set Table 3,It was previously reported that histological differentiation was related to LVI . ConsistInterestingly, according to the LASSO regression analysis, seven variables  were associated with LVI when log (λ) = -1.961 and λ = 0.0109 Table 4,, with an AUC of 0.833 in training set and 0.819 in the validation set The most important and final line of evidence for the use of the nomogram is based on the need to interpret individual requirements with regard to additional treatment or care. Therefore, to confirm the clinical utility, we determined the decisions assisted by this nomogram whether improved patients’ consequences by using DCA. The clinical outcomes based on the threshold probability can be known from this new method. False positive proportion was subtracted from the true positives proportion, and then the relative risk of false positive and false negative results was weighted to obtain the net benefit . It can In summary, tumor size, tumor invasion depth, tumor differentiation, and LNM were identified as significant predictive factors for LVI in patients with SESCC. Circumferential extension was also identified as a predictor for LVI by calculating the IDI and NRI. Furthermore, a nomogram scoring system was established using these five variables, making individualized LVI prediction easier and facilitating optimal treatment strategy selection for patients with SESCC. However, there are some limitations in this study. First, this was a retrospective study based on data from a single institution. This design could pose some potential biases including selection. Second, the sample size was relatively small, which restricted some of the analyses. Therefore, it is necessary to validate the results using data from multiple centers and a prospective study with big sample size is required to further confirm the reliability of the nomogram. Last but not least, our nomogram may improve and facilitate treatment strategy selection, which may lead to early diagnosis and prompt treatment initiation for patients with SESCC.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics committee of Zhejiang Cancer Hospital. The patients/participants provided their written informed consent to participate in this study.SW and RR conceived the idea and designed study. YT, JY, DZ, and ZC collected data. RR and QS analyzed the data. RR and SC drafted the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AT-rich interaction domain 1A (ARID1A) is a tumor suppressor gene that mutates in several cancer types, including breast cancer, ovarian cancer, and colorectal cancer (CRC). In colon adenocarcinoma (COAD), the low expression of ARID1A was reported but the molecular reason is unclear. We noticed that ARID1A low expression was associated with increased levels of miR-185 in the COAD. Therefore, this study aims to explore ncRNA-dependent mechanism that regulates ARID1A expression in COAD regarding miR-185. The expression of ARID1A was tested in COAD cell line under the effect of miR-185 mimics compared with inhibitor. The molecular features associated with loss of ARID1A and its association with tumor prognosis were analyzed using multi-platform data from The Cancer Genome Atlas (TCGA), and gene set enrichment analysis (GSEA) to identify potential signaling pathways associated with ARID1A alterations in colon cancer. Kaplan-Meier survival curve showed that a low level of ARID1A was closely related to low survival rate in patients with COAD. Results showed that inhibiting miR-185 expression in the COAD cell line significantly restored the expression of ARID1A. Further, the increased expression of ARID1A significantly improved the prolonged overall survival of COAD. We noticed that there is a possible relationship between ARID1A high expression and tumor microenvironment infiltrating immune cells. Furthermore, the increase of ARID1A in tumor cells enhanced the response of inflammatory chemokines. In conclusion, this study demonstrates that ARID1A is a direct target of miR-185 in COAD that regulates the immune modulations in the microenvironment of COAD. Colorectal cancer (CRC) is one of the most prevalent forms of cancer in the world, which is responsible for the most cancer deaths . SeveralThe post-transcriptional alteration of mRNAs is one of the most well-known mechanisms by which miRNAs regulate their target genes. Although the ARID1A deficiency is widely documented, there has been no research on ARID1A-dependent mRNA expression and post-transcriptional repression by non-coding miRNAs in COAD. To address this gap, we searched for miRNAs-targeted ARID1A in TCGA-based data tools and found a subset of miRNAs that may target ARID1A in colon cancer. We also uncovered the prognosis significance of low ARID1A levels in colon cancer, using RNA sequencing data from the TCGA database. We discovered that ARID1A predicted poor prognosis in colorectal cancer patients.https://www.tnmplot.com/). We used RNA sequencing and Gene Chip data to determine the differential expression of ARID1A in normal and tumor COAD tissues. The ARID1A expression profile was compared using Gene Chip data from tumor, normal, and metastasis tissues.TNMplot provides a comprehensive analysis of RNA sequence expression and Gene Chip data of tumor pan-cancer. We used the pan-cancer analysis page to evaluate the expression level of the ARID1A gene across all tissues in all available normal and tumor RNA Sew data (http://gepia2.cancer-pku.cn/#index). A statistical test from the TCGA database was used to analyze the correlation between ARID1A expression and clinical features in COAD patients, including age, gender, tumor stage, pathological stages, and tumor purity. Utilizing USCS Xian TCGA database http://xena.ucsc.edu/., we investigated the association between ARID1A and the standard clinic pathological variables in COAD. We further evaluated ARID1A’s prognostic importance using the previously published GEO dataset and discovered that a lower ARID1A level was correlated with a poor prognosis at a 5% statistical significance level.GEPIA 2 was used to study the prognosis of ARID1A in TCGA COAD . Cells were isolated after being transfected for 24 to 36 h, and after that, they were processed further. Total RNA was extracted from the cells using TRIzol Reagent .Human colon cancer cell lines HCT116 and LoVo were cultured in Dulbecco’s Modified Eagle medium (DMEM) or RPM1640 with medium 10% fetal bovine serum (FBS) at 37°C with 5% COcDNA was obtained using kit , which served as a qPCR template, and the qPCR reaction mix was prepared in triplicate. Primers ARID1A Forward” TTATGACAGAGTGAGGACGGAG, and ARID1A Reverse” TGCCTTGGGTGGAGAACTGAT mixed with SYBR green analysis reagent as recommended by the manufacturer (company). GAPDH, a housekeeping gene, was used to normalize gene expression in the same sample. Normalized Ct values were determined using the ΔΔCt method.The total protein derived from cells was extracted using RIPA lysis buffer with PMSF and cocktail protease inhibitors, quantified using the BCA method in lysis buffer (Beijing Solaribo Science & Technology). A total protein was separated using 8% SDS-PAGE and then transferred to a Millipore PVDF membrane. After blocking with 5% non-fat milk in Tris-buffered saline (1× TBS) containing 0.05% Tween-20, the membrane was incubated overnight at 4°C with rabbit antibodies against ARID1A , βactin . After washing, the membrane was incubated at 37°C for 2 h with a secondary antibody conjugated to horseradish peroxidase (Beijing Solaribo Science & Technology).http://cistrome.org/db/#/).We predicted ARID1A-targeted miRNAs using several Web-based TCGA databases, including Gene Set Cancer Analysis (GSCALite), CanEvolve, and linkedomics. Following that, we used the UALCAN database to detect ARID1A-targeted mir185 expression in COAD. Notably, mir185 was shown to target ARID1A using miRNA mimics and inhibitors. GraphPad software was used to analyze the ARID1A mRNA expression data and to visualize the results. The protein expression was evaluated by SDS-PAGE and Western blotting using the ARID1A antibody (USABIO TECHNOLOGY). The primary antibodies were detected with HRP-conjugated secondary antibodies, and target protein bands were visualized using LAS-500 imaging system. The study was replicated three times to ensure the reliability of the findings. Additionally, Cistrome database-based chip-seq data were used to predict candidate transcription factors that are most likely to bind and regulate ARID1A expression in COAD (http://www.linkedomics.org/login.php). Cancer Gene and Explorer Pathways were used in conjunction with LinkedOmics to investigate ARID1A-related function using TCGA and GEO gene expression data (https://cgpe.soic.iupui.edu/gsea_result/).To further elucidate ARID1A’s tumor-promoting mechanism in COAD, we sequenced 379 colon cancer tissues in linkedOmics database. LinkedOmics is a freely accessible portal that contains multi-omics data from all 32 TCGA cancer types, which includes genetic information such as DNA mutations, gene expression, and chromosome copy number variation (http://timer.cistrome.org/).We studied immune cell infiltration using the TIMER2 tool, which can directly analyze TCGA data and quantify the ratio of immune cells in the tumor and normal samples based on gene expression profiles, copy number, and mutation status. We established a link between the infiltration of sex immune cell types and ARID1A mRNA expression, mutation, and copy number alterations . Further, we analyzed gene expression of ARID1A in COAD metastases and found that ARID1A had a lower expression level in metastases compared to normal  –15. SeveIn the following two respects, the mechanism of downregulation of ARID1A in CRC was mainly explained. Firstly, we discovered that miR-185 binds to the ARID1A 3’ UTR, validating databases findings that miR-185 targets and regulates ARID1A expression. Additionally, miR-185 expression was inversely associated with ARID1A expression in CRC tissues. Similarly, gastric cancer samples exhibit absents or weak ARID1A protein expression even though there were no detectable ARID1A mutations . TFs couSeveral recent studies suggested that tumor microenvironmental cells play an essential role in the occurrence and progression of various cancers, including colon cancer . There wThe degree to which a gene impacts CTLs is reflected in the prediction of CTLs on patient clinical prognosis. Cox regression analysis showed that low CTL levels in patients with high ARID1A expression levels indicate a better prognosis. Whereas in patients with low ARID1A expression levels, high CTL levels indicate a worse prognosis. These findings are supported by another research that shows that increasing levels of ARID1A associated with a decrease in T-cell levels promotes cancer cell survival . We usedThe study investigated the clinical value of the ARID1A in TCGA colon adenocarcinoma (COAD) tissues and cell lines and discovered that mir-185 downregulates ARID1A in COAD and that lower ARID1A expression was associated with high overall survival (OS), disease-free survival (DFS) (P<.05), and is an independent prognostic factor in colon cancer. Also, a correlational analysis was presented based on TCGA colon cancer data between mRNA ARID1A expression and clinicopathological features, tumor-infiltrated immune cells, immune inhibitors, activators, and chemokines.The original contributions presented in the study are included in the article/SB conceived the ideas, design study, carried out the experiments, and wrote the manuscript. YQ, HW, ZZ, MZ, YG verified the analytical methods. Y-FZ supervised and review the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Early and rapid confirmation of dengue infections strengthens disease surveillance program and are critical to the success of vector control measures. Rapid diagnostics tests (RDTs) are increasingly used to confirm recent dengue infections due to their ease of use and short turnaround time for results. Several studies undertaken in dengue-endemic Southeast Asia have reported the performance of RDTs against enzyme-linked immunosorbent assay (ELISA), reverse transcriptase polymerase chain reaction (RT-PCR) and virus isolation methods. However, few studies have compared multiple RDTs for the detection of dengue NS1 antigen and IgM antibody in a single combo cassette. We evaluated six RDTs in Singapore for their utility in routine clinical testing to detect recent dengue infections.The evaluation comprised two phases. The first phase sought to determine each RDT’s specificity to dengue NS1 and IgM using zika and chikungunya virus supernatant and zika convalescent samples. RDTs that cross-reacted with zika or chikungunya were not further tested in phase 2. The second phase sought to determine the sensitivity and specificity of the remaining RDTs to dengue NS1 and IgM using pre-characterised dengue specimens and non-dengue/chikungunya febrile clinical specimens.None of the RDTs cross-reacted with zika IgM in Phase 1. Truquick and Quickprofile cross reacted with zika and chikungunya viruses and were not evaluated thereafter. Standard Q had the highest dengue NS1 and IgM sensitivity at 87.0% and 84.3% respectively whereas Bioline (68.5%) and Multisure (58.3%) had the lowest dengue NS1 and IgM sensitivity respectively. Combining dengue NS1/IgM detection results greatly improved the RDT ability to detect recent dengue infection; Standard Q had the highest sensitivity at 99.1% while Multisure had the lowest at 92.6%. All the RDTs were highly specific for dengue NS1 and IgM (96.7% to 100%). All the RDTs had high positive predictive values (98.4% to 100%) for NS1, IgM and combined NS1/IgM parameters whereas Standard Q had the highest negative predictive values at 68.2% (NS1), 63.8% (IgM) and 96.8% (NS1/IgM). For the RDTs, detection of NS1 declined from acute to convalescent phase of illness whereas IgM detection rate gradually increased over time.In our study, several RDTs were evaluated for their diagnostic accuracy and capability in detecting recent dengue infection. Standard Q demonstrated a high degree of diagnostic accuracy and capability in the detection of NS1 and IgM biomarkers. RDTs can provide rapid and accurate confirmation of recent dengue infections and augment dengue surveillance and control programmes. Further studies are required to assess the usefulness of these RDTs in other epidemiology settings. Dengue is one of the most important infectious diseases in tropical and subtropical regions of the world causing 100 to 200 million infections per year .There are four dengue serotypes (DENV-1 to 4) which cause illness in humans. The dengue virus infection can be asymptomatic or may cause undifferentiated febrile illness, dengue fever or dengue haemorrhagic fever including dengue shock syndrome. The clinical manifestation of dengue infection depends on factors such as the immune status, age and underlying medical condition of the host .Traditionally, dengue diagnosis can be achieved via virus isolation, immunological-based tests or the molecular-based polymerase chain reaction (PCR). However, these techniques are costly, have a long turnaround time and require specialised equipment and training . In receNS1, which is a highly conserved glycoprotein among Flaviviruses such as Zika, Dengue and Japanese encephalitis, can be found in day one and up to day nine after onset of fever in both primary and secondary dengue infected patients ,9. IgM iManufacturers have reported the performance of dengue RDTs, but past evaluations have shown varied, including poor performance, among different dengue RDTs ,12. ThisThe Environmental Health Institute (EHI) management committee chaired by Dr Lee-Ching Ng, Group Director of EHI, waived the need for informed consent as the archived specimens were fully anonymized prior to the start of the study. The ethical approval can be found under TS233.Aedes aegypti mosquito that inhabited urban areas. The dengue epidemic cycle in Singapore in the last two decades, oscillated between DENV-2 and DENV-1 even though all four DENV serotypes are reported , 91.67% DENV-2 [99/108], 2.78% DENV-3 [3/108] and 1.85% DENV-4 [2/108]. The fever duration of the positive specimens, including the subset of IgG positive serum specimens, ranged from 4 to 11 days and had a median fever duration of 7 days. A positive dengue NS1 and/or IgM result was used to confirm dengue infection in the combined dengue NS1/IgM test panel. In addition to the virus supernatants, 30 pre-characterised non-dengue and non-chikungunya specimens were collected from patients with undifferentiated febrile illness for specificity testing. The fever duration of the negative specimens ranged from 3 to 9 days and had a median fever duration of 5 days. The characteristics of the specimens used in the study is summarized in ™ NS1 ELISA, Panbio® Dengue IgG Indirect, IgG Capture and IgM Capture ELISAs. Viral isolation in C6/36 cells and confirmatory immunofluorescence testing for serotype identification were performed as described [The reference tests in the study included a two-stage RT-PCR comprising of an initial screening using SYBR green followed by the tetraplex probe-based serotype specific assay. The serological assays used were: Plateliaescribed ,21.The specimens were classified as a confirmed case for recent dengue infection when tested to be positive by PCR or NS1, or with IgM or IgG Indirect seroconversion, or with a four-fold rise in IgG Indirect in the convalescent specimen or were positive by viral isolation. Specimens that were negative by PCR, NS1, IgM but maybe IgG Indirect positive without four-fold rise in titre in the convalescent specimen were classified as negative for recent dengue infection. Chikungunya RT-PCR were performed on all the pre-characterised samples to screen for acute chikungunya infection .Sensitivity and specificity indices with a 95% confidence interval (CIs) were performed using the proportional test in R software. Positive predictive value (PPV) and negative predictive values (NPV) indices with a 95% confidence interval (CIs) were calculated as described .The phase 1 evaluation results are summarized in Tables Four kits were put through this phase of evaluation. Standard Q had the highest NS1 sensitivity at 87.0%, which is significantly higher than Bioline and careUS. Standard Q also had the highest IgM sensitivity at 84.3%, whereas Multisure had significantly lower IgM sensitivity compared to the other three RDTs. When we combined the results for NS1 and IgM detection, the sensitivity of the RDTs improved substantially. Standard Q had the highest sensitivity at 99.1% and was significantly higher than Multisure with a p-value of 0.04. The overall sensitivity of the RDTs are summarized in All RDTs were highly specific for dengue NS1 at 100%. For dengue IgM, the specificity of Multisure was 96.7% whereas the other three RDTs were highly specific at 100%. However, this difference was not statistically significant.The positive predictive values (PPVs) for the NS1, IgM and combined NS1/IgM parameters were all above 95%. In contrast, the negative predictive values (NPVs) ranged from 46.9% to 68.2% for NS1 and 39.2% to 63.8% for IgM. The NPVs range increased to 78.4% to 96.8% for combined NS1/IgM. Standard Q demonstrated the highest NPVs for NS1, IgM and combined NS1/IgM. The PPVs and NPVs of the RDTs are summarized in High number of NS1 positives were detected in the early phase of illness and subsequently declined over time. careUS saw the steepest decline in NS1 detection from 95.2% at days of illness ≤ 5 to 36.8% at days of illness ≥ 9 whereas Standard Q declined the least from 95.2% to 68.4% . IgM wasIn this study, we evaluated six dengue RDTs for their diagnostic accuracy and capability in detecting recent dengue infection. The RDT with the highest diagnostic accuracy and capability can be used for dengue testing thereby enhancing Singapore’s vector control effort.2 to 103 pfu/ml. The low specificity will potentially result in false dengue positive in countries where zika, chikungunya and dengue virus co-circulate and hamper the efforts in disease control, patient management and etiological investigation.In phase 1, Truquick and Quickprofile cross reacted with both zika and chikungunya virus supernatant at relatively low titre of 10The phase 2 results showed that Standard Q had the highest overall diagnostic accuracy for NS1 and IgM using specimens collected in Singapore, which have low incidences or no reports of other related flaviviruses such as Yellow fever and Japanese encephalitis.When IgM results were analysed in combination with NS1 results, the sensitivity of all the RDTs in determining a recent dengue infection reached at least 92.6%. This observation was consistent with several studies reported –25. HowePositive and negative predictive values are indicative of the RDT’s diagnostic capability in clinical setting where the true disease status of a patient is unknown. These values indicate the probability that the RDT will provide a correct diagnosis . All theThe effect of days of illness on the RDTs sensitivity to NS1, IgM and combined NS1/IgM were consistent with several studies reported –12,25. OThe specimen type used for this evaluation limited us. Out of the 108 pre-characterised specimens, 91.67% were DENV-2 hence, the dengue NS1 sensitivity for DENV-1, 3 and 4 may not be representative in this study. As described in a 2014 study, the Bio-Rad Platelia™ Dengue NS1 antigen capture kit was reported to have a lower sensitivity for DENV-4 circulating in Roraima, Brazil and for DENV-2 circulating in Southeast Asia . This suIn March 2020, Singapore reported two cases of COVID-19 positive patients who were misdiagnosed as dengue serology positive. The investigation revealed that the initial dengue serology results tested by careUS and Bioline were false positives . TherefoConsistent intra- and inter- lot performance are important indicators of the RDT’s quality. The Environmental Health Institute laboratory encountered intra-lot IgM variability when using a certain brand for routine clinical testing. Therefore, intra-/inter- lot variability should be tested when carrying out such evaluation to minimize the possibility of misdiagnosis as highlighted previously.In this study, we evaluated six dengue RDTs for their diagnostic accuracy and capability in detecting recent dengue infections. Truquick and Quickprofile cross reacted with zika and chikungunya virus while none of the RDTs cross reacted with zika IgM. Standard Q showed a high degree of sensitivity and specificity in the detection of dengue NS1 and IgM biomarkers and had the highest positive and negative predictive values, demonstrating its utility in enhancing dengue disease surveillance and control programmes. Further studies are required to assess the usefulness of the RDTs in other epidemiology settings. Clinicians must be aware that a negative result does not rule out dengue. Conversely, a positive dengue antibody result must be interpreted with symptoms and does not rule out the possibility of other diseases.S1 FigThe RDTs are colour coded and grouped into NS1, IgM or combined NS1/IgM categories. The bar charts indicate the sensitivity estimates and the solid black lines indicate the 95% confidence intervals for those estimates. (*) indicates a significant difference between 2 or more RDTs within each group.(DOCX)Click here for additional data file.S2 FigEffect of time (number of days of illness) on RDT sensitivity for (A) NS1, (B) IgM and (C) combined NS1/IgM.(DOCX)Click here for additional data file.S1 Table. Positive and negative predictive values of the RDTs to dengue(XLSX)Click here for additional data file.S1 Annex(DOCX)Click here for additional data file.


Kushida and Kushida found that FM radio waves from stations at distances over-the-horizon are received before earthquakes. Based on this finding, since the mid-1990’s, the Kushidas have been practicing “Earthquake Precursor Detection Experiment”. The performance of the Kushida method during 2000–2003 has been evaluated by checking their predictions against the actual seismicity. During the period, there were 92 Kushida predictions mentioning the possibility of M ≥ 5.5 event, whereas there were 49 M ≥ 5.5 earthquakes in the Japanese region. If the criteria for successful prediction are set as: the errors in date is less than one day, epicentral position is roughly within specified area, and error in M is less than 0.5, the success rate was 20% and the alarm rate was 12%. If we relax the criteria to: the errors in dates within 10 days, epicenter within additional 100 km of specified area and the magnitude error less than 1.0, the success rate was 40% and the alarm rate was 27%. These rates may look insufficient for a practical prediction method. Considering, however, the fact that no other short-term prediction has ever been made in Japan so far it is a significant achievement. Moreover, it was found that in almost all failed predictions, meaningful signals were detected although the interpretations were incorrect. This indicates that the method is promising provided further investigation is carried out. The same evaluation at the M ≥ 6.0 level showed that the general performance was similar to the M ≥ 5.5 level, except that both success rate and alarm rate were lower at the M ≥ 6.0 level. If this unexpected finding is real, it might be inherent to the methodology using scattering of short-wave length radio waves as suggested by M. Hayakawa and may contain important information in understanding the earthquake physics and LAI-coupling. The results of the present study indicate strongly that the earthquake prediction research using anomalous transmission of VHF FM radio waves should be enhanced in parallel with complementary research in other frequency ranges. What actually happened was an M5.6 event which shook Tokyo area with JMA seismic intensity 4. Although this alarm was a failure because of the large magnitude error, it raised certain interests both in scientific and general communities. We, having long been paying attention to the Kushida method since its inception, believe that the method is now worth thorough cross-checking. Actually, such cross-check experiments have recently been started at several universities, including Hokkaido,et al., 1999 At this time, it seems also important to evaluate the performance of the method objectively. Since their information is not open to non-members, objective checking has not been made since Yoshino We evaluated the performance of the Kushida method by scrutinizing all of their 560 New Letters issued during the period 2000–2003. Their predictions were compared against the actual seismic activity tabulated in the catalogue of earthquakes published by the Japan Meteorological Agency (JMA).S and the other is the alarm rate A defined by,Performance of earthquake prediction may be evaluated by two parameters. One is the success rate S, only predictions which mentioned the occurrence of M ≥ 5.5 earthquakes in their statement were selected from Kushida News Letters. (They will be abbreviated as “M ≥ 5.5 predictions”.) Then, we examined if they were successful by checking with the JMA catalogue. In doing so, we looked at M ≥ 4.5 earthquakes only, disregarding M < 4.5 earthquakes.First, to evaluate these parameters, it is necessary to define the population of predictions and earthquakes to deal with. It would be reasonable to focus our attention to large earthquakes and disregard small ones. For evaluating Here, a care was taken in counting the number of predictions. Kushida’s signals often show time evolutions, which are sometimes complicated. In such a case, statements in consecutive News Letters also change. As long as the changes were made before the predicted dates of the earthquake in question, they were lumped together and considered as one prediction. For example, prediction of the January 19, 2003 M5.6 earthquake off Tokai coast started several months before, i. e., July 11, 2002 (News Letter #971). Until the last prediction was made on December 28, 2002 (News Letter #1037), almost 20 statements, some of which were of different contents, were issued. We count them as one prediction. News Letter #1046 of January 21, 2003 shown in A, only M ≥ 5.5 earthquakes in Japan and its immediate environs were selected from the JMA catalogue and examined which of them were successfully predicted. Earthquakes deeper than 100 km, aftershocks of major earthquakes, earthquakes that occurred in the area outside the “detectability area”, and earthquakes that occurred while the system was out of order were excluded from the population. Non-detectability areas are empirically inferred. Faraway regions are obviously non-detectability areas, but some areas such as parts of the Pacific side of the Tohoku region were also designated non-detectable area. According to Kushida, the emitter-receiver-epicenter geometry, focal depth and water depth at sea may be key factors in dictating the detectability and non-detectability. However, geographic distribution of detectable and non-detectable areas has not been clearly delineated yet.For the evaluation of Second, it is necessary to set the criteria for success-failure decision. Successful predictions must specify the time, T, location, R, and magnitude, M, within some permissible error ranges, ΔT, ΔR, and ΔM. We adopted two levels of criteria as follows:It must be noted that, according to the above evaluation system, the lower limit of the magnitude of earthquakes for the success rate count can be as low as 4.5, whereas that for the alarm rate is 5.5, because M < 5.5 earthquakes were not the objects of alarm rate evaluation.A prediction is scored successful only when all three conditions are satisfied in each criteria. Criteria A are practically the same as the Kushidas themselves impose on their predictions. Since they are extremely stringent, we set the more relaxed alternative criteria B, which are still reasonably high.The rate varied year to year : the yeaS was low in 2000, the success rate for this swarm was 71%. This high rate, however, could be by chance because of the high swarm activity (see below).In the summer of 2000, there was a large swarm activity with 11 M ≥ 5.5 events in the Izu island region.There were large earthquakes which were not successfully predicted. The case history of some notable failures is as follows: It seems that some relevant signals were recognized but the interpretations were incorrect.The June 7, 2000 M6.2 Off the Western Ishikawa Prefecture earthquake No. 1 in .Starting from December 8, 1999 (News Letter #613), more than 10 News Letters carried its prediction. Of the two candidate positions, Kushida finally  discarded the Off the Western Ishikawa Prefecture (the right one) and adopted the inland candidate (Nagano Prefecture) wrongly because the signals looked similar with those of a previous inland earthquake. The results were ΔT = 0, ΔR = 200 km, and ΔM = 0.2. The large ΔR disqualified this prediction.The October 6, 2000 M7.3 Western Tottori earthquake No. 2 in .This was a major inland earthquake during the period. Starting from August 13, 2000 (News Letter #724), many News Letters carried its predictions with candidate epicenter at varied areas in Japan Sea, Philippine Sea and Kanto area. On October 6 immediately before the earthquake (News Letter #751), the signals were wrongly attributed to volcanic activity in the Izu region.The March 24, 2001 M6.7 Geiyo earthquake No. 3 in .This was also a major inland earthquake for the period. From February 1, 2001 (News Letter #796), more than ten News Letters dealt with this prediction with possible epicenter in Wakasa Bay, and western Chugoku (which was right). But on March 4 (News Letter #811), the signals were wrongly attributed to volcanic activity in the Izu region.The July 26, 2003 M6.4 Northern Miyagi Prefecture earthquake No. 4 in .It shook Sendai area with intensity 6 in JMA scale, but no prediction was made because the epicenter was out of detectability region according to News Letter #1132 .The September 20, 2003 M5.6 Southern Chiba Prefecture earthquake No. 5 in .Prediction started on February 15, 2003 (News Letter #1057), and a number of predictions followed inferring possible epicenter in Tokai region and Off the Kanto areas. Public announcement (in a popular weekly magazine), mentioned in the Introduction, was made on September 8, alarming the possibility of devastating M7.2 earthquake on around September 17, 2003. The results were ΔT = 3 days, ΔR ~100 km, and ΔM =1.6. The large ΔM disqualified this prediction.The September 26, 2003 M8.0 Off Tokachi earthquake No. 6 in .This was the largest event in the period. On August 3, 2003 (News Letter #1136), two locations were suggested for an earthquake; one in Japan Sea and the other Off Tokachi. On September 25 (one day before the earthquake), a brief prediction was made as “~M5–6 earthquake is expected at Off Tokachi in a few months”. Thus, it was a false prediction on account of both M and time error. Kushida remarks he was too busy with the September 20 earthquake to pay due attention to this earthquake.As shown in Another matter of potential importance of the Kushida method is that it seems to fail for larger earthquakes. Some case histories were depicted in the previous section. Exactly the same evaluation as done at the M ≥ 5.5 level has been made at the M ≥ 6.0. 2)The performance of the Kushida method during 2000–2003 has been evaluated at the M ≥ 5.5 and M ≥ 6.0 levels. During the period, there were 92 “M ≥ 5.5” and 44 “M ≥ 6.0” predictions, whereas there were 49 M ≥ 5.5 and 21 M ≥ 6.0 earthquakes. Under the criteria for successful prediction that ΔT less than 1 day, ΔR within specified area, and ΔM less than 0.5 Richter units, the success rate was 20% and 18% and the alarm rate was 12% and 5% for the two levels. If we allow the errors to 10 days, within additional 100 km of specified area and 1.0 Richter units, the success rates were 40% and 38% and the alarm rates were 27% and 19% respectively. These rates were much higher than chancy success. Moreover, it was found that in most of false predictions, meaningful signals were detected although their interpretation led unsuccessful results, indicating the possibility that their performance may well be improved by accumulating further experience. As mentioned above, it was also found that the performance of the Kushida method may be lower for earthquakes larger than 6 in magnitude. If this is a real fact, it may provide important information on the physics involved. Although the Kushida method is still far from perfect, it seems that the over-the-horizon transmission of VHF FM radio waves before earthquakes may well be real and serious efforts should be devoted to improve the methodology and clarify the underlying physics.


A 3-aminopropyl-triethoxysilane (APES) fiber-optic sensor based on a Mach–Zehnder interferometer (MZI) was demonstrated. The MZI was constructed with a core-offset fusion single mode fiber (SMF) structure with a length of 3.0 cm. As APES gradually attaches to the MZI, the external environment of the MZI changes, which in turn causes change in the MZI’s interference. That is the reason why we can obtain the relationships between the APES amount and resonance dip wavelength by measuring the transmission variations of the resonant dip wavelength of the MZI. The optimized amount of 1% APES for 3.0 cm MZI biosensors was 3 mL, whereas the optimized amount of 2% APES was 1.5 mL. In fiWhile the condensation reaction between APES and hydroxyl group is occurring on the surface of the MZI, APES will also react with water . To prevIn this paper, an optical fiber-based MZI for APES detection was proposed. The different amounts and concentrations of APES on the fiber surface can be monitored by the MZI’s interference patterns. The relationship between the amount of APES and the resonance dip wavelength was obtained. Optimized amounts of 1% APES and 2% APES for fiber-optic biosensors were also obtained.A schematic diagram of the APES fiber-optic sensor based on MZI is shown in ® SMF-28e+® BB. The core and cladding diameters of the SMF were 9 μm and 125 μm, respectively. The core-offset connect joints showing on the fusion splicer screen had no built-in rule scale. So, the cladding diameter has been marked as 125 um in In the paper, a developed core-offset fusion SMF structure was used for the advantages of low cost and easy operation. x axis direction of SMF1, SMF2 was shifted downward by 4 to 5 μm with a length of 3.0 cm. SMF2 and SMF3 also adopted core-offset fusion. SMF3 moved upward by 4 to 5 μm along the x axis direction of SMF2 [The core-offset structure was formed by using a commercial electric-arc fusion splicer (Fujikura FSM-60s). Along the  of SMF2 .For the core-offset structure, the light from SMF1 cladding is partly coupled to the SMF2 core to excite the core-mode light, and the rest of the light from SMF1 cladding enters the SMF2 cladding to excite the cladding-mode light. After propagating through SMF2, the component of core-mode light of SMF2 and component of cladding-mode light of SMF2 couple to the SMF3 core together, forming a type of MZI .In the MZI, the phase difference essed as ,(1)Δϕ=2It can be seen from Equation (3) that the smaller the difference between Ninety-eight percent 3-aminopropyl-triethoxysilane (APES)  was stored at room temperature (RT). Ninety-eight percent toluene and KOH used in this study were of analytical grade.First, the MZI was rinsed 3 times with absolute ethanol and deionized water, respectively, to eliminate residue and dirt on the surface of the optical fiber, then dried naturally. Second, it was immersed in 0.1 M KOH for 1 h to carry out hydroxylation and form a layer of film with hydroxylated group as shown in In order to verify that adhesion between the optical fiber and APES could be achieved in a short time, we recorded and analyzed the transmission spectra of MZI immersed in 1% APES toluene solution for 1 h. A comparative experiment was performed to verify that APES cannot be directly combined with bare optical fiber, but can be combined with hydroxyl-modified optical fiber. After cleaning the bare optical fiber with absolute ethanol and deionized water, 1% APES was directly dripped onto the MZI without hydroxyl group. Cleaning the MZI with deionized water again, the transmission spectra underwent a change consistent with that of the bare MZI in air, as shown in the This phenomenon showed that if the fiber is not modified with hydroxyl, APES cannot be directly combined with the bare optical fiber. The spectra changed after adding APES. While cleaning the MZI with deionized water, the transmission spectra underwent a change consistent with that of the bare MZI in air, which meant that APES adsorbed onto a fiber surface without hydroxyl could be removed.The refractive indices of 1% APES toluene solution and fiber cladding are 1.496 and 1.462, respectively. Therefore, when dripping 1% APES onto the optical fiber surface, the effective refractive index of the cladding mode The refractive indices of 2% APES toluene solution and fiber cladding are 1.493 and 1.462, respectively. When 1.5 mL 2% APES was dripped onto the MZI surface, the effective refractive index of cladding mode The transmission spectra  of 2% APES amounts ranging from 0 to 7.5 mL are shown in The 1% APES film on the optical fiber surface was examined using scanning electron microscopy (SEM) to verify that adhesion between the optical fiber and APES could be achieved with the dripping method. In summary, an optical fiber-based MZI for APES detection was proposed. When APES was dripped onto the hydroxyl-modified MZI, the condensation reaction affected the effective refractive index of the cladding mode, which in turn caused a change in the resonant wavelength’s transmission intensity. Measuring the transmission intensities of the MZI revealed that increasing the amount of APES caused the transmission intensities to increase at the beginning and tend toward stabilization with the gradual saturation of the condensation reaction. The optimal amount of 1% APES for fiber-optic biosensors was 3.0 mL, while the optimal amount of 2% APES was 1.5 mL. Given its advantage of easy fabrication, the core-offset-based MZI structure has potential applications for many types of fiber-optic sensors. In addition, in the field of fiber-optic sensing, APES-modified fiber-optic biosensors with amino groups can be widely used in combination with different functional groups for the immobilization of proteins or nucleic acids.


In 945 LURIC participants both markers were high (UHGH), in 935 both were low (ULGL), in 589 only eGFR (UHGL), and in 582 only sUmod (ULGH) was low. After balancing the groups for cardiovascular risk factors, hazard ratios (95%CI) for all-cause mortality as compared to UHGH were 2.03 (1.63–2.52), 1.43 (1.13–1.81), and 1.32 (1.03–1.69) for ULGL, UHGL, and ULGH, respectively. In VIVIT, HRs were 3.12 (1.38–7.08), 2.38 (1.01–5.61), and 2.06 (0.81–5.22). Adding uromodulin to risk prediction models that already included eGFR as a covariate slightly increased the Harrell's C and significantly improved the AUC in LURIC. In UHGL patients, hypertension, heart failure and upregulation of the renin-angiotensin-aldosterone-system seem to be the driving forces of disease development, whereas in ULGH patients metabolic disturbances might be key drivers of increased mortality. In conclusion, SUmod/eGFR subgroups mirror distinct metabolic and clinical patterns. Assessing sUmod additionally to creatinine or cystatin C has the potential to allow a more precise risk modeling and might improve risk stratification.Serum uromodulin (sUmod) shows a strong direct correlation with eGFR in patients with impaired kidney function and an inverse association with mortality. However, there are patients in whom only one of both markers is decreased. Therefore, we aimed to investigate the effect of marker discordance on mortality risk. sUmod and eGFR were available in 3,057 participants of the Ludwigshafen Risk and Cardiovascular Health study and 529 participants of the VIVIT study. Both studies are monocentric prospective studies of patients that had been referred for coronary angiography. Participants were categorized into four groups according to the median values of sUmod  and eGFR (LURIC: 84 ml/min/1.73 m UMOD locus with estimated glomerular filtration rate (eGFR) . Results from genome-wide association studies linking genetic variation at the e (eGFR) , chronice (eGFR) –3, artere (eGFR)  and diabe (eGFR) , as wells so far . Expresss so far . Studiess so far . In siliand KLF4 .In the case of pathophysiological conditions they act together to rapidly modulate Umod concentration. Due to its immunomodulatory and anti-inflammatory properties, sUmod concentrations might be actively increased in the setting of systemic illnesses to reduce systemic inflammation , 10. Thi2, independently from eGFR and albuminuria . In LURIC, also clinical cutoff criteria were applied to create the groups using cutpoints of 150 ng/ml for sUmod and 60 ml/min/1.73 m2 for eGFR. Continuous data are presented as the mean and standard deviation (SD) when normally distributed or as the median and 25th and 75th percentile for non-normally distributed variables. Categorical data are presented as percentages. Statistical differences between groups and continuous variables were determined using ANOVA. Non-normally distributed variables were log-transformed before entering analysis. Tukey's honest significant difference test was used to investigate differences between individual groups using family-wise correction for multiple testing. The chi-square test was used for categorical variables and differences between individual groups were examined using the “chisq.post.hoc” function implemented in the R package “fifer” v1.1 using the false-discovery-rate method for multiple testing correction.Study participants were categorized into tertiles of sUmod/eGFR or into four groups according to the median values of sUmod (146 ng/ml in LURIC and 156 ng/ml in VIVIT) and eGFR .All tests were two-sided and a  v4.0.3 .Regarding the survival advantage of the ULGH group compared to the ULGL group, the main cause probably is renal function, that is on average at an eGFR of 62 mL/min/1.73 mImportantly, rates of hypertension and heart failure are higher in UHGL than in ULGH in LURIC, suggesting that sodium and fluid overload occur that relate to high sUmod and cannot be balanced by a largely preserved renal function such as in the ULGH patient group.In VIVIT, there was no significant difference in the rates of hypertension between both groups. Information on heart failure was not available in this cohort but the concentration of NT-proBNP was almost three times higher in the ULGH group.2 in favor of the UHGL patient group.Conversely, when analyzing the higher mortality in UHGL vs. UHGH patients groups, a difference in mortality that is exceeding the difference between ULGH vs. UHGH by far, the obvious culprit is impaired renal function, that associates with higher rates of arterial hypertension, heart failure, and DM. The survival advantage when comparing UHGL with ULGL may be explained by protective, i.e., antiinflammatory and immunomodulatory effects of normal sUmod levels and by a somewhat better renal function. The difference in eGFR between both groups was 10 ml/min/1.73 m2 for eGFR in LURIC, we found significant differences between UHGL and ULGH regarding a number of vasoactive biomarkers that were not apparent in the analyses using the median groups  or to the Principal Investigator of the LURIC Study, Winfried März (winfried.maerz@luric-online.de) who are in charge of supervising ethical and legal aspects of the LURIC study. Finally, the authors confirm that they accessed these data upon approval by LURIC and that all other researchers can access the data upon approval in the same manner the authors did. Similarly, the data from VIVIT cannot be released to the public domain due to Austrian Data Protection Act, but access is also made available to researchers upon request and approval . Requests to access these datasets should be directed to Heinz Drexel; vivit@lkhf.at, winfried.maerz@luric-online.de.The data analyzed in this study is subject to the following licenses/restrictions: Due to the articles of Ludwigshafen Risk and Cardiovascular Health (LURIC) Study gGmbH, which needs to acknowledge the German Data Protection Act and the consent given by the study participants, data cannot be released to the public domain. The exploitation of the (LURIC) Study database is governed by the articles of the LURIC Study GmbH (non-profit LLC), registered under number HRB 7668 at the commercial registry of Freiburg in Breisgau, Germany. According to the articles of the organization, data access is made available to researchers upon request and approval. This procedure makes sure that rules of good scientific practice are followed and that credit is given to the people who have been in charge of the design and the organization of the study. Interested researchers are invited to address their request or proposal to Kai Grunwald ] and of Vorarlberg . The patients/participants provided their written informed consent to participate in this study.GD, MK, BY, and AL designed and performed research, made statistical analyses, and drafted the manuscript. BK, WM, and HD designed research and corrected the manuscript. JS drafted the manuscript. HS measured serum uromodulin and corrected the manuscript. All authors contributed to the article and approved the submitted version.LURIC was supported by the 7th Framework Program RiskyCAD (grant agreement number 305739) of the European Union and the H2020 Program TO_AITION (grant agreement number 848146) of the European Union. The work of WM and MK was supported as part of the Competence Cluster of Nutrition and Cardiovascular Health (nutriCARD) which was funded by the German Federal Ministry of Education and Research. The work of GD was supported by the European Union's Horizon 2020 research and innovation programme under the ERA-Net Cofund action N° 727565 (OCTOPUS project) and the German Ministry of Education and Research (grant number 01EA1801A). The funding sources were not involved in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.WM reports grants and personal fees from AMGEN, BASF, Sanofi, Siemens Diagnostics, Aegerion Pharmaceuticals, Astrazeneca, Danone Research, Numares, Pfizer, Hoffmann LaRoche: personal fees from MSD, Alexion; grants from Abbott Diagnostics, all outside the submitted work. WM and MK are employed with Synlab Holding Deutschland GmbH. JS declares a patent at the University Charite, Berlin pending, and no other conflict of interest. BK reports travel support a/o lecture fees a/o advisory board memberships from Astellas, Bayer, BMS, Boehringer Ingelheim, Chiesi, Hexal, Pfizer, Sanofi, Servier, Vifor all outside the submitted work. MK reports lecture fees from Bayer outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Objective: The number of older patients with dementia hospitalised in acute care hospitals increased and these patients underwent physical restrictions leading to a degeneration of essential mental and physical function. The dementia nursing competence e-learning program with audio-visual materials has been developed in acute care hospitals. Methods: An application form that explained the research was distributed to 1,944 registered nurses from seven hospitals, and 110 people applied. Nurses used an e-learning program for a month in May and practiced applying the knowledge learned from programs June through November 2020. The nurses completed a questionnaire survey at four periods: first (before program/baseline), second (after program), third (three months later), and fourth (six months later). In the second, third, and fourth periods, ‘Technical knowledge of the dementia nursing’ and ‘dementia nursing intervention’ were significantly improved as compared with the first. In the fourth period, ‘confidence of reduce of physical restriction’ showed significant improvement compared to the first. In ‘Ethical sensitivity scale of nurses’, the first of the four sub-scales significantly increased as compared with the first period. In ‘Self-assessment Scale of Nursing Practice for Elderly Patients with Cognitive Impairment with the Aim of Person-centred Care in Acute Care Hospitals’, the fourth sub-scale showed significant improvement compared to the first. In the ‘Personhood’ sub- scale of the Japanese version of Approaches to Dementia　Questionnaire, the fourth period showed a significant increase compared to the first. Conclusion: The results suggest that this program was effective and led to reduced physical restrictions in nursing practice.