Adaptation of evidence-based interventions upon implementation into new practice settings is universal, yet poorly understood. During a cross-site evaluation of the implementation of a proven intervention for pediatric asthma care coordination into 4 resource-challenged settings, we conducted in-depth interviews with site representatives, who reported how and why they modified intervention components. Interview notes were coded for themes. We focused on a single theme from a respondent who described the adaptation process as “backing” the intervention into ongoing services; we found evidence of a similar process at other sites. We labeled this process “retrofitting” to signify adaptation that consists of altering existing services to align with intervention components, rather than modifying the intervention to fit a new setting. Advantages of retrofitting may include allowing organizations to keep what works, capitalizing on existing support for program activities, elevating the role of local knowledge, and potentially promoting the sustainability of effective innovations. Practitioners are increasingly being called on to use evidence-based approaches , often aA common middle-ground solution is for intervention developers to designate certain program components as core  , while lClearly, much remains to be learned about optimizing the EBI adaptation process. One step in this direction is to deepen our understanding of the ways that implementing organizations adapt EBIs in the real world . On the La Red de Asma Infantíl de Puerto Rico ; and the neighborhood-based Addressing Asthma in Englewood Project . The cross-site evaluation of the implementation process focused on the EBI Yes We Can (YWC) because it was the one EBI that all sites implemented to a significant degree. YWC is a medical-social model of care that promotes optimal clinical care while deploying community health workers to provide asthma education, link families to health and social services, and facilitate family–clinician communication (www.cdc.gov/asthma/interventions/yes_we_can.htm).In 2010, sites funded by the Merck Childhood Asthma Network, Inc (MCAN) to implement EBIs to address asthma disparities among children  were invnication . The goanication , and thenication ,16. The As the cross-site evaluators studying the translation of established programs into new settings, our first challenge was to determine the core components of the programs, beginning with YWC. From the literature, we determined that no published evidence clearly demonstrated which YWC components were essential to producing the outcomes. Rather, as with most studies, the outcomes were associated with the intervention as a whole. We next examined the CDC online case study of this program. This description identifies 5 “readily distinguishable” program components, but it is unclear whether these should be considered “core” components. We then conducted separate interviews with 3 YWC developers. When asked, “What about the intervention really made a difference in the outcomes?” developers emphasized different elements; for example, one focused on characteristics and actions of the community health workers, whereas another emphasized the broader concept of pairing proper medical care with attention to social aspects that can impede asthma control. On the basis of the content of the published article , the CDCWe conducted 9 key informant interviews by telephone  with principal investigators (1 per site), project managers (1 per site), and field staff . Interview questions were developed by the evaluators  and incorporated implementation-related factors identified by Durlak and Dupre  satisfy funder requirements for EBI implementation, 2) enhance or expand the services they already provide, or 3) both. Of the 15 core components listed in www.merriam-webster.com/dictionary/retrofit). For example, a homeowner might retrofit a house to make it more energy-efficient by adding insulation, or replacing windows. Similarly, program providers might retrofit existing services in a certain program area  to make them more like those of an EBI. Just as it often makes more sense for homeowners to retrofit their existing house rather than build a new one, so might program providers modify what they are already doing rather than replace services to implement an EBI in its entirety. We believed that the term “retrofitting” was appropriate for the implementation-related phenomenon we sought to describe, as it denotes improving, via scientifically supported practices, something that already exists.A dictionary defines “to retrofit” as “to install new or modified parts or equipment in something previously manufactured or constructed” .The notion of overlapping EBI components with existing services can be found in several implementation science articles: a category from Stirman and colleagues’ program-modification typology, “Integrating the intervention into another framework — ie, selecting elements” ; Durlak  its fit , rather The difference between adaptation and retrofitting may merely be one of vantage point and degree. However, we suggest that recognition of the retrofitting phenomenon can inform various stages of the EBI pipeline: the design of programs and the trials to establish their efficacy; isolating core components and preparing EBIs for dissemination; conducting needs and capacity assessments of implementing organizations; training and technical support; and evaluations of the processes and outcomes of retrofitted programs and practices. We give brief examples of how this influence might occur:Program design and efficacy testing: A consideration of retrofitting may mean decreased frequency of designing programs “from scratch”  with increased attention to current practices and how they can be incrementally improved. Equitable community–academic partnerships may be especially valuable in this regard.Isolating core components: In a retrofitting-aware approach, current practice is seen as a foundation that can be improved with evidence-based modifications. Therefore, being able to accurately identify the “active ingredients” in a program (whether processes or principles) takes on special importance. The challenges we experienced in identifying YWC’s core components have been described (escribed . Althougescribed  for imprNeeds and capacity assessment: EBI implementation frameworks (ameworks ,21 typicTraining and technical support: Training and technical support that accommodate retrofitting would place greater emphasis on smoothly integrating EBI components into existing services and less on fidelity to the procedures used in the EBI’s original trial (al trial .Process and outcome evaluations of retrofitted programs and practices: Miller et al (er et al  pose an er et al . This aper et al , and 1 sSeveral limitations apply. First, the concept of retrofitting emerged from the qualitative data collected from a single evaluation, and we did not collect data specifically addressing this concept. In future studies, instruments could be developed to measure retrofitting more explicitly. Second, retrofitting may apply to only a limited number of circumstances; for example, it may be less relevant where the innovation under study changes a single existing practice as opposed to a multicomponent package of activities and processes. Third, our sample was small, consisting of 4 sites with different patient populations, recruitment approaches, and settings. Therefore, we were not able to correlate degree of retrofitting with important implementation-related outcomes such as reach, adoption, or sustainability. Nor were we able to associate retrofitting with asthma-related outcomes among participants. Notably, each program site reported improvements in asthma-related outcomes among program participants, offering tentative evidence that the retrofits contributed to, or at least did not hinder, the success of the programs .We found preliminary support for the validity of the retrofitting concept, although the explanatory or practical value of this concept is yet to be determined. The key assumption underlying the notion of retrofitting is that practitioners are doing things that work and that may benefit from evidence-based modifications rather than new interventions. This perspective aligns with calls for more practice-based evidence  and with


In addition to payments, desirable non-monetary program attributes and ecological feedbacks attract a wider range of potential participants into PES programs, including those who have more negative attitudes and lower trust. Designing programs that draw individuals into participating in PES programs is likely the most strategic path to reaching scale. Research should engage in new models of participatory research to understand these dynamics and to design programs that explicitly integrate a broad range of needs, values, and modes of implementation.Payments for ecosystem services programs have become common tools but most have failed to achieve wide-ranging conservation outcomes. The capacity for scale and impact increases when PES programs are designed through the lens of the potential participants, yet this has received little attention in research or practice. Our work with small-scale marine fisheries integrates the social science of PES programs and provides a framework for designing programs that focus  We offer a new way to think about participation in PES programs that employs a design perspective to enhance the capacity for a program to scale.Payment for ecosystem services (PES) and other conservation incentive programs have become increasingly common over the past two decades, but they have struggled to attract the necessary participation and scale to the extent needed to achieve broad-level impacts. Historically, architects of these programs have focused on factors that ensure and maximize the desired programmatic and environmental outcomes, such as conditionality, additionality, and cost-effectiveness –4. PES pWhile the needs of a species or ecosystem service receive substantial attention during the design phase of a PES program, human needs are largely assumed to be satisfied by conditional cash payments. However, the ability to scale a conservation program is substantially greater when PES programs also create reinforcing feedbacks via social norms and ecological outcomes that stimulate social contagion effects , 9. ViewOur work with fishing communities in Chile illustrates the potential for program desirability to create a scalable PES program. Chilean small-scale fisheries provide a relevant case study: a long-standing national policy gives the government the authority to assign territorial user rights (TURFs) to fisher associations for the exclusive acess to harvest benthic resources . Rights-There is a growing recognition of the potential benefits of TURF-reserves including increased species richness, biomass, and densities of reef fish and macro invertebrates as well as increased environmental stewardship and monitoring . A pilotOur research examined factors associated with program desirability that facilitate program scalability. By conducting social science research prior to undertaking efforts to scale the program, we sought to gain an understanding of the values, needs, and preferences of fishers that informs the program design in the scaling effort. We asked fishers across Chilean fisher associations to evaluate prototypes of TURF-reserve programs that differ on program characteristics related to program structure and the efficacy of achieving resource outcomes. By jointly considering payments and nonmonetary program factors, we can better understand how fishers perceive the overall value of participating in a program. We expect programs aligned with the preferences and needs of fishers will garner greater approval than those that are not. Further, we expect the greater participation occurs because desirable programs draw in fishers who have more negative attitudes and trust in the program.The sample frame included members of all 12 fishing associations with functioning TURFs in administrative Region V in the Central bioregion of Chile . We condThe TURF-reserve program was described to respondents as a marine biodiversity conservation program in which businesses, organizations, and agencies interested protecting marine biodiversity, either for philanthropic purposes or to offset environmental impacts elsewhere, would provide the fishing association with an annual cash payment to set aside 15 hectares of their fishing territory as a no-take protected area. We selected 15 hectares based on long-term ecological research demonstrating that a 15 hectare no-take marine protected area (MPA) in similar habitat can sustain a functioning marine ecosystem . Furthera priori, we asked fishers to indicate their approval of program prototypes, which involved the designation of a no-take zone within a fishing association’s TURF . Thus, we provided two scenarios for monitoring requirements: regular boat-based monitoring accompanied by stationary coastal video monitoring and the latter alone.Finally, because information feedbacks about collective efficacy of environmental protection programs are a key element in achieving collective action , we examWe employed a choice experiment approach to assess fisher preferences for programs . With thWe also considered the influence of three characteristics of small-scale fishers on participation: 1) attitudes toward a TURF-reserve program, including beliefs about outcomes of participating; 2) trust that the facilitating conditions exist for the program to be successfully implemented; and 3) livelihood dependence on fishing. Attitudes toward participating are a key predictor of behavior  and haveFoolish to Wise and Ineffective to Effective) and two evaluative indicators (Undesirable to Desirable and Bad to Good) [Risky to Safe). Because fishers may hold differing beliefs about how participating in the program might affect their income, we included their belief about the expected change in their income as a result of participating. Participants were asked if enrolling in the program would decrease, increase, or have no effect on their income.Employing attitude theory, we asked fishers to evaluate the merits of the TURF-reserve program based on two instrumental indicators (to Good) . We alsoDistrust entirely to Trust entirely). The target groups included fishing association leaders, consultants that often conduct assessments with fishing associations, police or security that might enforce poaching events, local government, fisheries officers, and fisheries scientists.Our indicators of trust focused on the enabling conditions present in the current social and political setting. Fishers indicated how much they trusted different groups who may be involved the TURF-reserve program  identity with one’s organization creates an enhanced willingness for members to cooperate in conservation actions , and truf ‘they’ .Almost no involvement to High involvement). They also provided an overall evaluation of their trust in the fishing association’s leaders.Trust in the association to engage in TURF-reserve program is also a function of an individual fisher’s involvement in the association, as well as the individual’s trust in the association’s leadership. We asked fishers to indicate their involvement in association issues in general, as well as in natural resource decisions  because this is the adoption rule used by fisher associations.i over j when Ui > Uj. The utility function is unobservable and instead we model the probability of choosing program i over j using a conditional logit formulation [M is all of the choice sets contained in the study, Vi and Vj are the observable utilities for each set of programs. Marginal effects were calculated at representative values of covariates using Stata (version 13), and we report them as predicted probabilities [Our model assumes that fishers are willing to approve a TURF-reserve program if the utility of participating is greater than not participating. Further, fishers will approve a program in which their overall preference for program structure and expected outcomes exceeds an alternative program. That is fishers prefer program mulation :P possible scenarios. This overall evaluation is based on the calculation of the probability of choice for one alternative over any other alternative(s), as:To examine the role of fisher characteristics and beliefs, we calculated predicted probabilities across payment levels for both desirable and undesirable programs while holding participant characteristics at representative values of low, medium, and high levels. Because these variables were measured on a continuous scale from 0 to 7, we selected the following: low = 1, medium = 3.5, high = 7. We rescaled the years fishing variable to fit a 0 to 7 scale. Combining these 3 levels each for attitude, trust, and livelihood dependence generates 27 . In the trust group, trust in police/security and fisheries officers were combined into a single indicator. The two collective identity indicators were combined as were the two measures of involvement. In the livelihoods group only two variables, Identity1 and Identity2 see  were comAll program characteristics, except monitoring requirements, have a significant impact on fishers’ approval of a TURF-reserve program see . An incrExpectations that the program will increase economically valuable loco and ecologically important reef fish have substantial influences on approval. If the TURF-reserve program increases the loco population by a moderate (10%) or large (20%) amount over the status quo, the odds of approval increases by 100% and 138%, respectively. A moderate increase in reef fish by 10% increases the odds of program approval by 50%. However, a large (20%) increase in reef fish over the status quo is not associated with an increase in program approval. This preference for only a moderate increase in reef fish may be in part because the primary reef fish species prey on benthic resources, leading to possible concerns about decreases in loco availability at high reef fish diversity .No differences in predicted participation exist between stationary coastal video monitoring with boat-based monitoring versus only coastal video monitoring. We suspect that this lack of a significant effect may be due to the fact that Chilean fishing associations commonly conduct boat-based anti-poaching monitoring within the TURFs .The relationship between participation and program desirability reflected by a combination of program factors and collective efficacy provides greater insight into designing PES programs to scale. We compared predicted approval for the most and least desirable programs. The most desirable program includes a 2-year renewable contract length, coastal video & boat-based surveillance, a large (20%) increase in the targeted resource, and a moderate (10%) increase in biodiversity. The least desirable program includes a 10-year renewable contract, coastal video surveillance, and no increase in the targeted resource and biodiversity.Programs with reduced commitments and that provide greater confidence in benefits have a greater potential to scale. A TURF-reserve program with the least desirable characteristics results in a maximum approval rating of 34% at the highest payment level  while thWhen species or habitat needs do not align with program participants’ needs, tradeoffs can be identified that retain the ability to assess the potential to scale PES programs. For instance, Chilean fishers are more likely to approve a desirable TURF-reserve program with a less-preferred 10-year contract than they are to approve an undesirable TURF-reserve program that contains a more preferred 2-year contract . ProgramThe success of scaling a PES program is also a function of the characteristics of potential participants. Individual attitudes toward the program affect participation, as does trust in the institutions involved and the degree to which potential participants are reliant on the natural resources involved .Payment levels offered to fishers have little influence in achieving majority approval if attitudes toward an undesirable program are negative. In fact, majority approval can only be achieved with the highest payment level when fishers have a positive attitude . In contrast, the relative change in approval of fishers with very positive attitudes was a factor of 1.77 with an absolute increase in probability from 34% to 61% (+27%). Similar relationships occur for trust. These results suggest that payments are important determinants of program approval, but by themselves are not sufficient to achieve threshold levels of participation necessary to drive landscape-scale marine conservation.Program desirability serves as a strong pull factor for a TURF-reserve program with respect to fishers’ trust that the enabling conditions exist for a program. For fishers with the highest degree of trust, majority approval is unachievable in an undesirable program, even at the highest payment level . In contHigh livelihood dependence on fishing has the greatest impact on approval of a TURF-reserve program with respect to program desirability. High dependence results in majority approval at all payment levels for both undesirable and desirable programs . For fislow-hanging fruit—favor the undesirable program, others tend to only approve desirable programs. For instance, the number of approved programs increases by 15% under desirable conditions because such programs are more likely to appeal to fishers with more negative attitudes, lower levels of trust, and to those who rely less on fishing for their livelihood Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Qualitative evaluation was carried out to understand the perceptions of the students participating in the Tier 1 Program of the P.A.T.H.S. Project. Five focus groups based on 43 students recruited from four schools were conducted to generate qualitative data to evaluate the program. With specific focus on how the informants described the program, results showed that the descriptors used were mainly positive in nature. When the informants were invited to name three metaphors that could stand for the program, the related metaphors were basically positive in nature. Finally, the program participants perceived many beneficial effects of the program in different psychosocial domains. Intra- and inter-rater reliability analyses revealed that the coding of the positive or negative nature of the responses was reliable. The present study provides qualitative support for the effectiveness of the Tier 1 Program of the Project P.A.T.H.S. in promoting holistic development in Chinese adolescents in Hong Kong.


The authors regret to report an unfortunate mistake in the Acknowledgements of the paper. It lists several projects that did not provide any financial support for this paper. The only project that provided the financial support for the work described in the paper is the Targeted Program “Research and Development on Priority Directions of Science and Technology in Russia, 2014–2021”, Contract no. 14.604.21.0101, Unique Identifier of the Applied Scientific Project: RFMEFI60414X0101.Therefore, the following statements are excluded from the Acknowledgements: "The work was partially supported (VP) in the framework of the Russian State Academies of Sciences Fundamental Research Program for 2013–2020. This work was also supported by the following grants of the EU FP7 program: “SysMedIBD” no. 305564, “RESOLVE” no. 305707 and “MIMOMICS” no. 305280."The correct version of the Acknowledgements is the following: “This work was done with the financial support of Federal Targeted Program “Research and Development on Priority Directions of Development of Science and Technology in Russia, 2014–2020”, Contract no. 14.604.21.0101, Unique Identifier of the Applied Scientific Project: RFMEFI60414X0101.”The authors would like to apologise for any inconvenience caused.


Background: Nurses are an essential human resource to ensure a healthy population and support the socio-economic development. However, little research has focused on the capacity development of nurses.Objective: The performance of a capacity development project for an undergraduate nursing program in Vietnam was reviewed to share lessons.Design: A descriptive case report.Setting: A baccalaureate nursing program in Vietnam from June 2014 to June 2016.Methods: A case report was analyzed in terms of the project's process, and the outcomes of 2 years' activities were evaluated.Results: Practice-based curriculum redesign and two basic nursing subjects were developed after five rounds of curriculum workshops. To improve application efficiency, two nursing experts were dispatched to provide instructions regarding the application of the new subjects. Three candidates were invited to complete their master's and doctoral studies in Korea. An advanced nursing education environment was supported with simulation labs equipped within a ubiquitous network. The result of experts' evaluation was excellent by every criterion of the Organization for Economic Co-operation and Development—Development Assistance Committee.Conclusions: The capacity development of a nursing program was possible through ownership, accountability, and results-based management. Gradual improvement in nursing academic and clinical capacity building based on research evidence can empower partner countries' nursing leadership. Introduction. Nurses are an essential human resource for strengthening the health system, as they help to ensure a healthy population, and support socio-economic development. The World Health Organization (WHO) identified six building blocks critical for a national health care system to result in improved health and effectiveness . The WHOThe term “capacity development” in this article refers to the process by which faculty and organizations get, improve, and maintain the skills and knowledge needed for curriculum development, teaching, and guiding nursing students in keeping with the global nursing educational standards . The effThe process of curriculum design is very important because it may provide a learning opportunity to the participants on philosophy, components, and the global direction. The analysis, design, development, and implementation (ADDIE) model is a linear approach to curriculum design; therefore, it is very easy to identify the process and activities. According to Voogt et al. , the ADDThis study aimed to review a 2-year project of capacity development for an undergraduate nursing curriculum in Vietnam, to share lessons learned and to discuss strategic cooperation direction, which influences nursing education policy and health care quality in Vietnam.Vietnam's health care system is predominantly a public-sector delivery system involving central and specialized hospitals, provincial and district hospitals, community health stations, and village health workers. This means that government regulations and policies can exert a powerful effect on the public sector. Vietnam's nursing education system has been developed over the last 20 years. There are two main programs to become a nurse: bachelor programs and 2-year programs. Four-year bachelor's programs are offered primarily in 10 national universities and three private universities; other nursing programs at the provincial level are 2-year junior college level programs.The author first visited Hue University of Medicine and Pharmacy (HueUMP) in July 2013 to request participation in the project for capacity building of hospital nurses in Vietnam, which was sponsored by the Korea International Foundation of Healthcare. Thereafter, the nursing program capacity of HueUMP was observed more closely in February 2014. Thirty-one hospital nurses were invited to HueUMP and participated in a 1-week train the trainer program held in the last week of February 2014, which was led by Korean and Vietnamese instructors. The infrastructure for the training program included one classroom and two nursing department practice labs at HueUMP. After the project, focus group interviews with the dean and vice-dean of the Faculty of Nursing were conducted. During these interviews, they requested that the nursing curriculum development be focused on competency and advanced educational infrastructure, parallel to the global trends. Therefore, a project team was formed to submit a 4-year project proposal to the National Research Foundation of Korea on the practice and competency-based capacity building of nursing undergraduate programs, and the proposal was approved in May 2014.HueUMP was established as a medical training institution and was administered by the Ministry of Health until 1993. The Ministry of Education and Training (MOET) took over supervision in 1994, and a full-time 4-year nursing program began in 2001, with 20 students enrolled per year, which increased to 150 students per year by 2015. Most of the nursing curriculum was designed based on a medical-oriented model and most of the faculty members were medical doctors. The first nursing master's program began at Hochiminh University of Medicine and Pharmacy in 2010, and the other two master's programs started in 2015 at Namdinh Nursing University and Vietnam National University in Hanoi.In a cross-sectional survey or needs assessment conducted by Nguyen et al. , more thThis study is a descriptive case report from the capacity development of an undergraduate nursing program at HueUMP, Vietnam which was implemented through official development assistance between Vietnam and South Korea.The setting was a baccalaureate nursing program including human resources and infrastructure of HueUMP, Vietnam between June 2014 and June 2016.The Faculty of Nursing at HueUMP consisted of 25 full-time and about 50 part-time faculty members. Systematic phases in previous development assistance projects by Hwang et al. , Shin etData were collected through the whole project process as shown in Figure In the analysis phase, situation analysis, and needs assessment were conducted three times. First, observational analysis was completed by hospital nurses in the trainer program during the last week of February. Second, in-depth interviews with deans and vice-deans of the Faculty of Nursing, HueUMP were conducted mid-April 2014. The analysis was conducted after the project launch from November to December 2014 using questionnaires.In the design phase, capacity development of a 4-year project was designed to upgrade a current HueUMP program to a competency-based nursing program. This was the first attempt at a comprehensive project, which strengthened the overall capacity of the nursing program; the activities of the Faculty of Nursing were otherwise limited to cooperation with short-term nursing faculty training and master's programs, support for nursing books, and co-practice for patients or communities by students from Finland, America, Australia, and the Netherlands.In the development phase, five areas of capacity development activities were identified: improvement of quality of teaching and learning with curriculum redesign, scholarship support, strengthening of the governance, construction of an advanced nursing education environment, and global network for nursing education. Throughout the development process, the researcher observed, and communicated with the participants and collected data.In the implementation phase, curriculum development workshops and faculty capacity building sessions were held in Korea and Vietnam. Renovation of education infrastructure, activities of the curriculum development committee, and evaluation were implemented in Vietnam. Related reports and comments were collected for this study.In the evaluation phase, the accomplishment of the first phase of the project was evaluated by the project team, using the nursing faculty's self-evaluation, followed by external nursing and medical experts' evaluation each year based on five criteria of the Development Assistance Committee of the Organization for Economic Co-operation and Development (OECD-DAC). The criteria were relevance, efficiency, effectiveness, impact, and sustainability in the guidelines for project and program evaluation by the Austrian Development Cooperation . Those eAll the project activities' outputs and 2 years' performance of projects were analyzed by areas of capacity development aligning with OECD-DAC evaluation criteria as shown in Figure Three invited training sessions for curriculum development were conducted with delegates of the Faculty of Nursing, HueUMP, and two visiting workshops by Korean nursing education experts at Cheju Halla University were conducted to expand faculty members' ability to redesign nursing curriculum, during summer and winter vacation in 2014 and 2016, to avoid routine academic program operations as shown in Table To monitor and provide active support onsite during the nursing program renovation and faculty capacity building, a retired nursing advisor was dispatched to encourage curriculum development committee activities. In addition, a retired nursing professor with profound experience in problem-based learning method and curriculum development was dispatched to mentor the application of new nursing subjects in problem-based learning packages and pilot testing with nursing students, at HueUMP as shown in Table For faculty training, several Korean nursing education experts' invited workshops were conducted to increase the quality of teaching and learning, such as creative thinking in nursing education, development of two problem-based learning packages, nursing statistics, and writing for research journals in English.The most critical problem for the undergraduate program was lack of faculty with a nursing background. From a long-term perspective, fostering faculty human resources is the most important capacity building aim at the personal and institutional level. Although a tripartite collaboration for Vietnam, with master's and doctoral programs in Thailand and support for tuition from the Korean government, was considered, there were many related political issues.In terms of the nursing faculty, clinical practice assistants were nurse graduates waiting for sponsorship for their master's and doctoral study, so the project team had a meeting and asked for funding from the Ministry of Education of Korea. Finally, at the request of HueUMP, three candidates were selected for 1 year of Korean language support and master's and doctoral nursing programs in Korea. All tuition was sponsored by the Global Korea Scholarship program under the Ministry of Education in Korea.After 1 year of Korean language education and approval of the qualification of proficiency in the Korean language, the candidates could enter the advanced nursing program in Korea. The first three candidates came to Korea in 2016 and started language training in the designated Korean language center.The curriculum redesign process should include the needs of nursing consumers  and internal consensus among faculty members, university authorities, and the MOET in Vietnam. In total, 20% of the nursing program was managed by the university authorities without the approval of the MOET. A nursing curriculum redesign committee was organized with 15 members: two external experts in clinical nursing, two nursing lecturers with nursing backgrounds, four nursing faculty and five university authorities, and two Korean nursing professors from the project. This committee met at least bimonthly to reach a consensus on the revised nursing curriculum. All of the invited training faculty in Korea were selected based on the criteria of gender and educational background in medicine or nursing on an equal basis.Through the project process, a bottom-up approach for new nursing subject development and top-down approach for policy support in the university and nationwide were reciprocally applied. All of the purchased educational equipment and the remodeled Hue-Halla nursing education center were managed by the project team, with the support of the University President, which was acknowledged in a national economics magazine.Based on a needs assessment and the study result of Kang et al. , a learn2 of space. This new building was constructed through funds from an American non-government organization called Atlantic Philanthropies. The discussion room was appropriate for small group teaching and learning using five desktops for group tables and one instructor control table. All of the learning labs and spaces were networked for ubiquitous learning and an inauguration ceremony of the Hue-Halla center was held in September 2015 after several months of pilot operation in each lab and space.Renovation of the nursing education environment was coordinated through mutual communication and based on agreement of projects. This included two nursing simulation training labs and three labs for basic life support and advanced life support training. In addition, a two-way computerized discussion room with a ubiquitous learning network was furnished on the fifth floor of the newly built learning resource center in 2015 with 355 mTo meet the future nursing leaders and potential faculty candidates, a special student program was developed through an educational needs assessment. In the needs assessment study by Kang et al. , Borich'Nurses in low-resource setting countries can be empowered when their basic educational capacity development and the culture of nursing practices become evidence-based. According to Shin et al. , the ICNCurriculum redesign and development is a strategic approach because it encompasses and meets global nursing education norms as well as human resource development and infrastructure refurbishment. According to Evans et al. , Kang etIn this study, the main areas of faculty training in previous research were upgrading knowledge and skills, teaching method, transforming theory into practice, research ability, leadership, and clinical education capacity as demonstrated in previous studies , 4, 6. TRegarding ownership and accountability, all the faculty members were welcome to contribute ideas and opinions about modules or nursing subject development and the application process. Another method of change governance was to set criteria to form a committee based on major and gender, to avoid male and non-nursing major dominant situations. However, there has been very little research on governance or administration training for nurses; it was good for capacity building through bottom-up and participatory approaches in project implementation. Koto et al.  and ShinIn terms of educational environment, Evans et al.  also indGlobal partnership is also another success factor for future sustainability; however, it was hard to identify networking with other countries in the present project activities. The majority of nursing programs in HueUMP have partner institutions worldwide, including other less-developed countries, and it is not difficult to meet global partners in those developing countries. According to Sies et al. , Tvelt eThis case report on a Vietnam nursing capacity building project was very similar to the situation of the Republic of Korea. The Republic of Korea was a very poor country in the 1950s after the Korean War, and Western assistance for socio-economic development helped to revive Korean society, especially with respect to scholarship programs. In an effort to improve human resource development, the first nursing doctoral program began in 1973 and changed to an advanced nursing curriculum model from a medical-oriented program after there were enough faculty members with the required qualifications to operate the program during the 1960s and 1970s. In terms of war and development processes, Korea's past experience could be a good model for Vietnam's advancement in all areas. Vietnam has seen change in its socio-economic level since the Doi-moi economic reform in 1986 and the Vietnam War in the 1970s. Though Vietnamese people possess positive potential, a fundamental shortage of human resource development and know-how for further advancement in higher education means that long-term cooperation in education is needed.Though the nursing profession usually lacks firm and powerful strategic representation in healthcare policy, according to Kang and Kim , in deveAccording to the evaluation of each criterion, project teams showed good communication with stakeholders in Vietnam and Korea and accomplished all of the performance indicators within two project years with the full use of the budget. The appropriateness of the project was guaranteed by Vietnam's socio-economic development plan, HueUMP's strategy of development, and Korea's high education know-how transfer vision. In particular, the project outcome was remarkable for resource networking through understanding and cooperation with global partners visiting the Hue region. The impact of the project was publicly promoted naturally to North Vietnam, and one faculty member visited HueUMP to witness what was achieved. To guarantee sustainability, the inclusion of educational policy or regulations and sharing project outcomes with faculties in other regions should be considered.However, there is a gap among healthcare professionals in their educational level and national standards, self-regulation, social position, difference between academic and clinical reality, and evidence-producing research ability. In developed countries, healthcare professionals have come to be recognized as a healthcare team and respect other professional fields; however, this culture is lacking in developing countries. Therefore, it takes time to increase the level of the nursing profession as a healthcare team in terms of educational and clinical capacity, based on the linkage of those capacity areas to research evidence.In conclusion, the priority success factor of project accomplishment was ownership and accountability of the partner institution or recipient organization. A nursing program in developed countries would not be appropriate to apply directly to developing countries, because of differences in culture, educational development level, and preparing faculty. Further research should consider trans-cultural and participatory approaches for sustainable development.SK substantially contributed to the study conception, design, and writing of the manuscript, as well as acquisition and interpretation of the data; TN contributed to the study design and writing of the manuscript; TH contributed to the acquisition and interpretation of the data.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.


In this article, two types of Hardy’s inequalities for the twisted convolution with Laguerre functions are studied. The proofs are mainly based on an estimate for the Heisenberg left-invariant vectors of the special Hermite functions deduced by the Heisenberg group approach. Then, by the cancelation condition of p-atom and Lemma p-atom. □From p.58 of  we see tFor For the term Now, for the term p-atom, we have Again assume In this paper we have used the Heisenberg method to prove two Hardy’s inequalities for the twisted convolution with Laguerre functions. We also note that the two classical Hardy’s inequalities can be written as


This study reports on the effectiveness and efficiency from the program funder’s perspective of the Suraj Social Franchise (SSF) voucher program in which private health-care providers in remote rural areas were identified, trained, upgraded, and certified to deliver family planning services to underserved women of reproductive age in 29 districts of Sindh and 3 districts of Punjab province, Pakistan between October 2013 and June 2016.A decision tree compared the cost of implementing SSF to the program funder and its effects of providing additional couple years of protection (CYPs) to targeted women, compared to business-as-usual. Costs included vouchers given to women to receive a free contraceptive method of their choice from the SSF provider. The vouchers were then reimbursed to the SSF provider by the program.A total of 168,206 married women of reproductive age (MWRA) received SSF vouchers between October 2013 and June 2016, costing $3,278,000 ($19.50/recipient). The average effectiveness of the program per voucher recipient was an additional 1.66 CYPs, giving an incremental cost-effectiveness of the program of $4.28 per CYP compared to not having the program (95% CI: $3.62–5.31).The result compares favorably to other interventions with similar objectives and appears affordable for the Pakistan national health-care system. It is therefore recommended to help address the unmet need for contraception among MWRA in these areas of Pakistan and is worthy of trial implementation in the country more widely. In 2012, the London Summit on Family Planning proposed the ambitious “FP2020” commitment to increase coverage of modern contraceptive methods (MCMs) for 120 million more women and girls by 2020 (Pakistan initiated family planning (FP) programs first in 1953 in the private sector and then in the 1960s in the public sector. However, the contraceptive prevalence rate increased by only 0.25% annually until 1990 when it rose more sharply but still remained behind other countries in the region . ImproviThe Unites States Agency for International Development (USAID) has supported interventions to increase coverage and improve the quality of FP/RH services in Pakistan. One intervention is the Suraj Social Franchise (SSF) implemented by MSI, in which private providers working in remote rural areas are identified, trained, certified and inducted into the franchise. MSI conducted a similar social franchise implementation in Mali from 2012 to 2015. It was successful in this setting in improving FP access, choice and use of contraception among the rural poor .In the Pakistan SSF program, the quality of FP/RH services delivered is improved by a continuous supportive supervision mechanism. From 2013, with support from USAID, Marie Stopes Society (MSS) Pakistan (MSI’s country affiliate) used vouchers that allowed married women of reproductive age (MWRA) to access FP/RH services that are free at the point of service . The purThe Unites States Agency for International Development has supported its implementing partner MSS to expand the social franchising network to promote RH/FP services to poor and underserved communities in 32 districts of Pakistan. The franchise is a partnership through which MSS gives training and supervision to private local health service providers, enabling them to provide high quality services to eligible low-income and otherwise disadvantaged women. The women are given vouchers, through door to door visits and support group meetings by the Field Health Educators, that they can redeem for RH/FP services without additional out-of-pocket costs. This facilitates demand for the services while still allowing women to choose their service provider from among the many who are participating in the program. Primary targets of the intervention are MWRA (aged 15–49 years) who live in rural and peri-urban areas with high unmet need of FP and identified as disadvantaged on the Progress out of Poverty Index tool .1All SSF facilities underwent third-party post-training evaluation to ensure they were adequately staffed and equipped to provide RH/FP services to national standards. One type of facility, SSF-As, provides short- and long-term contraception methods while SSF-A+ facilities have doctors on staff and available to provide contraceptive implants and tubal ligation in addition to the short- and long-term methods SSF-As can provide. Between 2013 and 2016, MSS inducted 255 SSF-As and 45 SSF-A+ to make a total of 301 SSFs in the 29 districts in Sindh and 3 districts in Punjab. They also provided technical assistance to those providers who required it to become eligible for inclusion as an SSF service delivery center. All contraceptive supplies were provided to women with vouchers and MSS provided ongoing mentoring and supervision at the facilities to maintain high standards of service delivery.A decision tree was constructed to model the program in operation in the 32 districts Figure . Such moThis study relied solely on data already collected during routine monitoring and evaluation of the program and was anonymized for use in this study. Because no additional data were required from human subjects, it was considered exempt from full Institutional Review Board application in the US by University Research Co. Inc. and in Pakistan by the MSS.We used activity-based costing to estimate the resources consumed in implementing this program. Inputs for program costs were compiled from the administrative records of the implementation available from MSS Pakistan. They were divided into (1) voucher management cost, (2) facility support costs, and (3) FP consultation costs.Social franchise program costs included all staff time, transportation, and administrative costs involved in getting the vouchers to the recipients.Facility support costs included the time taken by the project staff to conduct the training in participating facilities to ensure they meet the standards of quality service to be part of the program. It also included the cost of the upgrades in equipment and supplies to the facilities required to meet the standards, clinical monitoring and refresher training courses and supervisory visits. No building or infrastructure changes were made to the facilities.Consultation and service costs for those receiving FP services were mostly captured in the amount redeemed by the private health-care provider using the voucher for payment for the services they rendered. It did not include the cost of contraceptives supplied because these were not borne by the project but many of the supplies were received through the USAID Deliver Project.Baseline contraceptive prevalence was obtained from a study of coverage in rural Pakistan .Effectiveness measures for the program were primarily the uptake of contraceptive methods by MWRA who were eligible for the vouchers. This was measured by MSS as part of their routine monitoring and evaluation system. Secondary measured were couple years of protection (CYP). Both of these were derived from the records of contraceptive uptake determined from redemption of the vouchers. Records of these were recorded and collected in each of the participating providers. For example, if one married woman of reproductive age redeemed a voucher at a participating SSF-A for a tubal ligation, the service provided would be assigned 10 CYPs to represent the what is attributed to that form of contraception in CYPs , 12. We A total of 168,206 women of reproductive age received the FP vouchers in this program over the period between October 2013 and June 2016 for a total cost of $3,278,000. The average cost of the program per woman who received the services was $19.50. This includes the cost of upgrading the facilities providing the services divided by the total number of voucher recipients using services at those clinics. The average effectiveness of the program per voucher recipient was an additional 1.66 CYPs. This gives an incremental cost-effectiveness of the program of $4.28 per CYP compared to not having the program (95% CI: $3.62–5.31).One-way sensitivity analysis was conducted on all of the major input variables to determine their relative effect on the overall results. We increased the values one at a time by 10% to determine the change and magnitude of the incremental cost-effectiveness ratio Table . For exaOne-way sensitivity was also done on other variables in the model that were varied within their feasible range to determine their effect on the overall result Figure . For exaInterventions to increase access for women to MCMs in LMICs is important for reaching global health goals but there are few studies on the cost-effectiveness of such interventions . This stThe perspective taken for this evaluation was the funder’s—USAID—and it was conducted to inform decision-making by current and potential future external funders. If the analysis was considered from the societal perspective, other costs and consequences of the program would need to be considered. These include additional costs for the contraceptive supplies and the time cost for the MWRA seeking services. On the potential cost-saving side, there would be the decrease in expenditure of the health system due to fewer pregnancies occurring, especially unwanted or high-risk pregnancies that would be avoided when the problem of unmet contraceptive need is satisfied with implementation of the program. Considering the results from this study, it is expected that using this alternative perspective would make the program even more cost-effective than it appeared from the external funder’s point of view. Therefore, we recommend implementation of this program to the provincial or national government if external funding cannot be secured because of its affordability and efficiency and because its benefits accrue to society.From the tornado diagram and the one-way sensitivity analysis in Table It is possible that the volume of MWRAs could be increased improving outreach in the region targeted by the intervention or by increasing the geographical coverage without substantive changes in at least some of the administrative and capital costs to gain from economies of scale. It is also possible that some changes in the program, possibly with more behavior change communication, while respecting the client method of choice, to encourage permanent contraception methods or LARCs that last longer would increase the CYPs per consultation which would also improve the cost-effectiveness of the program .It is estimated that there are 50 abortions per 1,000 women in Pakistan or a total of 2.2 million annually . It is aLimitations in this evaluation include the dearth of baseline data available for contraceptive use among MWRA in the target population. Health information systems are weak in Pakistan , and accAnother weakness of the evaluation was the assumption that all services provided for by the voucher program and therefore counted as additional services, with their associated CYPs, attributed to the presence of the SSF. It is possible that a proportion of MWRAs receiving such services would have done so regardless of the SSF program. This assumption may have biased the result toward a more positive outcome: a lower cost per CYP than there may actually be.The cost-effectiveness of this implementation of the SSF in 29 districts in Sindh and 3 districts in Punjab between October 2013 and June 2016 was $4.27 per additional CYP compared to business-as-usual from the perspective of the funder. This compares favorably to other interventions with similar objectives and appears affordable for the Pakistan national health-care system. It is therefore recommended to help address the unmet need for contraception among MWRA in these areas of Pakistan by considering a trail implementation in the country more widely.This research involved the analysis of routinely collected data from patients that were completely anonymous. No additional data were collected. The research was therefore exempted from full Institutional Review Board review in both Pakistan and the United States.EB designed the study, wrote the protocol, performed the analysis, and wrote the first draft of the manuscript. WH and AG collected data on effectiveness of the program and provided information of implementation. SS and IB collected the cost data and helped perform the preliminary analysis on costs. MV designed and supported the overall evaluation and provided guidance on the program’s history and critical revisions to all versions of the manuscript. WH, AG, and SS all had critical input on the design of the study, protocol development, and all versions of the manuscript. All the authors approved the final version of the manuscript.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.


The antibody isotype IgM appears earlier than IgG, within days of onset of symptoms, and is important during the early stages of the adaptive immune response. Little is known about the functional role of IgM during infection with chikungunya virus (CHIKV), a recently reemerging arbovirus that has caused large global outbreaks. In this study, we studied antibody responses in 102 serum samples collected during CHIKV outbreaks in Malaysia. We described the neutralizing role of IgM at different times post-infection and examined the independent contributions of IgM and IgG towards the neutralizing capacity of human immune sera during the early phase of infection, including the differences in targets of neutralizing epitopes. Neutralizing IgM starts to appear as early as day 4 of symptoms, and their appearance from day 6 is associated with a reduction in viremia. IgM acts in a complementary manner with the early IgG, but plays the main neutralizing role up to a point between days 4 and 10 which varies between individuals. After this point, total neutralizing capacity is attributable almost entirely to the robust neutralizing IgG response. IgM preferentially binds and targets epitopes on the CHIKV surface E1-E2 glycoproteins, rather than individual E1 or E2. These findings provide insight into the early antibody responses to CHIKV, and have implications for design of diagnostic serological assays. Togaviridae. Its emergence has led to unprecedented global epidemics among immunological naïve populations across different countries in Asia, the Americas, Africa and Europe [Aedes mosquitoes serve as the main vectors in disease transmission. Phylogenetic analysis reveals the existence of three distinct CHIKV genotypes: West African, East/ Central/ South African (ECSA) and Asian, with the ECSA genotype causing the recent epidemics in India, the Indian Ocean and Southeast Asia, while the Asian genotype is responsible for the recent extensive outbreaks in the Americas and the Caribbean.Chikungunya virus (CHIKV) is an alphavirus from the family d Europe . Aedes m9 viral RNA copies per ml occur during early infection, and viremia may last for 5–7 days [Infection of CHIKV is characterized by abrupt fever, profound acute joint pain, myalgia and erythematous maculopapular rashes , 3. Othe5–7 days . Interfe5–7 days . General5–7 days .-/- or Rag2-/- (lacking mature lymphocytes) and μMT (B-cell deficient) mice resulted in persistent viremia accompanied by joint inflammation [Pre-clinical studies on mouse models have shown the importance of antibody-mediated immunity in controlling infection , 12. CHIammation , 13, 14.ammation . Immune ammation , 17.The functional role of infection-induced specific IgM against CHIKV is less well-characterized compared to immune IgG during acute and early convalescent phases of infection in mice and humans. Infection of athymic mice with the closely related alphavirus Semliki Forest virus revealed the role of IgM in clearing viremia, but not virus localized in the brain . InductiWe hypothesized that IgM is important to provide early immunoprotection  prior to appearance of the full IgG response. The objectives of this study were to assess the function of immune (infection-induced) IgM in CHIKV neutralization, and to compare the contribution of IgM and IgG towards neutralizing capacity of human immune sera. We found that neutralizing IgM starts to appear as early as day 4 after disease onset and its appearance is associated with a reduction of viremia starting from day 6. IgM has the dominant neutralizing role up to day 10, with variable but strong contributions by neutralizing IgG. The neutralizing IgM preferably targets epitopes on the CHIKV surface E1-E2 glycoproteins.This study was approved by the Medical Ethics Committee of the University Malaya Medical Centre (reference no. 20157–1467). Our institution does not require informed consent for retrospective studies of archived and anonymized samples.This study used two panels of serum samples. Panel A comprised 27 samples collected from patients attending University Malaya Medical Centre, Kuala Lumpur, during the 2008–2010 outbreak of CHIKV of East Central/ South African (ECSA) genotype. These were acute samples collected from viremic patients between day 1 and day 9 after disease onset. Viral loads had been quantified by real-time PCR targeting the E1 region in a previous study . In thisFor panel B, the neutralizing titers of IgM and IgG were determined separately in 79 samples to study the relative contributions to total neutralizing activity. Of these 79 samples, 39 serum samples were from the same 2008–2010 outbreak and known to contain neutralizing anti-CHIKV IgM and/or IgG . These sSerum samples from 15 healthy controls with no past infection of CHIKV were included as negative controls, confirmed by the absence of antibodies by serum neutralization assay. This made a total of 117 sera used in this study.Baby hamster kidney (BHK-21) cells (ATCC no. CCL-10) were maintained in Glasgow minimum essential medium (GMEM)  supplemented with 5% heat-inactivated fetal bovine serum , 10% Tryptose phosphate broth , 20 mM HEPES, 5mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies). Infected cells were maintained in GMEM containing 2% FBS.The virus strain used was MY/08/065 (GenBank accession number FN295485) at passage 3, a previously characterized ECSA virus isolated from a patient in Malaysia in 2008 . The virTwo types of virus antigens were used for this study. For Western blot and indirect IgG ELISA, the antigen was partially purified virus prepared by sucrose-cushion ultra-centrifugation. Virus pellet was treated with 1% Triton X-100 in TE buffer, clarified by centrifugation and stored in the presence of 50% glycerol at -20°C. For capture IgM ELISA, formalin-treated virus supernatant was utilized as antigen. Formalin (37%)  was added to a final concentration of 0.75%, with constant rotation at 4°C for 24 hours. This source of antigen was used within 3 days when kept at 4°C.Standard molecular cloning was performed to clone E1 glycoprotein  and E2 glycoprotein , without the transmembrane and cytoplasmic tail regions, into a pIEX-5 vector . Fusion rE1 and rE2 was generated by overlapping PCR to link both fragments with a short linker GGGS-His (8X)-GGGG. All the constructs were transfected into TriExSf9 cells (Novagen) by TransIT-Insect Transfection Reagent  . The rec6 pfu per well of formalin-treated virus supernatant diluted in 1% BSA-PBS, or purified recombinant rE1 or rE2 glycoprotein diluted in 1% BSA-PBST to a final concentration of 20 μg/ml. Anti-CHIKV antibody was diluted in 1% BSA-PBST to a final concentration of 1 μg/ml and added to the plate, which was then incubated for 30 min. For the ELISA using rE2 and formalin-treated virus as antigens, anti-E2 monoclonal antibody B-D2(C4) [A capture IgM ELISA format was developed to determine the presence of anti-CHIKV IgM. All the incubation steps were performed at 37°C for 1 hour, using 1% BSA-0.05% PBST as diluents for serum and antibodies. The plates were washed 4 times with 0.05% PBST after each incubation step, and 6 times after antigen , and monoclonal and secondary antibody incubation steps. The plate was coated with rabbit anti-human IgM  to a final concentration of 2.8 μg/ml and blocked with 3% BSA-0.05% PBST. Sera were diluted at 1:200 and added. Antigen was then added, and this was either 10B-D2(C4)  was the B-D2(C4)  was usedIndirect IgG ELISA was performed on all CHIKV immune sera at a dilution of 1:1000 with similar incubation conditions and washing steps (4 times). The plate was coated with 250 ng of whole virus antigen or 100 ng of rE2 and blocked with 3% BSA-0.05% PBST. Diluted sera were added to the plate and the bound antibodies were detected by addition of rabbit anti-human IgG-HRP  at 1:5000 dilution.The proteins were resolved with 12% SDS-PAGE under non-reducing or reducing conditions and electro-transferred onto a nitrocellulose membrane . The membrane was blocked with 10% skimmed milk in 0.05% PBS-Tween 20 (PBST). For IgM detection, the pooled sera were treated with RIDA RF-Absorbens  in 1% bovine serum albumin (BSA)-PBS prior to blotting. The immunoreactivity of recombinant CHIKV proteins and virus antigen were evaluated at 1:100 and 1:400 dilutions. The bound antigen-antibody complex was detected by goat anti-human IgM-HRP  at 1:5000 dilution in 1% BSA-0.05% PBST. The membrane was visualized by chemiluminescence  and images were acquired by BioSpectrum AC imaging system . Mouse anti-His tagged antibodies  were included as the control.4 cells in a 96-well CellCarrier-96 optic black plate  and further incubated for 1.5 hours at 37°C. The inocula were decanted and 2% FBS GMEM was added. The plate was fixed with 4% paraformaldehyde after 6 hours of incubation at 37°C, permeabilized with 0.25% Triton X-100 for 10 minutes, and immunostained using CHIKV monoclonal antibody clone B-D2(C4) [50) was expressed as the serum dilution that reduced infectivity by 50% using non-linear regression fitting in GraphPad Prism 5. For non-converged regressions, the neutralizing titer was set to 1.Seroneutralization was performed with a previously described immunofluoresence-based cell infection assay in BHK-21 cells , 35, 36.B-D2(C4)  at 1 μg/To assess the IgM neutralization activity, the human IgG antibody from heat-inactivated sera was first precipitated with RIDA RF-Absorbens (R-Biopharm). The absorption buffer was prepared in 1:10 dilution in 1× DPBS and serum was diluted 2-fold with absorption buffer from 1:100 to 1:1600. The IgG antibody from samples was precipitated at 37°C for 30 min prior to mixing with CHIKV. The cells were rinsed with 1× DPBS at the end of cell-virus mixture incubation prior to replenishment with maintenance medium. For determination of neutralizing activity solely due to IgG, the heat-inactivated sera were treated with 0.1M dithiothreitol (DTT) (Life Technologies) to a final concentration of 5mM, to inactivate IgM, and were incubated at 37°C for 1 hour prior to dilution from 1:100 to 1:6400.For seroneutralization using chimeric viruses, heat-inactivated serum was first treated to obtain anti-CHIKV IgM and IgG independently. Diluted sera were mixed with viruses pre-diluted with 2% FBS GMEM, with infection performed at an MOI of 50, followed by the steps described above. After 7 hours of incubation at 37°C, the plates were fixed and counter-stained with DAPI prior to acquisition of zsGreen fluorescence.P-value of <0.05 was considered significant. All the statistics were performed with GraphPad Prism 5.Data are presented as means ± standard deviation (SD) or means ± standard error of the mean (SEM). Differences between groups and controls were analyzed using appropriate statistical tests, as stated in the figure labels. A The IgM and IgG titers were measured after IgG precipitation or IgM inactivation steps to ensure that the neutralizing roles of IgM and IgG were examined independently. The efficacies of IgG precipitation and IgM inactivation were 96% and 80%, respectively . To unde50 IgM/NT50IgG was more than 1, indicating a predominant role for neutralizing IgM  and waned over time ; IgM waszing IgM . These szing IgM . The inacapacity . This int of IgM . Both loinst rE2 . These rTo further understand the neutralization characteristics of IgM, the target epitopes of IgM were investigated using the serum samples from panel B1 against the individual structural envelope glycoproteins. Immunoblotting analysis was performed under non-reducing conditions. At 1:100 serum dilution, anti-CHIKV IgM poorly recognized rE1 and rE2 glycoproteins with undefined bands, but strongly bound to the whole virus antigen, with a band of 50 kDa consistent with E1 or E2 . At highNeutralizing antibodies in viral immunity provide protection by clearing viremia. Other effector functions of IgM and IgG are complement activation, opsonization of antigens for phagocytosis and antibody-dependent cell-mediated cytotoxicity , 39. NeuIn this study, we defined the independent roles of anti-CHIKV IgM and IgG from immune sera, and showed that both antibody isotypes have neutralizing characteristics similar to previously reported humanized/mouse monoclonal antibodies –45. In oBetween days 4–10, there was individual variation in the relative contributions of IgM and IgG to overall neutralization capacity, with a few patients displaying predominant IgM and most others having predominant IgG. After day 10 from disease onset, IgM contributes minimally to overall neutralizing activity, as neutralizing IgG plays the dominant role. During this period of seroconversion (day 4 to 10), strong overall neutralizing capacity can be achieved with synergistic binding action of IgM and IgG against infectious virus . The earThere has been no prior study of IgM epitopes for CHIKV. Current commercial diagnostic serology kits to detect IgM in acute samples showed poor sensitivity when using recombinant E1 glycoprotein alone as the antigen, showing the importance of epitopes on E2 –68. ThisDevelopment of vaccine candidates also requires sufficient understanding of antibody responses to enable design of assays that reflect vaccine effectiveness. Our study offers several relevant insights. IgG ELISA is useful as it correlates with total neutralizing antibodies, which are predominantly IgG and are correlated with protection against symptomatic CHIKV . HoweverOur key findings were supported by additional study of acute and early convalescent samples from a 1999 outbreak of CHIKV (Asian genotype) in Klang , 74. TheIn conclusion, CHIKV-infected individuals develop neutralizing anti-IgM and anti-IgG during the early phase of infection, which mediate the clearing of viremia. Neutralizing IgM is particularly important up to day 10 of infection, when it acts in a complementary manner with the early IgG, after which a robust neutralizing IgG response consistently predominates. Anti-CHIKV IgM preferably recognizes epitopes on the CHIKV surface E1-E2 glycoproteins rather than E1 or E2 individually, which has implications for the design of diagnostic IgM assays.S1 FigP<0.01, Mann-Whitney U test. (B) ELISA to whole virus antigen was carried out to evaluate the efficacy of IgM inactivation by DTT from pooled acute sera at 1:100 dilution. The IgG level was maintained after treatment. Data are presented as mean + SD (n = 3). **P< 0.01, Mann-Whitney U test.(A) ELISA to whole virus antigen was carried out to evaluate the efficacy of IgG precipitation from pooled acute sera at 1:100 dilution. The IgM level was maintained after treatment. Data are presented as mean + SD (n = 3). **(PDF)Click here for additional data file.S2 FigP<0.05, **P<0.01, ***P<0.001 by two-way ANOVA with Bonferroni multiple comparisons test. Data are presented as mean ± SD.(A) The addition of LP1 antibody at 0.5 μg/ml improved the overall neutralizing capacities in the presence of IgM. Data are presented as means ± SD. Dotted lines represent the infectivity with 0.5 μg/ml LP1 antibody (IgG) in the absence of IgM. (B) The complementary effects of neutralizing IgM and IgG antibodies was also shown with different concentrations of neutralizing anti-LP1 IgG at 1 and 3 μg/ml, but not with non-neutralizing IgG  and rabbit antibody E2dA, which target linear epitopes on the surface of domain A, E2 glycoprotein). Dark green and purple dotted lines represent the infectivity with LP1 antibody treatment at 1 and 3 μg/ml, respectively, in the absence of IgM. Serum MY/ES10 was used in this experiment. All experiments shown were performed in triplicate from 1:100 to 1:1600 dilutions. Results are expressed as percentage of virus control. *(PDF)Click here for additional data file.S3 Fig50 IgM and NT50 IgG was assessed for different serum panels using Spearman’s rank correlation coefficients (ρ). P-values are shown; ns, not significant. Four samples from panel B1 were excluded from analysis as they were collected early within the seroconversion period, and NT50 IgM was predominant over NT50 IgG.The correlation between NT(PDF)Click here for additional data file.S4 FigP<0.05, **P<0.01, ***P<0.001 by two-way ANOVA with Bonferroni multiple comparisons test. Data are presented as mean ± SD for individual samples with low NT50 IgG and mean ± SEM for 11 samples with high NT50 IgG. (B) Schematic diagram showing the chimeras used in seroneutralization with comparison of infectivity using pooled serum samples. Data are presented as means ± SD from 4 independent experiments at a serum dilution of 1:100. *P<0.05, **P<0.01, ***P<0.001, Kruskal-Wallis test.(A) Neutralization of virus infectivity due to IgM or IgG was compared to neutralizing capacity due to total antibodies. Results are expressed as percentage of virus control. *(PDF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Cyprinus carpio) populations having high titers of viral neutralization in vitro after surviving an experimental infection with cyprinid herpes virus 3 (CyHV-3). The range of diversity of the induced antibodies was unexpectedly high, showing CyHV-3 infection-dependent, non-specific IgM-binding activity of a ~20-fold wider variety than that found in sera from healthy carp  with no anti-CyHV-3 neutralization titers. An inverse correlation between the IgM-binding levels in healthy versus infection-survivor/healthy ratios suggests that an infection-dependent feed back-like mechanism may control such clonal expansion. Surprisingly, among the infection-expanded levels, not only specific anti-frgIICyHV-3 and anti-CyHV-3 IgM-binding antibodies but also antibodies recognizing recombinant fragment epitopes from heterologous fish rhabdoviruses were detected in infection-survivor carp sera. Some alternative explanations for these findings in lower vertebrates are discussed.IgM antibody diversity induced by viral infection in teleost fish sera remains largely unexplored despite several studies performed on their transcript counterparts in lymphoid organs. Here, IgM binding to microarrays containing ~20,000 human proteins was used to study sera from carp ( Cyprinus carpio) that survive cyprinid herpes virus 3 (CyHV-3) infection when using indirect enzyme-linked immunosorbent assays (ELISAs) using purified whole virus  or infectious hematopoietic necrosis virus (IHNV) using indirect ELISAs  natural, non-specific IgM present in healthy sera should be optimally reduced to estimate specific and accurate IgM-binding levels for diagnostic purposes and (ii) fish infection-dependent IgM antibodies and B-cells may generate cross-reactivity properties characteristic of trained immunity, a possibility that has been previously unrecognized even in mammalians. Future work along these lines may help to understand how those complex fish non-specific IgM responses are generated and evolve, and whether or not they may have any importance in the prevention of other diseases.C. carpio) was kindly provided by Dr. Lee (Taiwan) and used as previously described  as previously described , 1 mM pyruvate, 2 mM glutamine, 50 µg/ml gentamicin, and 2.5 µg/ml fungizone. To replicate VHSV or CyHV-3, a similar cell culture medium containing 2% FCS was used at 14 or 25°C, respectively. For the in vivo infections, supernatants from infected cell monolayers were clarified by centrifugation at 4,000 × g for 30 min and stored in aliquots at −70°C. VHSV or CyHV-3 was purified by ultracentrifugation of supernatants from the corresponding infected cell monolayers as previously described  and kindly provided by Dr. Bergmann. Carp (50–250 g of body weight) were infected by immersion in 10 for 2 h . All the for 2 h . To obtaThe fish were handled in accordance with the National and European Guidelines and Regulations on Laboratory Animals Care. Fish work was approved by the corresponding INIAs Ethic Committee  at INIA aquarium installation number ES280790002069 and handled as provided with permission A/ES/16/I-32. The fish were monitored two to four times daily, and those with external hemorrhages were sacrificed by an overdose of methanesulfonate 3-aminobenzoic acid ethyl ester (MSS2) to minimize suffering. The fish were anesthetized by MSS2 and used for blood extraction. Blood samples were individually collected from the caudal vein, allowed to clot overnight at 4°C, and centrifuged. Supernatant sera were kept frozen at −20°C.n = 3) or healthy (n = 2) carp were selected among those having high or no titers of anti-CyHV-3 neutralizing antibodies and high or no anti-CyHV-3 IgM antibodies by indirect ELISA against purified CyHV-3  or healthy (n = 6) carp because of the high costs of these microarrays. According to the manufacture, the microarray covers ~75% of the annotated human genome and supplies a large array of different random epitopes. Human proteins are expressed as N-terminal GST-RGS-His6 fusion proteins that were purified and printed onto 75 mm × 25 mm SuperEpoxy 2 glass slides. The solid phases were blocked for 60 min with dilution buffer containing a limited amount of skimmed milk as described earlier. Sera pools were 100-fold diluted in the dilution buffer mentioned earlier, to a final assay volume of 3 ml and then incubated with the microarray. Secondary reagents were anti-mouse-IgG-546 and streptavidin-647 conjugated to fluorophores with excitation maxima at 546 and 647 nm, respectively. Anti-GST staining acted as a positive control and provided data on the relative amount of every protein on the microarray. As a negative control to estimate backgrounds, one additional microarray was treated identically to the experimental microarray but was incubated with only dilution buffer instead of a serum sample. Upon completion of the assay, the microarrays were scanned with excitation at 543 nm (for detection of IgM binding to the protein) and at 633 nm (for detection of GST-binding to every protein), and the fluorescence data were collected. GenePix software was used to align the human proteins spotted via microarray positioning. For each protein on the microarray, the fluorescence signals that were obtained from the corresponding protein on the negative control microarray that was incubated with reagents, but not with carp sera, were subtracted from the fluorescence signals that were obtained with the sera, yielding corrected protein-specific fluorescence signals. Following these corrections, mean fluorescence signals were calculated from the duplicate spots, values were log2 transformed, and signal significance was determined by following the manufacturer’s instructions (z-scores and interaction scores). The values and thresholds used for filtering were the relative SD of duplicate spots, which was <0.35, and the signal-to-noise-ratio of the sample signal which, was >2.5, to be considered significant.Antibody screening was performed on HuProt™ v3—Human Proteome Microarrays . The HuProt™ v3 Human Proteome Microarray provides yeast VHSV and frgIICyHV-3 were selected because of their immunodominance among the VHSV (CyHV-3 and the 56 amino acids of frg11VHSV resulted in only a 12% match (15 conserved positions randomly distributed throughout both sequences) , minimizing possible cross-reactivities among them. Recombinant frg11IHNV and frg11SVCV were similarly obtained from homologous frg11VHSV sequences in the corresponding glycoprotein genes within genomes NC001652 and DQ491000, respectively. The nucleotide frg sequences were chemically synthesized  and subcloned into pRSETa expression vectors that added poly-histidine tag tails to their amino-terminal ends. The plasmids were then transfected into E. coli BL21, cultured, and induced with IPTG followed by the purification of extracts by Ni2+ affinity chromatography as previously described  of the glycoprotein G of VHSV-07.71 strain (AJ233396) were performed as previously described . The seqthe VHSV  and CyHVescribed , 31. PurConcentrations of total IgM were estimated in carp sera by sandwich and/or indirect ELISA. For the sandwich ELISA protocol, plates were coated at 37°C to dryness with 2 µg/well of protein A-affinity purified anti-carp IgM MAb8   diluted To convert the absorbance values abovementioned assays to IgM concentrations, IgM standard curves were obtained by assaying different concentrations of purified IgM from carp sera. Carp IgM was purified from 10 ml of sera by either affinity chromatography on protein-A-Sepharose columns or precipitation with ammonium sulfate followed by Sephacryl-S300 chromatography. Purity of 80–90% was confirmed by SDS-denaturing gel electrophoresis stained with Coomassie blue and/or Western blotting using the corresponding anti-IgM MAbs (not shown). Serial dilutions of purified IgM created the corresponding standard calibration curves to convert absorbance values at 492–620 nm in micrograms of IgM/ml by Sigmoidal fit   containing 10 µg/well of skimmed milk (Sigma) for 60 min. On the other hand, sera were 100-fold diluted in dilution buffer containing 2 mg of protein/ml of E. coli extracts  and preincubated for 60 min. The amount of E. coli extracts was previously determined to reduce the absorbance of the healthy carp sera population to a mean of 0.2 U. Preincubated and -diluted carp sera (50 µl/well) were then incubated for 60 min in frg11VHSV solid phases, and the resulting supernatants were transferred to solid phases coated with frgIICyHV-3 and incubated for 60 min. To continue with the ELISA, both sera-treated frg11VHSV and frgIICyHV-3 solid phases were incubated with anti-carp IgM MAb8 and peroxidase-labeled rabbit anti-mouse IgGs  for 30 min. After three washes, the color reactions were developed by adding 1 mg/ml o-phenylenediamine in citrate buffer containing 3 mM H2O2. To avoid saturation of the increasing absorbance values with time, the color reactions were stopped after 7 min by adding 3 M H2SO4. Raw absorbances were measured by using 492–620 nm dual-wave length to correct for individual well differences. Individual raw absorbance values were then normalized to the calculated mean absorbance of the corresponding healthy sera population by the following formula: raw individual absorbance value × 0.2/mean absorbance of n healthy sera. To address individual carp variations, sera from populations n > 30 carp were tested individually, and each carp population was characterized by a polynomial fitted profile as described before (p < 0.05 (Student’s t-test).Polystyrene Maxisorb (Nunc) 96-well plates were coated with 50 µl of water containing 2 µg/well of purified recombinant frg11d-Lys coated wells  were used for CyHV-3 micro-neutralization assays by the high-throughput method . Strikingly, the results indicated the presence of significant IgM binding to all unrelated proteins tested in sera from both CyHV-3 infection-survivors and healthy carp (data not shown).To study the CyHV-3 infection dependence of non-specific IgM binding, eight sera from CyHV-3 infection-survivor carp having the highest CyHV-3 neutralization and anti-frgIItibodies , were fiE. coli extract preincubation. The results showed that each carp serum sample had different distributions of IgM-binding levels for each of the human proteins. However, despite those individual differences, the results also showed a direct correlation between infection-survivor and healthy carp IgM-binding levels  to increasing shifting differences  to maximal shift   but also to heterologous viral fragments (pathogen cross-reactive antibodies). To further explore the possible existence of such heterologous IgM binding, reactivities of carp sera populations were assayed after preincubation with E. coli extracts by using fragments from heterologous fish rhabdoviruses, and the results were analyzed as population profiles.Taken together, all of those results suggested the possibility that sera from CyHV-3 infection-survivors may contain infection-dependent IgM binding not only to homologous viral fragments . Furthermore, an additional excess of E. coli extracts or synthetic polyH tails in the buffers did not reduce the levels of CyHV-3 infection-dependent IgM binding (data not shown). The observed heterologous IgM-binding profile was not related to any previous infections either, since neither VHSV nor CyHV-3 neutralizing antibodies were detected in the carp sera from the different sources. On the other hand, carp was not susceptible to VHSV even when infected experimentally at 14°C, the optimal temperature for VHSV replication (data not shown).Two months after the carp population survived CyHV-3 infection, the sera IgM-binding profiles from the carp population to frg11To study any possible correlation between heterologous and specific IgM binding, their concentration levels in sera Figure B.in vitro neutralizing activities in the carp sera, high-throughput micro-neutralization assays were employed. The relative level of CyHV-3 neutralization and the relative quantity of CyHV-3-specific or non-specific IgM antibodies were not correlated in CyHV-3 infection-survivor carp , the exploration at the protein level surprisingly identified heterologous IgM epitope targets, such as those in frg11VHSV, and to frg11IHNV or frg11SVCV. Because of their higher abundances compared with other non-specific IgM binding, any IgM targeting frgIICyHV-3, frg11VHSV or other frgs could be part of the few dominant transcripts of IgM heavy chain clones previously discovered at the transcript level  IgM could be classified as a pathogen cross-reactivity property. Such cross-reactivity could be generated by several mechanisms. For instance, one possible explanation would be an antigen-independent B-cell polyclonal expansion such as those caused by mitogens, or by T-independent antigens  the CyHV-3 infection in carp induces not only specific but also non-specific IgM binding of broader reactivity and (ii) heterologous IgM-binding levels capable of recognizing unrelated whole viruses, such as VHSV, were among the non-specific IgM-binding levels generated by CyHV-3 infection.The fish were handled in accordance with the National and European Guidelines and Regulations on Laboratory Animals Care. Fish work was approved by the corresponding INIAs Ethic Committee  at INIA aquarium installation number ES280790002069 and handled as provided with permission A/ES/16/I-32. The fish were monitored two to four times daily, and those with external hemorrhages were sacrificed by an overdose of methanesulfonate 3-aminobenzoic acid ethyl ester (MSS2) to minimize suffering. The fish were anaesthetized by MSS2 and used for blood extraction. Blood samples were individually collected from the caudal vein, allowed to clot overnight at 4°C, and centrifuged. Supernatant sera were kept frozen at −20°C.JC designed, helped in the experimental work, and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many studies have now demonstrated the efficacy of text messaging in positively changing behaviours. We aimed to identify features and factors that explain the effectiveness of a successful text messaging program in terms of user engagement, perceived usefulness, behavior change and program delivery preferences.Mixed methods qualitative design combining four data sources; (i) analytic data extracted directly from the software system, (ii) participant survey, (iii) focus groups to identify barriers and enablers to implementation and mechanisms of effect and (iv) recruitment screening logs and text message responses to examine engagement. This evaluation was conducted within the TEXT ME trial—a parallel design, single-blind randomized controlled trial (RCT) of 710 patients with coronary heart disease (CHD). Qualitative data were interpreted using inductive thematic analysis.307/352 (87% response rate) of recruited patients with CHD completed the program evaluation survey at six months and 25 participated in a focus group. Factors increasing engagement included (i) ability to save and share messages, (ii) having the support of providers and family, (iii) a feeling of support through participation in the program, (iv) the program being initiated close to the time of a cardiovascular event, (v) personalization of the messages, (vi) opportunity for initial face-to-face contact with a provider and (vii) that program and content was perceived to be from a credible source. Clear themes relating to program delivery were that diet and physical activity messages were most valued, four messages per week was ideal and most participants felt program duration should be provided for at least for six months or longer.This study provides context and insight into the factors influencing consumer engagement with a text message program aimed at improving health-related behavior. The study suggests program components that may enhance potential success but will require integration at the development stage to optimize up-scaling.ACTRN12611000161921.Australia and New Zealand Clinical Trials Registry,  Coronary heart disease (CHD) remains the leading cause of death and disability worldwide. AlthoughIn recent years, mobile phone text-message based interventions have arisen as a potential means of modifying health behaviors. Recent sAs recognised within the UK Medical Research Council framework for the development and evaluation of complex interventions, assessment of fidelity and causal mechanisms optimises the likelihood of implementation in clinical practice. A recentWe undertook a mixed methods approach within the Tobacco, EXercise and dieT messages (TEXT ME) single-blind RCT that aimed to reduce cardiovascular risk in 710 patients with CHD.,23 ResulThe evaluation plan is summarised in We also examined the data for evidence of behavior change techniques and links with psychological theory based on a published taxonomy. As reporThe text messaging program and its development process are detailed elsewhere.,23,27 Th[NAME], try identifying the triggers that make you want a cigarette & plan to avoid them[NAME], for many it may take several attempts to quit, so keep tryingDid you know 90% of people don’t eat the recommended daily intake of vegetables (5 serves a day)?Try avoiding adding salt to your foods by using other spices or herbsHi [NAME], don’t forget physical activity is good for you! It reduces your risk of diabetes, heart attack, stroke, and their complicationsWalking is cheap. It can be done almost anywhere. All you need is comfortable shoes & clothinghttp://www.heartfoundation.org.au/Pages/default.aspxHave you got a chest pain Action Plan [NAME]? Find ideas at Studies show that stress, worry & loneliness can increase the risk of heart disease. Please talk to a health professional if you need help*Reproduced with permission from JAMA 2015.314(12):1. Copyright (2015) American Medical Association. All rights reserved. thinking about getting a snack… then suddenly I received a message of TEXT ME saying: Hi [name], feeling like a snack? A banana or a piece of fruit! It really gave a big brother feeling to me, how could they know I was thinking about picking a snack? I thought well forget it lets go and walked away with a smile on my face. I got busted” This process evaluation found that patients with CHD were engaged with and positive about a text message program aimed at supporting behaviour change. Results suggest that engagement was increased by several factors including the program being initiated close to the time of hospitalisation, the consistency of the messages with prior in-hospital education and management, having the opportunity for face-to-face contact at program commencement and the personalization of messages. We found features that impacted on behavioural change were the positive content, the sense of feeling supported, the delivery time being random and that most participants felt program duration could have been longer than six months. Through thematic analysis this research has identified several potential drivers of behavior change within a text messaging program and therefore facilitates understanding of components of the intervention that may increase effectiveness.The primary goal of this evaluation was to help with understanding why text message-based programs for people with CHD are effective, the ingredients for success and how engagement can be optimized so as to inform development of other text message programs aimed at behavioural change in patients with other type of chronic disease. Overall, an important role for program evaluations is to examine the quantity and quality of what was actually implemented in practice and why.,21 ThrouVery few studies have reported qualitative evaluations alongside a RCT of a text message-based intervention for improving health-related behavior. One study tested the effectiveness of prompts sent via text message in terms of improving BP management via adherence to clinic visits and treatment. The qualUnderstanding the theory associated with behavior change allows consideration of hypothesized causal processes involved. In the cWhile this mixed methods evaluation provides significant insight into the potential mechanisms of intervention effectiveness and opportunities for optimising engagement, the study is not without limitations. Firstly, the sample was recruited from within a RCT from a tertiary hospital which might limit the generalisability of the intervention, however this hospital has broad socioeconomic and multicultural representation. Secondly, our sample size for women was small and therefore examination of gender differences requires further investigation. It is acknowledged that we did not stratify for age and that there were more people with higher years of education participating in the focus groups and that our findings will vary between individuals and naturally certain groups should not be denied access to potential interventions. The qualitative nature of this research enables features to be examined in detail and allows consideration of human experience. Future research is needed to identify the effectiveness of various program design features. For example, to specifically determine whether it is ideal to offer one-way or two-way communication or whether this should be offered to participants by choice. In addition, future research could clearly define the ideal message delivery frequency, timing and the ideal program duration.This process evaluation found that patients with CHD were engaged with and positive about a text message program aimed at supporting behaviour change. Exploring the theoretical frameworks provides insight into the causal pathway between the text-message intervention, behavior change and impact on risk factor measures. Gaining an understanding of the factors that influence engagement with the program as well as the advantages and disadvantages of various program characteristics can inform the development of other programs, identifies the key elements important to maintain in larger-scale programs and gives clues to the directions of future research to improve programs and support their sustained effectiveness.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.