Surgical or bronchoscopic lung volume reduction (BLVR) techniques can be beneficial for heterogeneous emphysema. Post-processing software tools for lobar emphysema quantification are useful for patient and target lobe selection, treatment planning and post-interventional follow-up. We aimed to evaluate the inter-software variability of emphysema quantification using fully automated lobar segmentation prototypes.th percentile of lobar density (15th), emphysema volume (EV) and emphysema index (EI). Bland-Altman analysis  and linear random effects models were used for comparison between the software.66 patients with moderate to severe COPD who underwent CT for planning of BLVR were included. Emphysema quantification was performed using 2 modified versions of in-house software , as well as 1 commercial program 3 [Pulmo3D VA30A_HF2] and 1 pre-commercial prototype 4 [CT COPD ISP ver7.0]). The following parameters were computed for each segmented anatomical lung lobe and the whole lung: lobar volume (LV), mean lobar density (MLD), 15p<0.05). Program 3 and 4 showed the largest mean difference of 4% and the widest LoA of  for EI (p<0.001).Segmentation using programs 1, 3 and 4 was unsuccessful in 1 (1%), 7 (10%) and 5 (7%) patients, respectively. Program 2 could analyze all datasets. The 53 patients with successful segmentation by all 4 programs were included for further analysis. For LV, program 1 and 4 showed the largest mean difference of 72 ml and the widest LoA of  (Only a single software program was able to successfully analyze all scheduled data-sets. Although mean bias of LV and EV were relatively low in lobar quantification, ranges of disagreement were substantial in both of them. For longitudinal emphysema monitoring, not only scanning protocol but also quantification software needs to be kept constant. The largest difference for MLD was between program 1 and 4. The LoA was widest between program 2 and 4 for MLD. In Bland-Altman plot describing MLD, 95% confidence interval is narrower between program 3 and 4 than other pairs (data not shown), indicating better agreement between two programs. As for comparing MLD values between program 3 and 1, program 3 and 2, program 4 and 1, and program 4 and 2 (data not shown), 95% confidence interval is below the line of equality, indicating that program 1 and 2 overestimates MLD in all cases relatively to program 3 and 4 .The difference for MLD was significant between program 1 and 3, 1 and 4, 2 and 3, 2 and 4, and 3 and 4 . As for the 15th, the narrowest interval was depicted between program 1 and 2 (data not shown). Program 3 and 4 revealed the largest mean difference and the widest LoA for 15th.In case of 15p = 0.005, 0.005 and 0.02, respectively). The difference for EV was largest between program 1 and 4 with a mean difference of 61 ml. However, the widest LoA existed between program 3 and 4 .As for EV, there were significant differences between program 1 and 4, 2 and 4, and 3 and 4 . Program 3 and 4 showed the largest mean difference of 4% and the widest LoA of  for EI.There were significant differences for EI between program 1 and 4, 2 and 4, and 3 and 4 (The median standard deviation (inter-quartile range) of the EI amongst the lobes of each single patient as a marker of intra-patient variability was 9.86% (7.67–13.24) for program 1, 9.86% (7.11–13.38) for program 2, 8.99% (5.85–12.16) for program 3, and 9.67% (7.60–13.72) for program 4. The pairwise correlation of intra-patient variability between software pairs ranged from 0.95 (program 1 vs. program 4) to 1 (program 1 vs. program 2). We then used the median SD of the intra-patient EI to separate patients into groups with low and high intra-patient EI variability. Interestingly, the group with high intra-patient variability also showed wider LAO for inter-software variability oft he EI, which was up to 1.81 times higher than in the group with low intra-patient variability . This efAfter visual inspection by a thoracic radiologist considerable errors in lobar segmentation were found in 27 of 53 patients: program 1: 11 patients, program 2: 9 patients, program 3: 2 patients, program 4: 3 patients, both program 1 and 4: 2 patients). Notice that the datasets where the programs 1, 3 and 4 delivered complete unsuccessful segmentations  were not contained in these 27 datasets.The comparison of the measurement results between programs was thus repeated with the remaining 26 datasets  after reth, EV and EI became smaller after user interaction and removal of all datasets with obvious segmentation errors in one or more software tool, reflecting the improvement of inter-software agreement in the remaining pairs after this interaction  LoA, however, remains substantially wide, resulting in non-interchangeability of the results obtained from different software programs; (4) high intra-patient variability of EI resulted in a higher inter-software variability of EI.The main findings of the present study about emphysema quantification using fully automated lobar segmentation are as follows: (1) intra-program reproducibility was generally excellent in all four programs in moderate to severe emphysema patients with various degree of destroyed lungs and distorted course of a fissure due to severe emphysematous changes of adjacent lung parenchyma; (2) the mean difference of lobar LV, MLD, 15Compared to PFT or other clinical examinations, one of the main strength of quantitative MDCT in evaluating emphysema lies in offering regional information about the distribution of emphysema from lobar segmentation, which enables selection of the target lobe to be treated regionally e.g. by BLVR . Fully aThe clinical relevance of a diagnostic modality depends on its ability to provide reproducible results regardless of the influence of external factors . Along tIn the present study, we intended to evaluate the impact of selecting different software tools on fully automated lobar emphysema quantification by comparing the results from 4 different software programs. Firstly, even commercial programs were not able to process all provided data successfully. After exclusion of those error inducing data-sets, we found a high degree of correlation and linearity between results derived from the four software programs in our study. However, these high correlation values should not be incorrectly interpreted as a measure of the interchangeability between results from different programs. In Bland-Altman analysis, calculated values are located around the mean line within a 95% confidence interval for perfect agreement .In the present study, we did not observe good agreement between different programs mostly because LoA on Bland-Altman plots were not narrow enough to be considered as negligible from our radiological perspective. In general, program 1 and 2  showed better agreement compared with other pairs of software programs. Even between program 1 and 2 which are different version of the same program without and with application of advanced lung vessel segmentation, however, there existed some bias (mean difference) for all of the five parameters. For example, we could notice one extreme example of substantial improvement in LV measurement by program 2 compared with that by program 1, which is probably due to an additional algorithm considering pulmonary vessels . ProgramThere are some researchers who believe that EI directly reflects the phenomenon of destruction of lung tissue that occurs in emphysema, diminishing the partial volume effect of air and lung structures on each voxel of the affected zone, unlike percentile lung density . In the According to the previous study , potentiThe additional measurement variation in the present study probably comes from different lobar segmentation algorithms in the current study, resulting in different LV and subsequent different densitometry values. In patients with an inhomogeneous emphysema distribution who are generally more suitable for volume reduction surgery or BLVR, we found a higher inter-software variability, probably related to a stronger distortion of normal lobe anatomy posing additional challenges to lung lobe detection. Thus, patients with inhomogeneous emphysema are prone to a higher measurement variability of computational emphysema quantification.As we expected, we found that right upper lobe and right middle lobe were major sources of relative variability (considering fissure) due to sharing of the other fissure (minor fissure) by visual observation, in agreement with the previous study . Fissurein-vivo gold standard existing for lobar-based emphysema quantification. Measurements of emphysema quantification software programs are usually based on segmentation of airways on MDCT data sets and an algorithm that translates the segmented voxels into lung volumes. Depending on the voxels included in each segmentation, the result of density histograms can be produced in a different way although segmentation volumes are the same or very similar in amount. It is also known that the segmentation of the pulmonary lobes becomes cumbersome when the inter-lobar boundary is unclear [There are several limitations in the current study. First, there is no  unclear , which m unclear , 41. HowIn conclusion, we should not interpret the results from different software programs as interchangeable. The significant differences between software programs used for lobar emphysema quantification may lead to contradictory target lobe selection for BLVR in some cases. Another important issue is that different emphysema quantification programs or different versions of the same program may be used in different institutions, impairing comparability of study results. When performing follow-up studies in patients, the software tool should be kept exactly constant.S1 Fig(A and B) Coronal images of a 62 year old patient with FEV1 = 51% demonstrate distorted incomplete right minor fissure and relatively severe emphysematous change in LLL. (C~F) Sagittal images shows suboptimal segmentation of RML and LLL. RML = right middle lobe LLL = left lower lobe(DOCX)Click here for additional data file.S2 FigBland-Altman plots demonstrate inter-program comparison for EI. The central thick line indicated the mean difference and the upper and lower thin lines indicate upper and lower limits of agreement. EI = emphysema index(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Customer Relationship Management (CRM) with its various components has been considered as a tool causing customers’ loyalty. The present study aims to investigate the relationship between the various components of customer relationship management and patients’ loyalty to the place of their treatment.20 software.This cross sectional and descriptive-analytical study was conducted among nurses and hospitalized patients in inpatient wards in selected hospitals in 2014. Using the stratified random sampling method, 224 valid and reliable researcher-drafted questionnaires were completed for CRM by nurses and 359 questionnaires were completed by patients for patients’ loyalty in the studied wards. Data were analyzed using the SPSSThere was no statistically significant relationship between the level of patients’ loyalty and organizational indicators, information technology and knowledge management . However, there was a statistically significant relationship between loyalty and the dimensions of the service process , human resources  and CRM . The strength of these relationships were 34, 40 and 36 percent, respectively all of which were positive.Customer Relationship Management is a tool for improving influencing factors on patients’ satisfaction and loyalty. Therefore, attempts to implement customer relationship management as a process for improving hospitals performance and improving communication between service providers in hospitals and customers leading to enhance patients’ loyalty should be taken into account by managers and policy makers in the health sectors. Attracting and maintaining customers have long been believed to be of main aspects of activities in every organization. New forms of competition and structural changes in transactional processes have led to the creation of patterns for long term communication between stakeholders in market . HoweverIs said that CRM, as a new term, was widespread since the mid-1990s . This syThere are several factors influencing customers’ loyalty such as employee satisfaction, employee loyalty and the quality of services .Nowadays, the number of health care centers has increased along with the increased competition in the health care industry. This has resulted in establishing the customer relationship management system in order to meet diverse demands of customers. Due to the intensifying competition among health care centers, services tailored to customers are provided through analyzing their needs by the centers .The CRM for health care service providers is an approach through which they learn whatever related to their patients, their viewpoints and expectations in order to make relationship with them, provide timely information and follow up their related results so that they can carry out corrective measures, increase customer loyalty and gain more profit . Also, CA favorable relationship between health care providers and patients not only enhances patients’ satisfaction but also improves the quality of health . The impMost of the past attempts by hospitals related to the implementation of the CRM have been wrongly directed and supported . It is tNurse= 224 & S Patients=359). In each hospital, the number of nurses and patients were determined in terms of wards and this number of sample size proportional to the number of nurses and patients was distributed.This cross sectional and descriptive-analytical study conducted in 2014 aimed to investigate the relationship between customer relationship management and patients’ loyalty. The study was conducted in 34 inpatient wards in selected hospitals affiliated to Tehran University of medical sciences. Nurses (N=429) and patients (N=4127) in inpatient wards in the studied hospitals were the population of this study. In order to determine the sample size, at first the Morgan's table and stratified random sampling method were used and the total number of sample for all hospitals were calculated (S=583) , service-providing process (9 questions), human resources (12 questions), information technology (6 questions) and knowledge management (6 questions). This questionnaire was designed based on the 5-point Likert scale consisting of very high (5 point), high (4 point), moderately (3 point), low (2 point) and very low (1 point). The validity of the questionnaire which was based on the collective judgment of scholars and experts and by using the content validity index (CVI) was calculated to be 84%. Also, in order to assess the reliability of the questionnaire, the Cronbach's alpha was calculated to be 96%. As to measure patients’ loyalty, the questionnaire used by Tabibi et al.  was usedThe researcher distributed the CRM questionnaire among nurses working in morning, evening and night shifts in 34 inpatient wards in the studied hospitals. Also, the patients’ loyalty questionnaire was distributed among the hospitalized patients at the point of discharge in these 34 wards.The SPSS 20 software and descriptive and inferential statistical methods were used to analyze data. Descriptive statistics were used to draw the frequency distribution tables and inferential statistics were used to determine differences and the relationship between variables. The Kolmogorov-Smirnov test (KS-test) was used to assess the normality of the scores distribution. Also, Pearson's and Spearman's correlation test were performed to assess the correlation between variables. To address ethical and legal issues, a letter of permission was issued by the relevant university. Also, the participants were given sufficient explanations and they were assured that the questionnaires information will be kept confidential.Among the nurses, 96% were female, 50% were married and 96% had a Bachelor of Science degree in nursing; 88% were nursing experts and 9% were head nurses. The average age of the nurses was 30.54±5.1 years and the average years of work experience among them was 6.8±4.8 years. Among the patients, 52% were female, 85% were married, 18% had college degrees, 43% were housewives, 41% were salaried employees in the public and private sectors, 62% were over the age of 40. Among the 93% of those covered by the health insurance, 73% had basic health insurance and 27% of them were covered by complementary health insurance, besides the basic health insurance.Among the 34 studied wards, the mean score of loyalty was 3.43; the mean score of organizational indicators was 3.39; the mean score of service process was 3.48; the mean score of human resources was 3.77; the mean score of information technology was 3.44; the mean score of the knowledge management was 3.34 and the mean score of CRM was 3.52. The highest attainable score was 5.The Kolmogorov-Smirnov test showed that all the scores of loyalty, organizational indicators, service- providing process, human resources, information technology, knowledge management and the customer relationship management were normally distributed.The findings of The assessment of the correlation between the CRM components with each other and with CRM also indicates that there is also a significant relationship between all these components. This is while the dimensions of organizational indicators and the service-providing process showed the highest correlation with CRM (90 percent) and the dimension of human resources showed the lowest correlation with CRM (86 percent).The results of the study showed that there is a statistically significant difference between the level of loyalty among male and female in the studied population . However, there is no statistically significant correlation between demographic features including marital status, age, the status of insurance and the type of insurance, the level of education and the status of employment with patients’ loyalty. This issue indicates that the perception among patients in various groups in the studied population is nearly the same.There is no statistically significant relationship between the level of patients’ loyalty and organizational indicators, information technology and knowledge management. However, there is a statistically significant and positive relationship between loyalty and the dimensions of the service-providing process, human resources and the CRM. There is also significant relationship between CRM and all its components; while the dimensions of organizational indicators and the service-providing process showed the highest correlation with the CRM. In other words, there is a significant relationship between customer relationship management and patients’ loyalty, however, no relationship was observed in the areas of organizational indicators, information technology and knowledge management. It may be concluded that patients can well understand the two areas of human resources and service-providing process and express their opinion in this regard. However, the areas of organizational indicators, information technology and knowledge management are not observable and understandable for patients.There are strong correlations between customer relationship management and its dimensions .Consistent with these results, Foss also regarded the cooperation among various sections, as one of the components of organizational indicators, as a necessary approach for customer relationship management . DewhursThe results of the Pearson's correlation test show that there is a significant correlation between customer relationship management and patients’ loyalty to the medical ward.Cobelas et al. concluded that when a hospital meets a patient's needs at a higher level than he/she expects, then this patient will not change the hospital . AdelekeAlso, Galbreath and Rogers have considered ‘customization’, ‘customized communication’  and ‘providing after sales support services’ as the three main goals of customer relationship management . PaazineA significant relationship between the component of service-providing processes and loyalty was observed. Arab et al. have reported the process quality as one of the dimensions influencing loyalty . KesslerAlso, a significant relationship was observed between the components of human resources and loyalty. Ayimbillah Atinga et al.  considerDespite the significant relationship between CRM and patient loyalty, there is no significant relationship between three components  with patient loyalty. The cause should be sought in objectives and functions of these three components in hospitals. These three components, unlike the other two components , are not tangible and understandable for hospitalized patients, but patients have a direct and understandable relationship with human resources and service-providing processes. Hussain et al.  also ackCustomer relationship management can be applied to enhance patients’ loyalty; while, two areas of human resources and service-providing process can directly affect patient loyalty, three areas including organizational indicators, information technology and knowledge management are considered as factors influencing patient loyalty to medical wards indirectly.In order to improve customer relationship management, all its areas should go further in the same direction. In addition, human resources and their functions are considered as the most important dimensions of patient satisfaction and loyalty. It can therefore be concluded that in order to implement CRM, firstly managers should focus on human resources and service-providing processes. However, in long terms, they should know that improvements in these two dimensions requires strong support by knowledge management, information technology and appropriate organizational structures.LimitationsThe lack of a hospital in which the CRM has been completely implemented is a limitation of this study, so that each hospital had taken steps in achieving the CRM, so inpatient wards in participating hospitals were selected for the setting of the study.


Health care systems in sub-Saharan Africa, and globally, grapple with the problem of closing the gap between evidence-based health interventions and actual practice in health service settings. It is essential for health care systems, especially in low-resource settings, to increase capacity to implement evidence-based practices, by training professionals in implementation science. With support from the Medical Education Partnership Initiative, the University of Nairobi has developed a training program to build local capacity for implementation science.This paper describes how the University of Nairobi leveraged resources from the Medical Education Partnership to develop an institutional program that provides training and mentoring in implementation science, builds relationships between researchers and implementers, and identifies local research priorities for implementation science.The curriculum content includes core material in implementation science theory, methods, and experiences. The program adopts a team mentoring and supervision approach, in which fellows are matched with mentors at the University of Nairobi and partnering institutions: University of Washington, Seattle, and University of Maryland, Baltimore. A survey of program participants showed a high degree satisfaction with most aspects of the program, including the content, duration, and attachment sites. A key strength of the fellowship program is the partnership approach, which leverages innovative use of information technology to offer diverse perspectives, and a team model for mentorship and supervision.As health care systems and training institutions seek new approaches to increase capacity in implementation science, the University of Nairobi Implementation Science Fellowship program can be a model for health educators and administrators who wish to develop their program and curricula. Health care systems worldwide have failed to bridge the implementation gap between medical evidence and current health care practice, resulting in inefficient use of resources and sub-optimal quality of care . The neeImplementation science (IS) has been defined as the study of “the use of strategies to adopt and integrate evidence-based health interventions and change practice patterns within specific settings” . As concAccording to the NIH, progress in implementation science will require the training and mentorship of a new type of professional, advances in dissemination and diffusion of information on evidence-based interventions, strengthening linkages between scientists, program implementers, and policy makers, and agenda setting to identify local priorities . Prior tBuilding local capacity to provide on-going training in IS requires a consistent pool of motivated trainees, clinical sites to conduct research, and teachers for both the theory and the practical application of IS. Training institutions in sub-Saharan Africa, such as UON, are challenged by a lack of faculty with relevant expertise, limited funding, and a lack of attachment sites staffed with experienced mentors for practical training. These barriers are compounded by the need for highly motivated trainees who can afford to take time away from their employment for graduate level training which typically lasts 1 to 2 years. Employers, stakeholders, and potential trainees are also deterred by a lack of awareness of career prospects in implementation science .The objective of this article is to describe the approach used by the University of Nairobi to overcome these barriers and build local capacity for implementation science. This paper examines how UON leveraged resources from the Medical Education Partnership Initiative (MEPI), created an institutional program to provide training and mentoring in implementation science, built relationships between researchers and implementers, and identified local research priorities for implementation science. The goal of the training program is to improve the quality of health care in Kenya by producing implementation scientists with the capacity to design, implement, monitor, and evaluate evidence-based interventions in health care settings.This paper describes an Implementation Science Fellowship program that delivers a novel curriculum aimed at producing researchers skilled in implementation science. As part of our evaluation plan for the program, a questionnaire was developed as a tool to assess both process and outcomes of the program. The questionnaire was administered on a voluntary basis to fellows, faculty, mentors, and administrators from the partnering institutions, all of whom were provided with full information on the evaluation, including the purpose and nature of the evaluation. Prior to obtaining verbal consent, the participants were assured that their personal information would be kept confidential, and that their identity would not be revealed. Fellows were further assured that there would be no negative consequences for not participating. Questionnaires were designed to take not more than 45 min to complete to minimize loss of time. The study was approved by the KNH/UON Ethical Review Committee.Implementation science practice and training requires partnerships between researchers, health care providers, and policy makers, or more broadly between academia, health facilities, and government ministries. Establishing these partnerships served as the foundation for the program at the UON.The University of Nairobi (UON) has well-established partnerships with the University of Washington (UW) and the University of Maryland, Baltimore (UMB). The partnership with the University of Washington began in the context of the AIDS International Training and Research Program (AIRTP), an initiative whose goal is to support training and HIV-related research. UON’s collaboration with UMB was launched as a component of the AIDS Relief Program, a consortium funded through the US President’s Emergency Plan for AIDS Relief (PEPFAR) to provide treatment services to HIV-infected individuals in Kenya and other African, Caribbean, and Latin American countries . The thrWith funding in place, the leadership of the University then established key partnerships for the program. UON (University of Nairobi) trains its medical and health sciences students at the Kenyatta National Hospital (KNH). KNH is thus a natural partner for UON in most of its health-related initiatives. The mission of KNH “to optimize patient experience through innovative health care; facilitate training and research; and participate in national policy” articulates key tenets of implementation science. The hospital required little motivation to participate in the partnership. KNH is the setting for a large proportion of biomedical research in Kenya; however, few employees of KNH have led research. The prospect of strengthening the research capacity of KNH employees, through the IS training, was a major incentive for the hospital to participate.Implementation science is ideally conducted in diverse settings to establish the feasibility of evidence-based approaches in different settings, including and especially places of routine care for the general population. As such, the PRIME-K leaders participated in a consultative process with the Kenyan Ministry of Health (MOH) to identify hospitals and health care settings across the country to participate in the program. The Ministry of Health employees at the selected sites were incentivized to participate by providing them appointments as adjunct faculty status with the UON. Adjunct faculty would thus work collaboratively with UON faculty on research projects in addition to training UON students during community-based clinical rotations. Partnerships to enable the IS training program thus formed between the University of Nairobi, KNH, the MOH, and northern schools (UMB and UW).Careful consideration to feasibility, sustainability, and local relevance was given during the design of the training program, which required aligning with national priorities and resources. In Kenya, the Ministry of Health has prioritized training in implementation science. In its policy document entitled “Kenya AIDS Strategic Framework (KASF),” the Kenyan Ministry of Health lists “priority intervention areas” which include the “increase [of] evidence-based planning and the use of implementation science outcomes to optimize programming and policy changes” . ImplemeWith funding and partners in place for investing in a locally relevant training program, the UON leadership sought to establish a program that could ultimately be sustained locally through graduates of the program. Various training models such as a Masters, Diploma, or Fellowship program were considered. Master programs typically require a lot of time on campus, making it difficult to maintain full-time work at the same time. Given that the target audiences for the program were those already with a Master’s degree, this approach also seemed unlikely to attract sustained interest. The Diploma option, on the other hand, was likely too short and may not allow adequate development of skills. The Fellowship approach, allowing students to remain in their place of work while completing their studies during evening hours, weekends, and online, seemed the best pathway to ensure a sustained interest from students and yet develop strong competencies in implementation science. The one substantive time commitment is a 3-month period where student in the early years of the fellowship (before in-house capacity is built) would go to one of the US Universities for didactic training.The selection process was framed to achieve a balanced mix of fellows based on educational background, geographical place of work, and gender. Trainees were to be selected from UON, KNH, and MOH health facilities. The minimum qualification for entry was a Master’s degree in the health sciences including pharmacy, nursing, medicine, dentistry, and public health but not limited to health sciences. Applicants with a Master’s degree in biomedical sciences or social sciences were also eligible. Applicants were required to demonstrate motivation to pursue a career in implementation science, be able to attend training for 2 years if accepted, and provide a letter of support from their organization or institution. Since one of the program objectives was to expand the cohort of researchers at UON, KNH, and the MOH, preference was given to early-career applicants and those without established major research funding.The curriculum for the program was based on an existing model from a partner school but contextualized for Kenya. Implementation science is a new discipline and is only taught at a handful of university departments globally . As suchLeadership for the UON implementation science curriculum development was entrusted to a coordinating committee. In designing the educational framework for the fellowship program, the coordinating committee extensively consulted with UON partners and stakeholders and applied a combination of educational principles to develop the curriculum. In particular, competency-based medical education (CBME) and adult-learning principles underpin the curriculum. Other curriculum elements that were added and emphasized include innovative teaching strategies, content related to the Kenyan health care system, inter-professional team training, assessment of learning needs, mentoring, development of research skills, and evaluation of the processes .The core competencies of the program Table  were aliComments and suggestions were synthesized and discussed at two interactive workshops, where faculty, stakeholders (including MOH), and educational experts (including the Dean of the UON School of Education) participated, to further deliberate and refine the educational goals, competencies, curriculum content, and teaching strategies for the IS program. The final curriculum aims for a mastery of each of the competencies and skills Table  through The curriculum content includes core materials in implementation science theory, methods, and experiences Table  taught bFor individual research projects, fellows identify research topics in implementation science that are relevant to the local context or to the host organization of the fellow such as the MOH health care facilities. Fellows then develop proposals and undertake data collection, analysis, and report writing with guidance from mentors and supervisors. The program has adopted a team mentoring and supervision approach, in which a fellow is matched with a local mentor, a local research supervisor, and an additional mentor or supervisor from the partner institutions (either UW or UMB). In certain cases, the mentor may also be the research supervisor. A key innovation in our program is organization of “mentoring workshops,” at which the fellows, the local mentors, and supervisors get further opportunities to interact and establish and assess progress towards the objectives.The first cohort of fellows admitted to the program includes six individuals from diverse backgrounds including public health (2), pharmacy (2), medicine (1), and nursing (1). One successful applicant was unable to join the program for personal reasons. Of the five participants, two were female. The second cohort has five trainees, with professional qualifications in public health (1), pharmacy (2), dental sciences (1), and nursing (1) Table Table 3ChThe first cohort of fellows has developed projects in the areas of both dissemination and implementation. The research topics include (i) determinants of contribution of research in Kenyan universities in informing National HIV policies and practice, (ii) evaluating and improving the quality of obstetric and neonatal care at Kenyan urban and rural health care facilities, (iii) sustainability and cost-effectiveness of antimalarial medicines program in a Kenyan county, (iv) correlates of organizational cultural factors and clinicians’ practice with low early mortality of patients initiated on ART in Kenya, and (v) fidelity of anticoagulants prescription practice to the guidelines.Because of this training, I have been selected by my organization to participate in reviews of micro-research project proposals at my work place.”The training program incorporates a monitoring and evaluation framework to measure progress towards the attainment of the program goals. The program administered a questionnaire to fellows in years 1 and 2 to solicit feedback and to assess various aspects of the training program including perceived value and progress towards desired objectives. Analysis of survey results revealed a high degree of fellows’ satisfaction with most aspects of the program, and most fellows indicated that they would recommend the program to their colleagues Table . Most feThe career development opportunities for fellows who have completed the program are promising. Four fellows have embarked on research careers by enrolling in PhD programs in dissemination and implementation science research, three fellows have secured promotions, and one became a Departmental Chairperson. Three fellows have secured further funding to enable them continue with research.Most (85 %) fellows identified implementation science research as a component of their work activity. Fellows have disseminated their research to general and specialist audiences at international conferences and published their work in peer-reviewed journals. One fellow regularly contributes a newspaper column with implementation science themes to a general audience. Fellows also contribute to broader research activities such as through membership in departmental research committees and adjunct professorships in partner institutions.Representatives of the UON describe the fellowship program as a faculty recruitment and retention strategy by providing opportunities for growth and recognition. All of the fellowship recipients are currently still at UON . One fellow changed employment from a Ministry of Health hospital to a university teaching hospital, and another contributes as faculty in the Afya Bora Fellowship program. A key finding is that the fellows are regarded by representatives of UON as increasing the knowledge base and capacity of the UON to conduct research in an emerging field and enhancing institutional recognition through academic output.Fellows and institutional representatives reported that the training program expanded their network. Most (90 %) fellows reported retaining contact with mentors and supervisors in the partnering institutions. Recently, UON implementation science researchers built on the program and the linkages with partnering institutions and MOH to win a new NIH grant in implementation science. UON has a growing community of implementation science researchers in diverse fields including mental health, child health, nursing, pharmacy, and dental sciences.The strengths of the fellowship program include leveraging existing partnerships with the University of Washington (UW) and the University of Maryland, Baltimore (UMB) Fig. . The colSustainable sources of funding are another challenge for the program. The fellowship program was funded by the Medical Education Initiative (MEPI), an initiative of the President’s Emergency Plans for AIDS Relief (PEPFAR), also supported by the US National Institutes of Health (NIH). This time-bound source of funding covers tuition and students’ stipend. Fortunately, sources of funding for Kenyan implementation science researchers are increasingly becoming available. Funding is available from local agencies, such as the Consortium for National Health Research and the Ministry of Health . There aThe persistent gap between evidence-based health interventions and actual health care delivery particularly in low-resource settings underscores the critical need to develop a workforce skilled in implementation science. Such training will serve to strengthen the capacity for health care systems to move evidence-based interventions into diverse health care and community settings, improving both the efficiency and the effectiveness of health services.In this article, we have described an Implementation Science Fellowship program that delivers a novel curriculum aimed at producing researchers in implementation science. The fellowship program applies a partnership approach that offers diverse experiences and perspectives and innovatively leverages information technology and a unique mentorship approach. Early findings suggest that this program has had a positive impact both on the participants and the host institution and could thus serve as a model to stimulate further expansion of this nascent field, especially in low-resource settings.


QST—FST comparisons, (i) no effect of selection (QST ≈ FST); (ii) diversifying selection (QST > FST); (iii) convergent selection (QST < FST) [51–52].There is an error in the third sentence of the sixth paragraph of the Introduction. The correct sentence is: There are three widely accepted scenarios of QST estimates were calculated for each measured trait using the equation QST = VPOP/(VACC+VPOP) where VPOP is the estimated variance component for the population effect and VACC is the estimated variance component for the accession (= genotype) effect.There is an error in the fourth sentence of the second paragraph of the Assessment of quantitative trait variation subsection of the Materials and methods. The correct sentence is: The


Just as HIV prevention programs need to be tailored to the local epidemic, so should evaluations be country-owned and country-led to ensure use of those results in decision making and policy. The objective of this paper is to describe the process undertaken in Ghana to develop a national evaluation plan for the Ghana national strategy for key populations.This was a participatory process that involved meetings between the Ghana AIDS Commission (GAC), other partners in Ghana working to prevent HIV among key populations, and MEASURE Evaluation. The process included three two-day, highly structured yet participatory meetings over the course of 12 months during which participants shared information about on-going and planned data and identified research questions and methods.An evaluation plan was prepared to inform stakeholders about which data collection activities need to be prioritized for funding, who would implement the study, the timing of data collection, the research question the data will help answer, and the analysis methods. The plan discusses various methods that can be used including the recommendation for the study design using multiple data sources. It has an evaluation conceptual model, proposed analyses, proposed definition of independent variables, estimated costs for filling data gaps, roles and responsibilities of stakeholders to carry out the plan, and considerations for ethics, data sharing and authorship.The experience demonstrates that it is possible to design an evaluation responsive to national strategies and priorities with country leadership, regardless of stakeholders' experiences with evaluations. This process may be replicable elsewhere, where stakeholders want to plan and implement an evaluation of a large-scale program at the national or subnational level that is responsive to national priorities and part of a comprehensive monitoring and evaluation system. A wide array of HIV prevention approaches exist, and HIV prevention programs should be tailored to local epidemic to best respond to the diverse needs of people at risk of HIV. There is international endorsement for combination (comprehensive) HIV prevention programs that include behavioural , biomedical , and structural interventions  ,2. From Evaluations are conducted to obtain evidence that can inform judgments about a program's performance, to improve the effectiveness of programming, for program accountability and transparency, and to inform decisions about future policies and programming including scale up . Just asRigorous evaluations of comprehensive HIV prevention programs have faced a number of challenges in their implementation. The pathway through which comprehensive programs influence health outcomes is complex. Because of the nature of HIV prevention programs they are targeted where the need is greatest, thereby complicating the identification of groups that serve as comparisons or control. Similarly, in countries there are a number of programs operating making it difficult to find untouched comparison groups .In response to the uncertain and incomplete evidence to inform HIV prevention programs, the Joint United Nations Programme on HIV/AIDS (UNAIDS) following a call from the UNAIDS Monitoring Evaluation Reference Group (MERG) issued guidance in 2010 on evaluating HIV prevention programs . The doc1.MEASURE Evaluation was interested to see how this guidance informs a practical approach at the country level; an approach that puts evaluation priorities and design decisions in the hands of country decision makers, makes better use of available M&E data for evaluation purposes, and better aligns data production with information needs. Thus, in the summer of 2011 the Ghana AIDS Commission (GAC) and MEASURE Evaluation launched a process to develop an evaluation plan for an evaluation of the effectiveness the national program to prevent HIV among key populations, specifically men who have sex with men (MSM) and female sex workers (FSW), with support from the U.S. Agency for International Development (USAID)2.The HIV epidemic in Ghana is heavily concentrated among key populations with high risk sexual behaviours such as female sex workers and men who have sex with men. This was based on survey evidence that key population groups faced HIV prevalence six to 12 times higher than the general population ,11. The Inform goals, objectives, and activities for the next national strategic planning cycle starting in 2016.Inform program implementation guidance to partners and standardized service delivery across partners.Provide generalizable information about effective HIV prevention program to other countries.The GAC and TWG want to conduct an evaluation so that they can understand the effectiveness of the strategic and operational plans for programs to prevent HIV among key populations in order to:http://www.cpc.unc.edu/measure/publications/sr-13-75. The process undertaken in Ghana will be of interest to program planners, managers, and implementers in other countries and across health areas who want to plan and implement an evaluation responsive to national priorities, of a large scale program at the national or subnational level, and that is part of a comprehensive M&E system.The purpose of this paper is to describe the process undertaken in Ghana to develop the document, the Evaluation Plan for the Ghana National Strategy for Key Populations  availablThe process undertaken in Ghana to develop the evaluation plan was a participatory one that involved meetings between the GAC members, TWG members, and MEASURE Evaluation staff. After the GAC, MEASURE Evaluation, and USAID reached an agreement to launch the process, the methods consisted of three two-day meetings and a final meeting of the TWG to approve the evaluation plan. This occurred over the course of 12 months, from September 2011 to September 2012. After each two-day meeting, a meeting report was prepared and necessary inputs into the evaluation plan process were developed and shared with participants to review before the next meeting. The methods to developing the evaluation plan are described in more detail below.Each of the three meetings was called by the GAC and an invitation went out to all members of the TWG from the GAC, and the GAC convened the meetings. Both the GAC and MEASURE Evaluation staff participated in developing the meeting objectives and agendas, contributing to meeting content, and facilitating the meetings. MEASURE Evaluation was responsible for producing meeting reports and documentation necessary to produce the evaluation plan. USAID funds via the MEASURE Evaluation project supported the cost of the meeting venue, per diem and transport for participants from outside Accra, and MEASURE Evaluation staff time.Meeting participants included representatives from GAC, Ghana Health Service, and the National AIDS Control Program (NACP); donors such as USAID, U.S. Centres for Disease Control and Prevention (CDC), and German International Cooperation (GIZ); multinational organizations such as UNAIDS and United Nations Population Fund (UNFPA); local universities such as the University of Ghana School of Public Health; international nongovernmental organizations (NGOs) such as FHI 360; local government sectors such as police and prisons; and local civil society organizations providing services and support to key populations such as Centre for Population Education and Human Rights Ghana (CEPEHRG), West African Programme to Combat AIDS and STIs (WAPCAS), Prolink Organisation, and Maritime Precious Life Foundation. Meeting participants also participated in providing meeting content through both presentations and in working groups.Strategic Guidance for Evaluating HIV Prevention Programs .Operational Guidelines for Monitoring and Evaluating HIV Programmes for Female Sex Workers, Men who have Sex with Men and Transgender People .The first two-day meeting was held in Accra in September 2011. The purpose of the meeting was to gain an understanding of the ongoing and planned monitoring, evaluation, and research activities related to key populations going on in Ghana and to plan for continuous, coordination and effective evaluation of key populations programming. Two main documents were used to guide the content of the meeting:Serve as a framework to organize on-going M&E activities.Reveal data gaps.Facilitate articulation of information needs relative to decision points.Facilitate planning to fill data gaps; and, ultimately.Obtain a thorough understanding of the HIV epidemic and programmatic response.Both of these documents contain the framework, “the public health questions approach to HIV M&E” (PHQA) . We usedIn the PHQA, each of the eight components needs to be addressed in order to have a comprehensive M&E system, and each step is aligned with related research questions and data collection needs.The activities carried out in this first meeting included presentations by participants on their various researches and monitoring and evaluation activities on-going in Ghana in order to take stock of the current and planned research and M&E activities. The details about the M&E and research activities presented during the meeting were mapped to the steps in the PHQA.Other activities at this first meeting included presentations on the UNAIDS approach to evaluating HIV programs , a generUsing existing documents to construct a logic model for the national HIV prevention program went a long way. However, the model was incomplete particularly in terms of what were considered the key program outcomes. Also lacking was an articulated program theory of change. Thus, we formed small groups to provide inputs on the theoretical program impact pathways and the main process, output and outcome measure from the logic model. We also had discussed the indicators and data sources for those measures.A second meeting was held in March 2012 in Accra. At this meeting, the objective was to obtain details necessary to draft the evaluation plan for the national key populations program, including identifying research questions; identifying the appropriate and feasible study designs; verifying on-going M&E activities; and discussing the main program impact pathways and gaps in data, including defining program “reach”.One of the first steps to designing an evaluation is to identify the research questions . At the With deeper knowledge about the on-going and planned M&E and research data, it became clearer that a distinction between plausibility and probability study designs was needed. Plausibility designs are appropriate when random assignment into intervention and control groups is not feasible and when the program being evaluated includes interventions of known effectiveness . In GhanParticipants also discussed the definitions of the main independent variables, particularly program “reach”. The goal of the national key populations' strategy is to reach 80% of key population at risk of HIV by 2015. To be able to measure whether the goal was accomplished, an operational definition of “reach” is needed at the national level to know whether a person was reached by the program. The concept of “program reach” is comprised of different types of service contacts . An examFeedback related to the study rationale, design and analysis plan.Input into priority study measures andRoles and responsibilities for action items for carrying out proposed data collection activities including identifying sources of funding for data gaps and responsibly parties to follow up on action steps.In between the second and third meetings, the Evaluation Plan for the Ghana National Strategy for Key Populations was drafted. The objective of that final two-day meeting in June 2012 was to obtain TWG member input and details necessary to finalize the draft evaluation plan for the national program to prevent HIV infections among key populations. Discussion points included:Information obtained during the third meeting was used to revise and finalize the evaluation plan, which was submitted to the TWG in September 2012 and presented at the Ghana National HIV and AIDS Research Conference in September 2013. The evaluation plan was subsequently posted to MEASURE Evaluation's Web site and a onetime printing of 800 copies was made for stakeholders in Ghana.Planned and on-going M&E and research activities mapped to the eight steps of the PHQA framework and the anticipated result of those activities was an important output. For example, for Step 1, “Know your epidemic: What is the size and nature of the problem” the on-going M&E activity was an integrated biological and behavioural surveillance survey (IBBSS) with MSM and FSW. Anticipated results from that activity were size estimates, denominator for coverage estimates, HIV prevalence estimates, and behavioural data.Information needs and data gaps were identified. For example, participants identified the need to better understand the role of substance abuse among MSM and risk behaviours. This information could be obtained through formative and qualitative studies. The identified gaps and information needs were relatively exhaustive, and although not all will be used in the evaluation plan, they are now documented and may be useful for other efforts.A logic model of defined activities, outputs, outcomes and impact for the national key population program was produced.Primary and secondary research questions were defined and prioritized by participants.Research methods and date sources needed to be able to execute the study design and carry out data analyses to answer the research questions were identified.Information was gathered about main outcome variables and data sources to help answer the research questions.Preliminary definitions of the main independent variables, program “reach”, and “exposure” were identified.A timeline for implementing data collection and analysis in order to yield results in time for the next strategic planning process was developed.The main outputs from the meetings included the following:Are changes in outcomes  due to the implementation of services and program components?Are there changes in behavioural outcomes and HIV prevalence and incidence over time?To what extent are planned MSM and FSW program activities realized/implemented and with improved quality?The research questions prioritized by participants for the evaluation plan were as follows:To be able to answer these questions, data from a planned survey in 2015 will help answer whether being reached by the program is associated with outcomes compared with those not reached by the program. Survey data need to be complemented by contextual data to reveal possible factors that may have modified the relationship between program reach and outcomes. Data from the surveys at two time points (one in 2011 and one planned for 2015) are necessary to understand changes in outcomes over time. Analysis of existing routine program data will help understand trends in program implementation and coverage in terms of condom promotion, HIV testing, etc. Data from process evaluations will help clarify the extent to which programs are being implemented as planned and whether quality improves over time. Comparing results from the different data sources can help show whether changes in outcomes were occurring at the same time that program reach was intensifying. Based on the study design, however, it will not be possible to say with certainty that the program resulted (or “caused”) these changes.The ultimate result from these meetings was an evaluation plan that serves as a roadmap for collecting important data for programmers which helps in identifying gaps, improving quality and scaling up the programs without diluting quality of programs. The plan informs stakeholders about which data collection activities need to be prioritized for funding, whose role it is to implement the study, the timing of data collection, the research question the data will help answer, and the analysis methods . The evaOverall, the evaluation plan has given added value to the work of the Ghana AIDS Commission in ways that were unanticipated originally. The plan has been catalytic in galvanizing support for key population related surveys and evaluation especially, in the area of resource mobilization. As a result, partners have made resources available for population size estimation and a planned 2015 IBBSS among MSM, FSW and their clients, and people who inject drugs. These studies will measure trends in the HIV epidemic and other sexually transmitted infections and track the outcomes of the implementation of the key population national strategy, a key objective of the evaluation plan. The evaluation plan has strengthened the GAC's efforts at results-based management practices where rigorous analysis and evidence-based approaches ensure program effectiveness and better health outcomes.This paper presents the experience and process in Ghana of designing an evaluation plan. The experience demonstrates that with stakeholder agreement, funding, and parties committed to carrying out the process, it is possible to design an evaluation plan that is responsive to national strategies and priorities. Although this process relied on some donor funding and external facilitation, this process respected the GAC's leadership and facilitated their ability to exercise their leadership. The process is informed by international recommendations ,9 and reThere were a number of factors that facilitated our ability to launch this activity in Ghana. There was strong national leadership, a clear national strategic plan and goals, and an active national level TWG composed of government, donors, and program implementers. The timing of developing the evaluation plan coincided with the GAC's desire to build evidence of the effectiveness of the strategic and operational plans to inform future strategic plans, to develop guidance to partners implementing programs, and to contribute to the global evidence base about what works for HIV prevention programs for key populations. Although there were no obvious control groups, there were on-going population based surveys that could serve as baselines.We hypothesize that this process may be applicable elsewhere, but will likely be successful in a context with facilitating characteristics such as strong national leadership and national strategic plans that have legitimacy with and buy-in from other stakeholders. Although the timeframe and workshop agendas were highly specific to the Ghana context, resources cited in this document, particularly the UNAIDS guidelines and the PHQA framework, are publically available to help tailor the process to other settings.The main limitation is that the process to develop the evaluation plan has not been demonstrated to be fully successful; only when the plan is used to carry out an actual evaluation will it be a success. It is important to note, however, that as a result of the evaluation planning processes, one activity has already been prioritized and funded, a performance evaluation of the national key populations program . ConfirmOther potential limitations include the time and the cost. The evaluation plan was created over the course of one year. The time frame was dependent on stakeholder's availability, as it is hard to find times for national level stakeholders to free themselves up for two full working days given their other responsibilities. On the other hand, the evaluation planning period occurred early in the strategic planning cycle, allowing for sufficient time for needed data to be collected and analysed.The approach incurred financial costs for per diems and venue rentals, for example, as well as opportunity costs. When people attended the meetings, this took them away from their other job responsibilities. Covering the financial costs can be planned for in annual budgets, and the activity of planning and conducting and evaluation can be integrated into stakeholders' work plan cycles. This has to be planned for in advance, however.The experience in Ghana demonstrates an approach to planning evaluations at the national level that includes a program theory or logic model, links decisions that need to be made to planning for data collection, and uses existing data sources where possible. To our knowledge this was the first time that all parties working on key populations programs and M&E and research in Ghana had a chance to meet and discuss the full picture of activities taking place. It is possible to have a process to evaluate HIV prevention programs that is participatory, regardless of stakeholders' level and experience with evaluation.


Plasmodium falciparum binds the Fc-region of human IgM molecules, resulting in a coating of IgM on the surface of infected erythrocytes. IgM binding is a property of P. falciparum strains showing virulence-related phenotypes such as erythrocyte rosetting. The parasite ligands for IgM binding are members of the diverse P. falciparum Erythrocyte Membrane Protein One (PfEMP1) family. However, little is known about the amino acid sequence requirements for IgM binding. Here we studied an IgM binding domain from a rosette-mediating PfEMP1 variant, DBL4ζ of TM284var1, and found that the minimal IgM binding region mapped to the central region of the DBL domain, comprising all of subdomain 2 and adjoining parts of subdomains 1 and 3. Site-directed mutagenesis of charged amino acids within subdomain 2, predicted by molecular modelling to form the IgM binding site, showed no marked effect on IgM binding properties. Overall, this study identifies the minimal IgM binding region of a PfEMP1 domain, and indicates that the existing homology model of PfEMP1-IgM interaction is incorrect. Further work is needed to identify the specific interaction site for IgM within the minimal binding region of PfEMP1.Binding of host immunoglobulin is a common immune evasion mechanism demonstrated by microbial pathogens. Previous work showed that the malaria parasite  Each parasite has approximately 60 var genes in its genome, with only one transcribed at a time per iRBC var gene transcription leads to a change in the PfEMP1 variant expressed on the surface of the iRBC and is responsible for antigenic variation of malaria parasites x) and CIDR , based on sequence similarity P. falciparum phenotypes such as rosetting with uninfected RBC in severe childhood malaria Many pathogens have evolved to bind to a common site on the Fc portion of immunoglobulin, however, the consequences of such interactions are largely unexplored P. falciparum line TM284R+, which is a culture-adapted parasite derived from a Thai patient with cerebral malaria Previously we studied an IgM binding rosetting P. falciparum genotypes In our previous work, we localised the PfEMP1-IgM binding interaction site to the Cμ3-Cμ4 region of IgM Fc, and showed that the same site on IgM is used by multiple different 22.1x objective. The total number of COS-7 cells per field was counted using the auto-fluorescence of the cell nuclei to identify individual cells. The precise number of cells counted differed in experiments with varying cell confluence (80–100% confluent), but at least 500 COS-7 cells per slide were counted in all cases. Positive cells were defined as those showing fluorescence over the whole COS-7 cell surface as indicated in Fig. S1 and in our previously published work Deletion constructs based on TM284var1 (Genbank accession number JQ684046) DBL4ζ were amplified and cloned into the pRE4 vector as described previously 2.2The homology model of TM284var1 DBL4ζ was constructed with the automated homology modelling tools in DeepView v.3.7 2.3Pfx Platinum polymerase according to the manufacturer's protocol (Invitrogen). The primers used for the E1663R mutant were 5′-caatgggagaaaacacgaaatgaagcacaaaaa-3′ and 5′-tttttgtgcttcatttcgtgttttctcccattg-3′. The primers used for R1764E mutant were 5′-ttcctttttgtaaaagaaggaaaaggagatgga-3′ and 5′-tccatctccttttccttcttttacaaaaaggaa-3′. The first PCR amplified two fragments from the wild type construct that was used as a template. The two fragments contained overlapping complimentary ends and included the mutation that would result in an amino acid substitution. The second PCR used primers specific for the outer borders  to amplify the overlapping fragments made from the first PCR. These primers were 5′-aaggatccaactgtgctgaaaaggttgct-3′  and 5′- aagctagcttacttacatttacaagcattacc-3′ . The resulting PCR product was cloned into the pET15b-modified vector Escherichia coli Origami B cells (Novagen) were transformed and grown to O.D. of 1.2 at 600 nm. Bacterial cultures were induced with a final concentration of 1 mM IPTG and grown overnight at 25 °C in an orbital shaker. Bacterial pellets were harvested, sonicated and protein was purified from soluble lysate using Ni-NTA metal affinity chromatography. Fractions containing protein were combined, concentrated using Amicon Ultra centrifugal filters (Millipore) and further purified by size exclusion chromatography on a Superdex 200 (16/60) column . Fractions were collected and the presence of protein at the expected molecular weight of approximately 58kDa was assessed by SDS-PAGE (see below). Fractions were concentrated and stored at −80 °C prior to use in circular dichroism (CD) or Surface Plasmon Resonance (SPR).Site-directed mutagenesis of TM284var1 DBL4ζ was carried out by two-step PCRs using mutagenic primers and 2.4The purity of the eluted protein was assessed by SDS-PAGE. Five μg of purified protein was prepared in loading buffer under non-reducing and reducing (5% β-mercaptoethanol) conditions and heated to 80 °C for 10 min. Five μl of broad range pre-stained marker (NEB) was run alongside the recombinant DBL4ζ proteins. Electrophoresis was carried out on 4–12% bis-Tris polyacrylamide gradient gels with MES SDS running buffer, and the gels were stained with Simply Blue SafeStain using the manufacturer’s protocols (Invitrogen). For the western blot, 5 μg wild type and mutant DBL domains were run on a 4–12% bis-Tris polyacrylamide gradient gel with MOPS SDS running buffer (Life Technologies). Replicate gels were either stained using Simply Blue SafeStain (Invitrogen) or transferred onto a PVDF membrane using the iBlot gel transfer device (Life Technologies). The membrane was probed with a Penta His HRP-conjugated antibody , and developed using Super Signal West Pico (Thermo Scientific).2.5http://dichroweb.cryst.bbk.ac.uk/html/userguide.shtml).DBL4ζ recombinant proteins were dialysed in 50 mM phosphate buffer (pH 7.2) overnight at 4 °C and concentration adjusted to 0.1 mg/ml. CD spectra were recorded with 300 μl of DBL4ζ proteins using a Chiracan-plus spectrometer (Applied Photophysics) at 25 °C. A cell of 0.05 cm path length was used and measurements were recorded at 1 nm intervals between 190 and 260 nm at 1s averaging time for each point. Ten consecutive measurements were averaged and corrected against buffer alone. Results were normalized to mean molar differential coefficient per amino acid residue  at a flow rate of 25 μl/min. After each run the chip was regenerated with 10 mM Glycine-HCl (pH 2.5). The responses of specific binding to IgM-coated channels were calculated by subtracting the response obtained from binding to an uncoated lane monitored simultaneously. The sensograms were fitted using a 1:1 Langmuir kinetic model and the ProteOn Manager software was used to derive the values for ka, kd and KD.IgM  (25 μg/ml) was immobilized onto the surface of a GLC sensor chip  at pH 5, using the manufacturer’s amine-coupling kit and protocol. The binding of DBL4ζ wild type and mutants to human IgM was measured using a ProteOn XP36 biosensor instrument (BioRad). Recombinant proteins in 10 mM Tris pH 7.4, containing 140 mM NaCl, 2 mM CaCl33.1We previously showed that recombinant TM284var1 DBL4ζ (amino acids Glu1481-Thr1952) containing 16 cysteine residues (construct 1) binds to human IgM DBL domains are composed of a core scaffold of alpha-helical bundles stabilised by disulphide bonds, and consist of three subdomains 3.2We previously used homology modelling of the TM284var1 DBL4ζ domain E. coli and tested the ability of each mutant to bind human IgM. In each mutant the key residue(s) were mutated to residues showing the opposite charge. The proteins tested were single amino acid mutants E1663R and R1764E, double mutant E1663R, R1764E and quadruple mutant E1663R, E1665R, R1764E, E1779K. All proteins were expressed as soluble his-tagged proteins in E. coli and were purified using nickel affinity chromatography followed by size exclusion chromatography. The proteins were assessed by SDS-PAGE and in each case showed a major band about 56 kDa under non-reducing conditions, with a shift upon reduction consistent with the presence of disulfide bonds in the recombinant proteins  in the nanomolar range for all proteins . This suggests that a fraction of the R1764E protein preparation bound irreversibly to the chip, probably due to small amounts of aggregates. Critically, however, the rest of the protein bound with similar kinetics to the wild type protein. Overall, the SPR data show that the mutated residues do not play a major role in binding human IgM, although further work would be needed to exclude minor effects.Surface Plasmon Resonance (SPR) was used to assess the binding of single (E1663R and R1764E), double  and quadruple  mutants to immobilized IgM. All mutants bound IgM similarly to the wild type , with biproteins . There wn curves . Fitting4In this study, we identified the minimal IgM binding region of the DBL4ζ domain of TM284var1, and tested the function of amino acid residues predicted from a PfEMP1-IgM homology model to be involved in binding IgM. We used deletion mutants to identify the minimal IgM binding region comprising all of subdomain 2 of DBL4ζ, along with adjoining regions of subdomains 1 and 3, containing seven cysteine residues in total. However, testing of charged amino acids within subdomain 2 that were predicted from homology modelling to form part of the interaction site with IgM, showed no large effect on IgM binding when the specific amino acids were mutated to ones bearing the opposite charge. Therefore the existing model is not supported by experimental data, and needs to be revised.Recent structural studies of DBL-receptor complexes have shown that although the DBL core scaffold composed of alpha helices is very similar between different DBL structures, there is extensive variation in the surface exposed loops linking the helices KD values. Despite the uncertainty introduced by the lack of completely pure protein, it is clear from the SPR data that all the mutant proteins retained the ability to bind human IgM at similar concentrations as the wild type. Repeated experiments with more highly purified protein preparations would be needed to determine whether there are any minor differences in binding affinity between the mutants and the wild type.One of the limitations of the current study was the difficulty in preparing completely pure recombinant protein. Even after size exclusion chromatography, multiple protein species were seen on SDS-PAGE. Despite this, the DBL4ζ proteins showed the characteristic secondary structure of DBL domains by CD, and all proteins bound human IgM at nanomolar concentrations by SPR. Western blotting indicated that the >50 kDa proteins were his-tagged, likely representing the full-length protein and fragments with limited proteolytic degradation at the C-terminus. The experiments shown in P. falciparum proteins also results in effects on host Fcμ receptors or B cell receptors remains unknown Plasmodium host-parasite interactions and severe malaria.Recent work has provided some insights into the function of PfEMP1-IgM interactions. The binding of IgM to the VAR2CSA PfEMP1 variant, that plays a key role in sequestration of iRBC in the placenta in pregnancy malaria, has been shown to be an immune evasion mechanism that masks the iRBC from being targeted by parasite-specific IgG antibodies


Radial endobronchial ultrasound with a guide sheath (EBUS-GS) has improved the diagnostic outcomes of peripheral lung lesions. However, to our knowledge, reports on the use of EBUS-GS for diagnosis of cavitary lesions are unavailable. Therefore, this study aimed to assess the effectiveness and safety of EBUS-GS for diagnosis of peripheral cavitary lung lesions (PCLLs).This study was a single-institution retrospective review of PCLLs examined by using EBUS-GS between July 2013 and October 2015. The diagnostic results of different EBUS-GS samples, including cytologic, histopathologic, and microbiologic samples, were analysed separately.P = 0.03). Although pulmonary infection occurred after the procedure in 1 patient (2.0 %), no other complications, including pneumothorax or significant haemorrhage, were reported.Of 696 radial EBUS procedures performed during the study period, 50 were performed for examination of PCLLs. The overall diagnostic yield for EBUS-GS was 80 % (40/50). Regarding 27 malignant lesions, the diagnostic yields for cytologic and histopathologic samples were 63.0 % (17/27) and 74.1 % (20/27), respectively. Regarding 23 benign lesions, the diagnostic yields for histopathologic and microbiologic samples were 69.6 % (16/23) and 47.8 % (11/23), respectively. Uni- and multivariate analyses indicated that the EBUS probe being within the lesion was the only factor significantly associated with increased diagnostic yield (odds ratio, 7.04; EBUS-GS was found to be an effective and safe procedure for diagnosis of PCLLs. A pulmonary cavity is defined as a gas-filled area within a pulmonary consolidation, mass, or nodule that may or may not contain a fluid level and is surrounded by a wall, usually of varied thickness . VariousAccording to the guidelines of the American College of Chest Physicians, the sensitivity of transbronchial biopsy (TBB) for diagnosis of peripheral lung lesions is 63 % for lesions >20 mm, and only 34 % for lesions <20 mm , 6. WithHowever, reports on the use of R-EBUS for diagnosis of cavitary lung lesions with a relatively high probability of infectious disease are limited . BecauseThis study was a retrospective review of a prospectively maintained database and medical records at our institution. The Institutional Review Board of Osaka Prefectural Medical Center for Respiratory and Allergic Diseases approved this study without the need to obtain informed consent. Written informed consent for bronchoscopy was obtained from all eligible patients.Patient information  and lesion characteristics were collected prospectively in the database before diagnostic bronchoscopy. Lesion characteristics, including type , maximum diameter, target bronchus, and presence of bronchus sign, were evaluated on thin-section chest CT images during regular team discussions that included board-certified bronchoscopists, pulmonologists, and medical oncologists. VBN  was used when the target bronchus was small and difficult to trace .PCLLs examined by using EBUS-GS at our institution between July 2013 and October 2015 were included in this study. Cases in which ultrathin bronchoscopes  were additionally used were excluded. A PCLL was defined as a bronchoscopically invisible lung lesion that had an internal gas-filled area and was surrounded by pulmonary parenchyma . MaximumAll EBUS-GS procedures were performed by expert bronchoscopists or trainees with sufficient experience with conventional bronchoscopy and EBUS-GS assistance. During all the procedures, the trainees were supervised by the experts.EBUS-GS procedures were carried out by using one of the following bronchoscopes (Olympus Ltd.): BF-1T260, BF-P260, BF-F260, or BF-Y0053, a new middle-range bronchoscope with a 5.1-mm outer diameter and a 2.6-mm working channel . A guideUpon insertion of the scope to reach the target bronchus, the guide sheath together with the R-EBUS probe was inserted through the working channel and advanced under fluoroscopic guidance. EBUS images before transbronchial sampling were categorized into 3 patterns: ‘within’ (the probe was located in a bronchus that was inside the lesion), ‘adjacent to’ , or ‘invisible’ (the probe was not able to reach the lesion) . After lMycobacterium tuberculosis (TB), and cultures including TB. Although additional bronchial wash was performed in 2 patients, those results were not assessed in this study.Brush and needle smears and tissue stumps on glass slides were sent for cytologic examination. Tissue samples obtained by using forceps were sent for histopathologic examination. After each sampling, the brush, needles, and forceps were rinsed with 5 mL normal saline (device wash) . After aFinal diagnosis was determined based on either one or a combination of the results of EBUS-GS, EBUS-TBNA, surgery, or clinical follow-up for at least 3 months. A lesion was designated as inflammation if it showed no significant pathologic or clinical findings, and it decreased in size on follow-up CT.For malignant lesions, class IV/V results on cytology or specific malignant findings on histopathology were considered as diagnostic. For benign lesions, specific benign findings on histopathology  or positive cultures on microbiology were considered as diagnostic. Histopathologic results were reviewed by 2 investigators (MH and KK). Overall, EBUS-GS was considered as diagnostic when at least 1 of the cytologic, histopathologic, or microbiologic results was diagnostic.All bronchoscopies were performed in inpatient settings. After every procedure, chest radiography was performed and oral antibiotics (cefcapene pivoxil 300 mg/day for 3 days) were administered. A major complication was defined as an event that necessitated premature termination of the procedure or a symptomatic postprocedure sequela, including pneumothorax, haemorrhage, infection, air embolism, or other untoward life-threatening outcome .P value of <0.05 was considered to indicate statistical significance. All statistical analyses were performed by using EZR , a graphical user interface for R  [Descriptive statistics are presented as frequency, percentage, and median (range). Univariate analyses were performed by using Fisher’s exact test for categorical data. Multivariate logistic regression analysis was performed to determine the factors associated with increased diagnostic yield. Variables for the multivariate analysis model were chosen from the results of the univariate analyses and experimentally predicted factors, such as lesion size and bronchus sign. A 2-tailed Austria) .Among 696 R-EBUS procedures performed between July 2013 and October 2015 at our institution, 50 PCLLs examined by using EBUS-GS were included in this study . Multivariate logistic regression analyses using the possible cofounders of lesion size, bronchus sign and EBUS probe ‘within’ also revealed that the only significant factor affecting diagnostic yield was EBUS probe ‘within’  . Univariate analyses indicated that PCLLs in which the EBUS probe could be placed within the lesion had a significantly higher diagnostic yield compared with when the EBUS probe was adjacent to the lesion or invisible  for PCLLs with a low risk of significant complications (2.0 %). Second, the EBUS-GS sampling method combined with cytologic, histopathologic, and microbiologic samples was effective for diagnosis of both benign and malignant PCLLs.A cavity is the result of such pathologic processes as necrosis, cystic dilation of pulmonary structures, and displacement of lung tissue by cystic structures . In addiCurrently, EBUS-GS is becoming a standard procedure for diagnosis of peripheral lung lesions based on its useful and safe profile. Therefore, confirmation of its diagnostic utility for PCLLs is also demanded in clinical practice. In the present study, we first reported a high diagnostic yield (80 %) for EBUS-GS for PCLLs. This result is comparable to the diagnostic yield for R-EBUS for peripheral lung lesions reported in recent studies. Therefore, EBUS-GS can be considered as a standard procedure for diagnosis of peripheral lung lesions and PCLLs , 21–23. The complication rate in the present study was 2.0 % (pulmonary infection), which is lower than that reported in previous studies on the use of TTNA for diagnosis of cavitary lung lesions , 20. TheThis study also demonstrated that the EBUS-GS sampling method combined with cytologic, histopathologic, and microbiologic samples was effective for diagnosis of both benign and malignant PCLLs. In the present study, diagnostic tissue samples were obtained by using TBB for 74.1 % of malignant lesions and 69.6 % of benign lesions. Recently, histopathologic diagnosis of lung cancer is becoming important for definitive treatment including chemotherapy and tyrosine kinase inhibitors . MoreoveAspergillus culture was not performed routinely. Because the culture sensitivities of these diseases using bronchoscopy samples are not satisfactory [In the present study, microbiologic samples demonstrated a diagnostic yield of 47.8 % for benign diseases; the diagnostic yield was particularly high for mycobacterium infection, which is the main cause of a cavitary lung lesion . Microbiology results were negative for infectious cases with lung abscess or Aspergillosis, though anaerobic or sfactory , 30, rousfactory , 31–33.The present study has several limitations. First, this was a retrospective, nonrandomised, single-institution study with a small sample size. Second, the procedures were not performed by the same operator. Thus, it remains unclear whether the results can be generalized to other institutions.In conclusion, we presented the first report on EBUS-GS for PCLLs based on our clinical experience. Our results indicate that EBUS-GS is a safe and effective procedure for diagnosis of PCLLs. Sufficient cytologic, histopathologic, and microbiologic samples can be obtained for both benign and malignant PCLLs. Further multicenter prospective studies are recommended in the future.The Institutional Review Board of Osaka Prefectural Medical Center for Respiratory and Allergic Diseases approved this study without the need to obtain informed consent.The dataset supporting the conclusions of this article is presented within the article. The detailed clinical data set is not publically available to protect research subject privacy and confidentiality.