Dear editor,The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2)-related acute respiratory distress syndrome (ARDS) is associated with an elevated coagulation activation pattern  and a hiHere, we describe the feasibility, safety, and diagnostic accuracy of endobronchial ultrasound (EBUS) to detect PE in patients with severe SARS-CoV-2 ARDS requiring vv-ECMO. Between April 15 and May 1, 2020, eleven patients were included. The procedure was performed using a 6.7-mm-outer-diameter, real-time, bronchoscope  with a 7.5-MHz linear ultrasound transducer  equipped with color-Doppler. For each patient, EBUS procedure followed the same roadmap  years old, with a body mass index of 29 [28–31] kg/m2  without PE on the EBUS had left segmental pulmonary embolism on CTPA, which was not accessible to the EBUS.Diagnostic correspondence between EBUS and CTPA is depicted in Table This case series of EBUS to diagnose PE in severe SARS-CoV-2 ARDS patients requiring vv-ECMO suggests that the EBUS procedure is safe and reliable to detect lobar and even segmental PE at bedside. Given the high risk of pulmonary embolism in patients with severe ARDS due to COVID-19, this minimally invasive diagnostic approach seems to be a useful and appropriate diagnostic tool to avoid the multiple adverse effects of CTPA in these severe and often unstable patients. The diagnostic performance of this innovative and promising technique needs now to be confronted to CTPA in larger prospective study and other clinical situations, especially for the analysis of segmental arteries.


Intensive Care (ICU) involves extended and long lasting support of vital functions and organs. However, current training programs of ICU residents mainly focus on extended support of vital functions and barely involve training on cost-awareness and outcome. We incorporated an educational program on high-value cost-conscious care for residents and fellows on our ICU and measured the effect of education.A cohort study with factorial survey design, in which ICU residents and fellows were asked to evaluate clinical vignettes, was performed on the mixed surgical-medical ICU of the Amsterdam University Medical Centre. Residents were offered an educational program focusing on outcome and costs of ICU care. Before and after the program they filled out a questionnaire, which consisted of 23 vignettes, in which known predictors of outcome of community acquired pneumonia (CAP), pancreatitis, acute respiratory distress syndrome (ARDS) and cardiac arrest were presented, together with varying patient factors , acute kidney failure (AKI) and haemato-oncological malignancy). Participants were asked to either admit the patient or estimate mortality.BMI, haemato-oncological malignancy and severity of pancreatitis were discriminative for admission to ICU in clinical vignettes on pancreatitis and CAP. After education, only severity of pancreatitis was judged as discriminative. Before the intervention only location of cardiac arrest  was distinctive for mortality, afterwards this changed to presence of haemato-oncological malignancy.We incorporated an educational program on high-value cost-conscious care in the training of ICU physicians. Based on our vignette study, we conclude that the improvement of knowledge of costs and prognosis after this program was limited. With a growing elderly population, technical innovations, and new treatment possibilities, healthcare is facing new challenges in high quality but cost- effective care. The Netherlands has one of the best healthcare systems in the world, however, maintaining this position is expensive, accounting for about 10% of the Dutch Gross National Product in 2017 . The difCareful decisions on the use of critical care can only be made when physicians are fully acquainted with the potential clinical outcomes as well as the costs of ICU care. However, the level of awareness regarding the costs of common prescription drugs , and commonly used materials in the ICU is often lacking, as described by Hernu et al.  MoreoverThe Awareness project was launched in the Netherlands in 2017–2018 by the Federation of Medical Specialists and Maastricht University, to try to incorporate awareness on value and expenditure in addition to the CANMED competencies in all post graduate medical training programs. Its goals were to preserve high quality care, while at the same time creating more awareness regarding the appropriateness of care and raise cost-consciousness . This isAn educational program for ICU residents and fellows enhancing knowledge on outcome and costs of ICU care was carried out at the Intensive Care Unit of the Amsterdam University Medical Centre. The present study aims to assess the effect of this educational program on the knowledge of treatment outcomes and associated costs of ICU support.In this cohort study we used a factorial survey design in which participants were asked to evaluate clinical vignettes. Hereby importance of factors influencing decision making could be assessed. A vignette is a brief, written case history of a fictitious patient that is based on a realistic clinical situation. In the vignettes the factors of interest (in our study possible predictors of ICU outcome) were varied between the different vignettes, in which each combination was unique. We made combinations between categories of predictors, in which only one factor changed between each vignette. These vignettes were presented to the participant, residents and fellows in our ICU, in an online questionnaire using the Survey Monkey website . Formal The online questionnaire consisted of 23 clinical vignettes, in which four common ICU admission diagnoses were presented; community acquired pneumonia (CAP), pancreatitis, acute respiratory distress syndrome (ARDS) and cardiac arrest. Admission diagnoses were discussed between authors and chosen for because CAP and OHCA are among the five most common admission diagnosis in the Netherlands in 2018  and our With the complete set of factors, 324 vignettes for community acquired pneumonia (2x2x3x3x3x3), 432 vignettes for pancreatitis (2x2x3x3x4x3), 288 vignettes for ARDS (2x2x3x3x2x4) and 288 vignettes for cardiac arrest (2x2x3x3x2x2x2) were created. Completing all 1332 vignettes would be too time-consuming for participants. In each vignette only one factor changed to the following vignette. A total of 23 clinically vignettes  were selected by a team of experts/ICU clinicians based on their realistic scenarios. The vignettes were written by one of the investigators (LW) and discussed by the other investigators (MM and CB) on clinical accuracy and realism. The complete questionnaire can be found in Supplement For the vignettes on CAP and pancreatitis, participants were asked to choose between admission or no admission to the ICU, taking into account chances of survival and treatment. For all vignettes with ARDS or cardiac arrest participants were asked to estimate mortality during first 30 days of ICU stay. The options were < 40% mortality, 40–80% mortality or > 80% mortality.Also, five questions were added on the cost of five products, which are frequently used in the ICU  scan of the head, platelet transfusion (one unit), immune-modulating enteral feeding, forced-air heating blanket). Each question was multiple choice and respondents were asked to pick the right price for the product.Demographics of the participants were collected at the start of the questionnaire. Recorded variables included working experience in ICU care, previous experience in residency or fellowship and primary postgraduate medical education programs. The first questionnaire was carried out in January 2018, the second, after the educational intervention, in April 2018. The questionnaire was carried out in January 2018, and again, after the educational intervention, in April 2018.This study was conducted at the Intensive care department of the Amsterdam University Medical Center, location Academic Medical Center, at the University of Amsterdam. The department is a 34-bed mixed medical-surgical ICU, where residents of various postgraduate medical education programs are trained . In addition, each year 7 fellows are trained to become an intensivist as a subspecialty of their training in anaesthesiology, internal medicine, cardiology or neurology.The study population consisted of ICU residents with medical training in anaesthesiology, internal medicine, emergency medicine, neurosurgery, cardiothoracic surgery or general surgery, and fellows in Intensive Care Medicine, with a medical specialization in internal medicine, anaesthesiology or neurology. All physicians were employed on the ICU of the Amsterdam Medical Centre, a tertiary clinic, during the study period.Between January and April 2018 an educational program on outcome and cost of ICU treatment was implemented in the regular educational program. This program consisted of flipped classroom sessions on outcomes of patients admitted to the ICU with a certain illness, comorbidity or patient characteristic; lectures on costs of ICU care and cost reduction and organization of ICU and a weekly quiz . The fliThe weekly quiz with a fictional but realistic case and a question on the actual costs of a certain investigation, medication or material was sent to all participants every Monday. The aim was to draw more attention to the project and stimulate discussion among participants.Potential modifiers of the measured effect of the intervention are previous experience in ICU care, outcome of ICU care, cost and already gathered knowledge on high-value cost-conscious care during general medical training or residency. For this reason, we included all residents and fellows employed at our ICU during the intervention and asked about their level of experience in ICU care. There was risk of inclusion bias, since participation was voluntary and this could select only participants interested in the subject of the study. To prevent selection bias we briefed all residents and fellows about the study and potential of high-value cost-conscious care education to gain interest in the study. We tried to minimize loss of follow up by asking participants to complete the final questionnaire on several occasions.Descriptive statistics were used to describe the characteristics of the participating residents. We used logistic regression to determine the first outcome, relative importance of the factors for ICU admission, in which ICU admission was set as determinant and the factors as independent variables. Data are shown as odds ratios (OR) with 95% confidence interval.For the second outcome, the importance of the factors for the estimate of mortality, we used univariate multi-nominal regressing, with percentage of survival as determinant and the factors as independent variables. Data are shown as odds ratios (OR) with 95% confidence interval.The third outcome, the estimation of costs of products used regularly in ICU, was noted a percentage different from the true cost. Results are shown as means with standard error of the mean (SEM). A students unpaired t-test was performed to compare answers before and after education.IBM Spss version 25 was used for the statistical analyses and a p value of < 0.05 was considered statistically significant.All residents and fellows employed at our ICU during the intervention were included in the study. The pre-education online questionnaire was completed by 32 participants (100% of those who attended) and the post-education questionnaire by 27 participants (84%). Loss to follow up of 5 participants was due to termination of the internship at ICU, after which they changed hospitals. There was no missing data. All the participants answered all the questions in the questionnaire. The majority of respondents had a background in anaesthesiology (56% and respectively 56%) and Internal medicine (25% and respectively 26%). Nine respondents had over 1 year of experience in Intensive Care, while four had no experience at all  and immune-modulating enteral food  both significantly showed reduced deviation in regard to the true product cost after education.All participants filled out all the questions on costs of ICU products. After the educational program the questions on costs showed reduced deviation of the true costs, except arterial blood gas analysis Fig. . The devWe implemented an educational program for residents and fellows on the outcome of ICU care in a tertiary academic ICU in the Netherlands. The program was designed to enhance knowledge about outcome and mortality of the most common ICU diagnoses and comorbidities. The program consisted of flipped class room sessions on outcome of care, classical lectures on benchmarking ICUs, organization of ICU and costs of ICU care, and a weekly quiz on costs of products often used in our ICU.We used a clinical vignette study to evaluate the learning effect of our educational program. A vignette study can show which factors are pivotal in the decision whether to admit a certain patient to the ICU.Participating physicians showed to make different decisions pre- versus post-education. A BMI over 25 and presence of a haematological malignancy were considered an important factor for ICU admission pre-education, however this effect was not measured after the educational program. In the teaching program residents learned that a BMI > 25 can be protective in certain situations –23. In aThe current project was carried out as a part of the Dutch Awareness project, which is a successor to the Choosing Wisely campaign, launched by the American Boards of Internal Medicine (ABIM) Foundation in 2012 . The infThere are several limitations to our study. For feasibility reasons the number of clinical vignettes was set to 23. In creating the vignettes only one of the factors could change between each case; this limited the number of factors used in our vignettes. Also, the range in answers was smaller than expected; this resulted in insufficient data to perform statistical analysis for some factors or categories. Future studies should therefore include a larger number of participants, so more vignettes can be developed and divided between participants.The residents completing the questionnaire probably also experienced a learning effect from their ICU internship next to the supplied additional educational program. We cannot differentiate between the acquired knowledge from the educational program and the effect of their day to day job . Also, while this program was scheduled during normal working hours, some residents could not attend all classes. To this extent, all lectures were available online to the whole group, also those who did not attend the class in person. However, we do not have any data on which lectures were reviewed by those who did not attend. Hence, we were not able to relate the level of participation to the eventual learning effect of the program. The decision to admit a patient to ICU is often based on clinical judgement, combined with knowledge on predictors of outcome. Moreover, overuse and inappropriate use of ICU care should be limited as much as possible. Participants were asked to choose between ICU- admission or withholding admission in some of the vignettes, taking account the expected benefit of ICU care. This might be a hard choice. The consensus between ICU physicians whether a patient will benefit from ICU admission is poor, as was shown by Valley et al.  At most,The basic knowledge on ICU mortality and contributing factors is limited in residents and fellows at our ICU and there is a need to improve this knowledge to improve cost-conscious decision making in an era of rising health care costs. Our study shows the possible implications of an educational program focusing on knowledge about ICU admission. Based on our vignette study, we conclude that the improvement of knowledge of costs and prognosis after this program was limited. With this information we would like to improve our educational program and assess the outcomes within a larger sample size and over a longer period.Additional file 1. Translation complete vignette questionnaire.


The accurate identification of venous thromboembolism prophylaxis implementation barriers is an important part of prophylaxis prevention. However, in China, data to help identify these barriers is limited. This study has two objectives: 1) to determine the knowledge, attitudes, and practices (KAPs) of healthcare professionals regarding graduated compression stockings (GCS) since the launch of the National Program for the Prevention and Management of Pulmonary Embolism (PE) and Deep Venous Thrombosis (DVT) in October 2018 and 2) to identify the obstacles and assist the program.This was a cross-sectional study of 5070 healthcare professionals in China. We used exploratory factor and reliability analyses to evaluate the researcher-designed questionnaire’s reliability and validity. The formal questionnaire, which included demographic data, knowledge, attitudes, and clinical practice patterns, was distributed to healthcare professionals.Of the 5070 respondents, 32.5% had a good knowledge of GCS, 78.5% had a positive attitude towards their use, and 34.0% exhibited normative behavior when applying them. The KAPs of healthcare professionals towards GCS were significantly correlated with one another. Binary logistic regression suggested that the training received by healthcare professionals was an important factor affecting their knowledge regarding GCS usage.The training provided for the use of GCS in China cannot meet medical staff needs and deserves more attention from policy makers. This represents an obstacle for venous thromboembolism prophylaxis, which restricts the effective implementation of the National Program for Prevention and Management of PE and DVT. Venous thromboembolism (VTE) is the third-leading cause of cardiovascular-associated deaths worldwide . EffectiThe National Program for the Prevention and Management of Pulmonary Embolism (PE) and Deep Venous Thrombosis (DVT) was launched in October 2018 in China. This program aims to improve the overall ability of hospitals to prevent, diagnose, and treat VTE. In this study, we aimed to determine the knowledge, attitude, and practices (KAPs) conducted by healthcare professionals regarding graduated compression stockings (GCS) since the launch of the National Program to identify obstacles and assist this program. To the best of our knowledge, our study is the first to explore the KAPs regarding the use of GCS by healthcare professionals accredited by the National Program and provide a basis for follow-up work.This was a non-interventional, anonymized, self-administered, one-time web-based survey for healthcare professionals in China. Based on the National Program for Prevention and Management of PE and DVT, this survey was conducted from November 19 to December 5, 2019.The questionnaire items were designed by experts from the working group for the National Program for Prevention and Management of PE and DVT. An original questionnaire that included 31 items was developed based on a preliminary survey of 928 cases to evaluate its reliability and validity. In the pre-investigation, researchers screened all items and created a formal questionnaire through exploratory factor analysis (EFA) using IBM SPSS Statistics for Windows version 25.0 . The questionnaire, which had a high Cronbach’s alpha coefficient (0.952), was designed by experts from the National Program , which generated a QR code. The QR code was then distributed to the certifying agency’s liaisons through the National Program’s platform. The participants used the QR code through the WeChat application , and responded to and submitted the questionnaire.This was a closed survey. The researchers uploaded the questionnaire to the Sojump online platform . Exploratory factor analysis and reliability analysis were used to evaluate the reliability and validity of this researcher-designed questionnaire. Descriptive statistical analysis was used to summarize the demographic characteristics of the 5070 healthcare professionals. Continuous data were expressed as means and standard deviations and were compared using Student’s Demographic characteristics are shown in Table Figure r = 0.463, p < 0.01). Furthermore, there was a significantly positive strong correlation between knowledge and practice , and a significantly positive moderate correlation between attitude and practice .Table p < 0.05; p < 0.01) and included gender, age, hospital level, service years, administrative duties, and GCS usage training , most of whom worked in tertiary hospitals (89.2%). China is a developing country with an uneven distribution of medical resources. Generally speaking, medical personnel with high-level knowledge are concentrated in tertiary hospitals. As such, we are concerned about secondary and community hospitals. Hence, the project team faces great work challenges and more doctors are needed to be involved in future research. In addition, a larger study sample is required.The knowledge of healthcare professionals regarding GCS clinical usage is not profound, and this represents a barrier for VTE prophylaxis in China. Training for the proper use of GCS in China has not yet met medical staff needs and deserves primary attention from policy makers. The substantial gap in the implementation of the program in the real world may be overcame by gaining the attention of practitioners as well as that of hospital management, which may result in the enhancement of medical education and management regarding GCS clinical usage.Additional file 1.


Earthquakes are natural disasters that cause damage in a wide range of regions and represent a complex system that does not have a clear causal relationship with specific observable factors. This research analyzes the earthquake activities on the Korean Peninsula with respect to spatial and temporal factors. Using logarithmic regression analysis, we showed that the relationship between the location of the earthquake and its frequency in these locations follows a power law distribution. In addition, we showed that since 1998 the average earthquake magnitude has decreased from 3.0143 to 2.5433 and the frequency has risen by 3.98 times. Finally, the spatial analysis revealed significantly concentrated earthquake activities in a few particular areas and showed that earthquake occurrence points have shifted southeast. This research showed the change in earthquake dynamics and concentration of earthquake activities in particular regions over time. This finding implies the necessity of further research on spatially-derived earthquake policies on the change of earthquake dynamics. This complex system can be found in both social7 and natural11 phenomena, and there are studies analyzing such complex systems using the power law15 and complex networks21. Therefore, to investigate the earthquake phenomena of the Korean Peninsula, this study examines the change of earthquake patterns with time and the distribution of the power law according to space and magnitude.Damage caused by earthquakes is difficult to predict22. With an average of 41.65 earthquakes stronger than 23. However, in 2016 and 2017, the number of earthquakes stronger than 24. These consecutive strong earthquakes have increased public interest in earthquakes25.The selected region for this research, the Korean Peninsula, is located in the stable intraplate region of East Asia26 and their aftershocks, and Gutenberg proved that if the earthquake magnitude is small, the frequency increases in a constant ratio27. Using identified earthquake power laws from previous studies, Serra and Corral compared the truncated gamma and tapered Gutenberg–Richter distributions to the simple Gutenberg–Richter power law distribution. They proved that truncated gamma distribution shows significant improvement over the simple power law when fitting the earthquake moment distribution of the whole Earth28.Various earthquake studies have been conducted to analyze their complexity. Omori validated the power law distribution between earthquakes29. Liu conducted research on the spatiotemporal complexity of earthquakes by applying a complex network to sections divided according to rock mass, which are based on earthquake and ground mass data. As a result, the connectivity distribution of the earthquake network showed a power law distribution between the average maximum magnitude of a region and frequency30. Earthquake networks have also been used to consider the spatial factors of earthquakes33. Moreover, previous studies have analyzed earthquake networks based on their sequence35, and Abe concluded that earthquake studies based on time distribution were necessary, using data from different regions36.Spatial factors are the key elements to understanding the original form of the earthquake, the damage caused by the earthquake, and earthquake-related social policymaking39. In particular, Kim and Choi studied active faults, concluding that further studies are needed to produce active fault maps41. According to a study of the Yangsan fault zone, which resulted in the largest earthquake on the Korean Peninsula, four significant deformation episodes occurred in the fault, and the dextral slip faulting in the late Paleogene had a significant effect on the deformation. Due to the deformation of the Yangsan fault, it was temporarily reactivated42.In Korea, studies are focused around Gyeongju and Pohang, where the largest earthquake occurred so farThis research considers spatiotemporal perspectives. We identified the relationship between the spatial-location and the frequency of earthquakes and its activities in space, along with an analysis of earthquake activity changes according to time. Spatial data was created based on the longitude and latitude of earthquakes that occurred in the Korean Peninsula. While previous studies divided the space into equal areas based on the rock mass, this study creates rectangular grid-sections based on the extreme latitude and longitude points of the earthquake. An analysis of the relationship between the grid-sections and the frequency of earthquakes in these grid-sections is conducted. In addition, the Gutenberg–Richter law is established between the magnitude and frequency of earthquakes by period, and the change in earthquake activity by period is identified to assess the overall change of earthquake activity within the Korean Peninsula.First, to verify that the earthquakes are intensively concentrated in a few areas, the Korean Peninsula was equally divided using a 40 × 40 uniform grid forming 1,600 spatial reference points. Then, the frequency of earthquakes per grid-section and the distribution of each grid-section was verified using logarithmic regression.43. Although 1,733 earthquakes greater than R2), between the grid-section and the frequency of earthquakes was 0.7674, showing a high correlation between space and frequency. This suggests that the earthquake frequency of each grid-section and the number of grid sections follow a power law distribution. In other words, statistically, we prove that even if there are a large number of earthquakes, they do not occur spatially uniform, but rather tend to concentrate in specific regions.More than two-thirds of the Korean peninsula consist of granite and metamorphic rock. The granite mainly consists of Jurassic and Cretaceous granite, Daebo granite, and Bulguksa granite. Metamorphic rocks are mainly composed of gneiss and include shale, sandstone, and limestone. The primary sedimentary rocks are shale, sandstone, conglomerate, and limestone, and they are mainly distributed in the Gyeongsang Basin, including Gyeongsang-do Province. In the Cretaceous basins, including the Gyeongsang Basin, sediments, volcanic rocks, and tuff appearTo identify changes in the frequency of earthquakes by magnitude, the overall observation window 1978–2018, was divided into 10-year periods, and the establishment of the Gutenberg–Richter law was examined in each period and the total window.Figures Because there was a significant difference in the coefficient of determination of the power law, coefficients of determination from before and after 1998, a visualization, and further analysis of frequency and magnitude were established for these two periods.Even though the earthquake activity observation period was divided in half, the earthquake activity record since 1998 accounted for about 80% of all earthquake activities. This is depicted in Fig. In this study, the earthquake phenomena of the Korean Peninsula were analyzed in consideration of spatiotemporal factors. To take into account spatial factors, the Korean Peninsula was divided into 1,600 equal sections using a grid. The logarithmic regression then verified that the relation between the grid section where the earthquake occurred and the frequency(number) of earthquakes in that grid-section follows the power law distribution. In another analysis, the earthquake observation period was divided to consider the temporal factors and to determine whether or not the Gutenberg–Richter law was established by period. As a result, earthquakes occurring after 1998 showed 1.7372 times higher coefficients of determination of the power law distribution than before, and the t-test confirmed that the magnitude of earthquakes occurring after 1998 was smaller than of those occurring earlier. In other words, according to the power law distribution between the grid section where the earthquake occurred and the frequency of earthquakes in that grid-section, the earthquakes are concentrated in specific regions. Hence, it was confirmed that many earthquakes occurred in Gyeongsang-do Province. In addition, since 1998, the power law distribution between the magnitude and frequency of earthquakes had a high coefficient of determination, and the average number of earthquakes has increased significantly. This increase in the number of earthquakes resulted in a number of small-scale earthquakes; also a few large-scale earthquakes occurred. In particular, since 1998, five out of seven earthquakes of The earthquake spatial data of this study were generated in rectangular grid-sections based on latitude and longitude. This is different from previous studies that have formed earthquake networks. This study presents ideas from the spatial point of view of the earthquakes. We argue that follow-up studies need to consider spatial factors due to the concentration of earthquakes in specific regions. Moreover, we revealed that the pattern of earthquakes differs significantly over time windows, which may be useful for earthquake-related social policymaking and follow-up studies.This study used the earthquake magnitude and location data of the Korean Peninsula from 1978 to June 2018 from the Weather Data Open Portal of the Korea Meteorological Administration. There is a total of 264 earthquake measuring stations in the Republic of Korea. The types of stations are classified into 95 broadband seismometers, 27 short-period seismometers, and 142 accelerometers. The collected data includes the date, magnitude, epicenter, and latitude and longitude of the earthquakes. Of the 4,107 datasets, 31 datasets with missing information on location or magnitude of earthquakes were removed. In addition, since the earthquake data from 1978 to 1998 were collected by analog observations of earthquakes with a magnitude greater than Table To consider spatial factors, the entire area of the Korean Peninsula was divided into a uniform grid with 40 rows and 40 columns. This is depicted in Fig. The distribution of the frequency of earthquakes in each space and the number of spaces corresponding to a specific number of events were confirmed by logarithmic regression Eq. . The depThe logarithmic transformed regression and t-test equations used in the two analyses are as follows:y is a dependent variable and x is an independent variable. αi s a regression coefficient for estimating the y intercept, ε is the error term, and n is the number of data. β is a coefficient that reflects the influence of log(x) and measures the change rate of y according to the fine change rate of x. In Eq. (A and B denote each group, S is the standard deviation, and In Eq. , y is a . In Eq. , t is th


Lessons from polio eradication efforts and the Global Polio Eradication Initiative (GPEI) are useful for improving health service delivery and outcomes globally. The Synthesis and Translation of Research and Innovations from Polio Eradication (STRIPE) is a multi-phase project which aims to map, package and disseminate knowledge from polio eradication initiatives as academic and training programs. This paper discusses initial findings from the knowledge mapping around polio eradication activities across a multi-country context.The knowledge mapping phase (January 2018 – December 2019) encompassed four research activities , health system analyses). This paper utilized a sequential mixed method design combining data from the survey and KIIs. The survey included individuals involved in polio eradication between 1988 and 2019, and described the contexts, implementation strategies, intended and unintended outcomes of polio eradication activities across levels. KIIs were conducted among a nested sample in seven countries  and at the global level to further explore these domains.The survey generated 3955 unique responses, mainly sub-national actors representing experience in over 74 countries; 194 KIIs were conducted. External factors including social, political, and economic factors were the most frequently cited barriers to eradication, followed by the process of implementing activities, including program execution, planning, monitoring, and stakeholder engagement. Key informants described common strategies for addressing these barriers, e.g. generating political will, engaging communities, capacity-building in planning and measurement, and adapting delivery strategies. The polio program positively affected health systems by investing in system structures and governance, however, long-term effects have been mixed as some countries have struggled to institutionalize program assets.Understanding the implementing context is critical for identifying threats and opportunities to global health programs. Common implementation strategies emerged across countries; however, these strategies were only effective where organizational and individual capacity were sufficient, and where strategies were appropriately tailored to the sociopolitical context. To maximize gains, readiness assessments at different levels should predate future global health programs and initiatives should consider system integration earlier to ensure program institutionalization and minimize system distortions. Between 1988 and 2017, over 15 billion US dollars have been spent towards efforts to eradicate polio globally  health post but with no health workers … and trainings were not given to health workers who manage [sic] those refrigerators.” – Subnational actor, Ethiopia.Considering program execution, the KIIs suggest a strong link between external and internal barriers to implementation. Respondents repeatedly described how external factors  made it more challenging to implement activities as intended (i.e. issues related to program execution). Likewise, health systems gaps made it difficult to carry out activities according to plan. Human resource issues were the most often cited health systems gap across countries, including challenges ranging from health worker shortages, maldistribution, low pay, lack of supervision, and health worker fatigue. Other health systems challenges which required addressing were supply chain issues, e.g. OPV and IPV stockouts and lack of cold chain infrastructure, as well as lack of surveillance and laboratory capacity. These issues were exacerbated in remote, hard-to-reach areas that required additional human resources and posed significant logistical challenges. Describing the confluence of these issues one respondent said:Some of the strategies discussed for addressing external barriers including ongoing stakeholders’ engagement and political advocacy were also successfully applied to address issues related to executing the program. Other strategies to address these issues were multifaceted and included addressing issues around specific health system inputs, as well as improving management processes. One strategy commonly cited was assessing organizational ability and readiness in order to identify barriers that may impede implementation, as well as strengths that could be used in the implementation effort. This allowed implementers to optimize organizational structure, for example, right-sizing team composition with clear roles for the vaccinator, supervisor, and community leaders, and making real-time adjustments as needed, such as shifting the outreach plan where staffing was insufficient, or shifting vaccine supply between posts where stockouts were occurring in critical areas (as defined by polio program managers). Another key strategy was to alter incentive and disincentive structures for providers. Polio workers often received additional or higher pay, particularly for campaign work, and volunteer staff received in-kind incentives, although this strategy led to demotivation of health workers not involved in polio campaigns – a negative and unintended outcome. Conversely, new supervisory structures were put in place to ensure health workers’ accountability. Finally, significant investment was made to recruit, designate and train leaders across the functional areas of the program and health system. These strategies led to improved organizational capacity and health worker motivation which improved polio program execution; however, the sustainability of those gains was dependent on the degree of integration of polio structures within the broader health system and in some cases drew health workers away from routine service delivery.“with the enumeration, we found out that most of the records were not true. The first enumeration we did, someone brought a paper: ‘household 30 children.’ I said ‘haba!!”- Subnational actor, Nigeria.Challenges related to program evaluating and reflecting , and planning were cited by somewhat fewer survey respondents. Per the KIIs, however, strategies deployed in these areas were some of the most effective facilitators of program activities, and thus contributed to addressing process barriers broadly, and also improving program execution. Examples of barriers in these areas included: lack of monitoring and evaluation tools, structures and processes, and lack of precision planning. These barriers operated at the organizational and individual levels, and the strategies used to address them were found to be effective where applied across contexts. The KIIs illuminated how these challenges were related to information systems and data quality, as well as management and governance issues. Some respondents explained difficulties in even establishing a denominator for campaign and outreach activities to determine coverage indices and assess performance:Other respondents described gaps in individual and organizational capacity to ensure data quality and effectively utilize available data that needed to be addressed; similarly, at the organizational level respondents described the lack of governance structures to support ongoing performance management and accountability.Strategies to address these issues included developing mechanisms for feedback, monitoring and evaluation, building robust record systems to capture outcomes, and conducting cyclical small tests of change to refine implementation strategies. On the planning side, working with health staff at the sub-national level to create detailed micro-plans and improve enumeration (sometimes via use of GIS and satellite technology) was universally cited as a key strategy that improved implementation. Micro-plans also put special populations, inaccessible groups and hesitant communities at the center of implementation plans, a tactic which proved critical for ensuring delivery to hard-to-reach populations. Building record systems was an important first step to improving monitoring and evaluation, but those systems proved most functional where ongoing and active engagement with monitoring data occurred. Respondents explained how regular planning sessions became a central program management component. Examples included “situation room” style meetings operationalized via emergency operational centers to examine the level of pre-implementation activities for campaigns, weekly meetings to refine polio surveillance efforts, post-campaign evaluations, and multi-day brainstorming meetings with the objective of creating a roadmap for implementation agreed upon by all stakeholders.Individual, organizational and program characteristics accounted for the remainder of barriers to implementation indicated in the survey, as indicated by 18.1%, 11.1% and 9.3% of identified barriers, respectively. These categories covered a broad array of barriers, but included issues related to knowledge and beliefs , poor implementation readiness , and limited program adaptability (program characteristics). The challenges identified under these categories illustrate how the implementing context and implementation process discussed above are inherently interrelated to the program’s inputs, functions, and actors.Among respondents who cited individual characteristics as the largest barrier to implementation, in nearly two-thirds of responses , an individual’s knowledge and beliefs about the activity  was cited as the most significant issue. The KII data suggests this may be a reflection of the social factors that underlie issues related to low acceptability of the polio program and low vaccine uptake. As discussed above, information and communication strategies such as tailored messaging and creating narratives that resonate with community values had to be deployed, in part, at the individual level in order to increase individuals’ knowledge about the polio program, and to resolve issues of distrust and fear held by household decision-makers.“In the instance of GPEI the money is not going to the government, it’s going to WHO and UNICEF and they are doing jobs that the government should have been doing but they’re basically disincentivizing the government from doing them, surveillance for example, it’s a hard sell to get any government to fund surveillance … we talk about WHO and UNICEF as implementing partners, but they are substituting for government services in that public health system and to get out of that will be very difficult.” – Global respondent.Regarding organizational settings, implementation readiness was the most commonly cited barrier to program implementation . Readiness refers to the availability of resources and capacities, and access to knowledge and information, as well as the level of leadership engagement to ensure the successful delivery of a health program. These challenges occurred at the organizational and policy levels to varying degrees across contexts, depending in large part on the overall strength of the health system. The health system and management gaps discussed above were discussed in detail by KII respondents as indicators of readiness, as were the availability and flow of financial resources. Where resources emanated from was sometimes discussed in relation to the degree of country ownership of the program. One global level respondent explained the potential distorting effect of overreliance on external resources explaining,Even where program functions were driven by external partners, the level of leadership engagement played a significant role in facilitating program implementation by generating program support and guiding program adaptations to fit the context and health system. From the survey, inability to adapt, tailor or refine the program to meet local needs was the most commonly cited challenge related to the polio program itself (40.2%), and reflects both policy and organizational level constraints.… one of the things that has been criticized is that there was not enough documentation. Obviously from time to time, we talked about it, but it was necessary to document enough so that [others could] appropriate this experience. But locally the actors in the field also developed approaches adapted to their environments … when they had the training, [they could] draw inspiration from others, from what had been done elsewhere, but as far as possible, they took into account local realities.” – National actor, DRC.“Where adaptive strategies were successfully applied, they were largely in response to difficulties in reaching hard-to-reach populations , changing epidemiology and vaccine strategy , and responding to cultural norms . Many adaptive strategies developed over the course of polio eradication had applicability across contexts, for example utilizing environmental surveillance in areas with poor AFP surveillance rates and creating temporary delivery outposts in hard-to-reach areas. By their nature, however, not all strategies developed to address barriers could be universally applied, and indeed, many were specific to the social and political norms of a locality. Door marking, for example, was a monitoring strategy developed by the polio program that was utilized in many contexts to track vaccinations, however who was responsible for marking varied by context, and in the case of Afghanistan, the activity was halted because of objections from local authorities. Reflecting on missed opportunities to apply lessons learned across regions, one respondent in DRC explained:This STRIPE study found that across country contexts and phases, factors external to the polio eradication activities, including political, social, and economic factors, significantly hindered implementation of program activities. Examples of these external factors include lack of social acceptance of polio vaccination due to a confluence of interacting factors at the community level, including mistrust of government and external actors, limited awareness among stakeholders, fatigue with polio-related activities, lack of political will, lack of frameworks conducive to coordinating multi-stakeholder actions, and insecurity and conflict. These external factors played a prominent role because the process of program implementation  is largely dependent on them, as well as on the organizational and individual level capacities of various implementers across the socioecological levels. Health systems gaps including human resource shortages, supply chain challenges, and inadequate surveillance systems further hindered the process of program implementation, especially in countries with weak health systems and among hard-to-reach populations.Over time, the GPEI developed a core set of implementation strategies to address these barriers and facilitate implementation of polio eradication activities, including strategies for social engagement and mobilization, coordinating information and communication at individual and community levels; strategic advocacy at various policy levels; and other health systems strategies such as incentivizing health workers, strengthening data and surveillance systems, micro-planning and establishing emergency operational centers for rapid decision-making. The degree of success of these various strategies depended on the organizational and individual capacities in the different contexts where polio programs were implemented.While there were positive externalities around the deployment of these strategies  and building local capacity (e.g. micro-planning), some of these strategies had negative unintended outcomes in certain contexts. These negative unintended outcomes included: distorting the local health systems, especially around the incentive structure of local health workers; inadvertently de-prioritizing other health problems and health system issues in the process of coalescing action and support behind polio eradication goals; creating an unsustainable development of health systems given local resources and priorities, particularly around health system infrastructure and human development; creating an overreliance on external funding and decision-making apparatus at the expense of developing local capacities and resources; and compounding mistrust of government and external actors in some settings.The STRIPE study findings align very much with the conclusions of other studies or reports that have examined global polio eradication efforts –9. For eOur analyses suggest a few key lessons learned from the polio eradication experience which should inform future global health programs.First, future eradication or elimination programs must recognize issues related to external factors earlier on, and actively strategize around them on a continuous basis and throughout the life of the program. The extent to which external factors have affected polio eradication activities suggests that global policymakers underestimated the role that differences in political ideology may play in shaping a global health program. It also suggests a limited understanding of the nature of incentives that shape individual, proxy, and collective agency, and reflects an oversimplification of complex and intersecting issues involving politics, culture, and economic disadvantage for different population groups, and how they may change dynamically over time. This lack of understanding and appreciation of the broader political, social and economic contexts surrounding most communities has been and continues to be a major barrier to achieving polio eradication goals.Second, implementers should conduct careful pre-program analysis of the broader political, social and economic contexts to identify both threats and opportunities to successful program implementation. Had the polio initiative done so, some of the implementation barriers described could have been foreseen and the implementation strategies pre-planned to save time and cost. Instead, many implementation strategies that were deployed under the polio program to address implementation barriers were reactionary as opposed to being preemptive, especially at the national and sub-national levels. Moving forward, readiness assessments of local communities and health systems should predate any global health program such as the GPEI. Such assessments should not only focus on understanding requirements for polio program implementation in a specific context on the supply-side , but should be extended to understanding social practices, customs, and politics at the local level that could influence the success of the program on the demand-side. Results from these readiness assessments could be used for more effective program planning, to target implementation strategies, and to guide capacity-building activities at various levels, including efforts to build leadership and capacity in management and supervision, to address critical social issues at community levels, and to address other health system gaps.Third, a set of core principles for implementing complex public health programs emerged across contexts in this study that we feel have relevant application to other health programs. These include: ongoing stakeholder engagement based upon mutual respect, coordinating efforts to build political will and accountability over different phases of the program and at different socioecological levels, systematic adaptation of service delivery activities to local contexts, and establishing sound planning, management and monitoring and evaluation practices which measure implementation outcomes (e.g. acceptability of the various key activities with different stakeholders) in addition to endpoint outcomes (e.g. vaccine coverage under the polio program). It is especially important to identify and work through “gatekeepers” at the community level from the outset of any implementation effort, understand how and when to engage these “gatekeepers,” know the target population and risk factors, and assess ongoing population movement and dynamic changes to the population risk profiles.Fourth, in order to minimize negative unintended outcomes, policymakers need to give adequate attention to participatory approaches which can facilitate implementation processes across socioecological levels, especially at national and sub-national levels , 29. TheFinally, delivery of lifesaving interventions, no matter how effective, involves individual choices and behaviors, and such choices and behaviors may not always be rational but rather influenced by factors unconnected to disease risks and intervention. This is a reasonable assumption that should underlie any future global disease eradication or control program. Hence, efforts to implement such programs should be accompanied with efforts to build social capital which may address factors outside of the intervention delivery and which may control behaviors and choices at individual and community levels .The lessons learned under global polio eradication efforts described in this paper are some of the first lessons in a series of findings that the STRIPE study aims to contribute to in order to improve the implementation, effectiveness and efficiency of future lifesaving interventions and health system strengthening activities in global health. These lessons are significant for improving health services delivery, overall health outcomes and quality of life of large populations globally. The project also hopes to contribute to the science of implementation and provide real-life data and experiences for formulating theories and frameworks for understanding why interventions may or may not work for their intended purposes, as well as strategies to ensure effective delivery of efficacious interventions under different contexts.The implementation of various polio eradication initiatives, including the GPEI, provides important lessons for implementing future lifesaving programs and health systems strengthening activities globally. Understanding the implementing context of any program is critical for identifying both threats and opportunities to program implementation. Systematic efforts to unpack contextual factors, target strategies to perceived barriers, and understand the readiness of recipient communities and health systems should be prioritized before implementation. These systematic efforts are especially relevant at the socioecological levels closest to the point of delivery of the intervention. While polio eradication initiatives were largely successful in deploying various implementation strategies to address barriers to effective implementation of the program, and these strategies yielded positive external benefits, the strategies were only successful where organizational and individual capacity were sufficient, and where they were appropriately tailored to the social, political and administrative context. There were also negative unintended outcomes of the polio eradication initiatives and/or of their related implementation strategies. These negative unintended outcomes could have been minimized with adequate attention to participatory approaches and efforts to build social capital alongside program delivery at national and sub-national levels.Additional file 1.Additional file 2.


BCR-ABL1 molecular detection using quantitative PCR (qPCR) methods is the golden standard of chronic myeloid leukemia (CML) monitoring. However, due to variable sensitivity of qPCR assays across laboratories, alternative methods are tested. Digital PCR (dPCR) has been suggested as a robust and reproducible option. Here we present a comparison of droplet dPCR with routinely used reverse-transcription qPCR (RT-qPCR) and automated GeneXpert systems. Detection limit of dPCR was above 3 BCR-ABL1 copies, although due to background amplification the resulting sensitivity was 0.01% BCR-ABL1 (MR4.0). Nevertheless, in comparison with GeneXpert, dPCR categorized more than 50% of the patients into different MR groups, showing a potential for improved BCR-ABL1 detection. •-ABL1 copies.dPCR assays attain MR4.0 sensitivity due to a blank amplification 3 BCR•Quantification principle of RT-qPCR and dPCR introduces bias in copy numbers.•BCR-ABL1 measurement in >50% MR3.0-MR4.5 CML patients.dPCR provides more sensitive  However, it often shows insufficient sensitivity and inconsistent detection of minimal BCR-ABL1 levels [Specific targeting of the Bcr-Abl1 enzyme by tyrosine kinase inhibitors (TKI) revolutionized the management of chronic myeloid leukemia (CML) to the point that TKIs can offer near normal life expectancy for CML patients . Current1 levels . Despite1 levels ,4.BCR-ABL1 monitoring suggest that dPCR has increased sensitivity up to MR5.5, although higher false positivity rate (FPR) could lower the detection limit of this method [Digital PCR (dPCR) offers high reproducibility, precision and increased sensitivity for rare target detection . Recent s method . TherefoBCR-ABL1 and ABL1 assays and to evaluate the suitability of dPCR for molecular monitoring of CML patients with low or undetectable levels of BCR-ABL1 in comparison with routinely used methods.The objectives of our study were to evaluate the performance of droplet dPCR using the Europe Against Cancer (EAC) standardized 22.1BCR-ABL1 negative controls. All samples were obtained from the Department of Internal Medicine, Haematology and Oncology, University Hospital Brno. Characteristics of CML patients are listed in A retrospective analysis was performed on 70 clinical samples from chronic phase CML patients and 15 samples from healthy volunteers used as 2.2BCR-ABL1 transcript by RT-qPCR in accordance with the manufacturer’s recommendations.ERM AD623 BCR-ABL pDNA (Sigma-Aldrich) plasmid set  was used2.3All qPCR measurements were performed at the Centre of Molecular Biology and Gene Therapy, University Hospital Brno. Two methods, routinely performed to assess treatment response, were used: conventional RT-qPCR and the GeneXpert-based assay.BCR-ABL1 in K562 cells was performed on an Applied Biosystems 7300 Real-Time PCR System. Cepheid Xpert BCR-ABL Monitor test was used for quantification of BCR-ABL1 in clinical CML samples, according to the manufacturer’s recommendations. Reverse transcription and qPCR reaction conditions are described in detail in Supplementary Data and RT-qPCR quantification of 2.4All dPCR measurements were performed at the Central European Institute of Technology of Masaryk University Brno, Department of Molecular Medicine on Bio-Rad QX200 Droplet Digital PCR System (dPCR) according to the manufacturer’s recommendations. Reaction conditions are described in Supplementary Data and 2.5The formulae for calculation of FPR, limit of blank (LOB) and limit of detection (LOD) are concluded in Supplementary Data. Statistical analyses were generated using GraphPad Prism v.9 software. P values < 0.05 were considered significant.33.1BCR-ABL1 and ABL1 copies across the dilution range of the ERM AD623 standard down to 10 copies, with R2BCR-ABL1 = 0.998 and R2ABL1 = 0.999, respectively  was 4% with LOB = 1.4 copies/sample, and the FPR of BCR-ABL1 negative controls  was 6% with LOB = 3.2 copies/sample , and the samples from s/sample . A detecBCR-ABL1 in 3 independent dilution series. We used a cDNA sample from a CML patient with 10% BCR-ABL1IS, which was serially diluted into BCR ABL1 negative control. Digital PCR detected BCR-ABL1 transcript all sample dilutions down to 0.0001% BCR-ABL1IS, however after correction to LOD (3.3 copies/sample), the final sensitivity was corresponding to MR4.0 (0.01% BCR-ABL1IS)  .Fig. 1Se3.2BCR-ABL1IS. Despite overall correlation of ratios (BCR-ABL1IS), RT-qPCR detected significantly more BCR-ABL1 copies than dPCR (P < 0.0001). Conversely, in the sample with low transcript levels (0.0032% BCR-ABL1IS), RT-qPCR quantified significantly less BCR-ABL1 copies compared to dPCR (P = 0.0477). Moreover, in all sample categories dPCR detected significantly less ABL1 copies (P < 0.0001)  . As a reR ratios .ABL1 copies than dPCR in these samples   (n = 12). RT-qPCR still quantified significantly more by dPCR) . The BCR3.3BCR-ABL1IS transcript level as follows: ≤10% (n = 3), ≤1% (n = 3), ≤0.1% (n = 14), ≤0.01% (n = 11) and ≤0.0032% (n = 13). We observed significant differences in the ratios measured by dPCR and GeneXpert in the patients with low transcript levels   , which a 2=0.49; C. OveralBCR-ABL1IS) 3-way comparison of BCR-ABL1 rations was performed as shown in RT-qPCR = 0.0049; PdPCR = 0.0010).In addition, in 12/44 patients .Finally, in 40% (10/25) of CML patients, evaluated by GeneXpert as anscript . However4BCR-ABL1 quantification has already been demonstrated, showing its superiority compared to RT-qPCR (LOD up to MR5.5) [BCR-ABL1 negative controls, not distinguishable from the lowest copy numbers in positive samples. Therefore, final sensitivity was decreased to MR4.0.The sensitivity of dPCR for o MR5.5) . In our BCR-ABL1 detection have been validated on dPCR in the studies of Franke and Maier [GUS and BCR control gene assays, or GUS alone, resulted in sensitivity of MR5.5 [BCR-ABL1 and ABL1 dPCR assays with low-background, 98% specificity, and sensitivity of MR5.0 [The assessment of the false positivity and the detection limit is especially important for the assays, monitoring minimal residual disease. The EAC assays for nd Maier ,10, revend Maier  describend Maier ,8,13. Inof MR5.5 . Most reBCR-ABL1 and ABL1 copy numbers quantified in K562 cell line and in CML patients by dPCR significantly differed from those detected by RT-qPCR. Franke et al. obtained similar results for samples with ≤0.1 BCR-ABL1% measured by dPCR, with significant redistribution of the patients into MR categories relative to RT-qPCR [ABL1 copies measured by two methods  [-ABL1% and BCR-ABL1 negative samples dPCR yielded more sensitive measurements, GeneXpert assay used for this comparison was validated to MR4.0 [BCR-ABL1IS). Still, more than half of the patients in MR3.0 - MR4.0 were categorized into different MR classes by dPCR. Similarly, Folta reported consistent MR levels only in 29/50 samples in a comparison of dPCR with more sensitive GeneXpert Ultra kit with sensitivity of MR4.5. [Herein, the  RT-qPCR . In addiby dPCR) . Since tto MR4.0 , which mf MR4.5. -ABL1 copies/sample, which corresponded to sensitivity of conventional quantitative methods. Nevertheless, dPCR categorized more than 50% of the CML patients into different MR categories compared to quantitative GeneXpert. CML patients in deep molecular response considered for TKI cessation could benefit from more sensitive monitoring using well optimized dPCR assays, however an international standardization independent from RT-qPCR would be required to promote dPCR as an alternative method.Our study demonstrated that droplet dPCR, tested with standard EAC assays, provided a detection limit of above 3 BCRDagmar Smitalova: Investigation, Writing – original draft, preparation. Dana Dvorakova: Resources, Conceptualization. Zdenek Racil: Resources, Writing – review & editing. Marianna Romzova: Conceptualization, Methodology, Supervision.Authors DS, DD, ZR, and MR have no competing interests.


Scientific Reports 10.1038/s41598-019-53703-3, published online 26 November 2019Correction to: The information in this Article is incomplete. The text in the Acknowledgements,“Funding was provided by EPA STAR and ARCS Foundation fellowships and the US Army Corps of Engineers’ Upper Mississippi River Restoration Program to K.J Jankowski; NSF and the Harriet Bullitt Professorship to D.E. Schindler; and the UW Alaska Salmon Program.”should read:“Funding was provided by Environmental Protection Agency STAR and ARCS Foundation fellowships and the U.S. Army Corps of Engineers’ Upper Mississippi River Restoration Program to K.J Jankowski; National Science Foundation and the Harriet Bullitt Professorship to D.E. Schindler; and the University of Washington Alaska Salmon Program. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.”In addition, the Data availability section was omitted from the Additional Information section:“Data availability: Data will be made available upon request to corresponding author.”Finally, the following text should be included in the Supplemental Material:“Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.”


Breakpoint Cluster Region (BCR) sequence and the Abelson (ABL1) gene. Despite BCR-ABL1 being one of the most studied oncogenic proteins, several of its partners, acting either as positive or negative BCR-ABL1 regulators, are still unknown. To identify the new components involved in BCR-ABL1 transforming activity, we conducted an extensive genetic screening using different Drosophila mutant strains. We identified several putative candidate genes that may be involved either in sustaining CML or in its progression. For the first time, we identified in Drosophila and confirmed in CML patients a tight connection between the BCR-ABL1 protein and Rab family members. Our data strongly suggest that Drosophila is a powerful tool to dissect BCR-ABL1 etiology and to identify unknown pathways, thus providing the basis for the development of new potential therapeutic strategies for the treatment of CML patients.Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome that encodes for the tyrosine kinase protein BCR-ABL1 from the Breakpoint Cluster Region (BCR) sequence and the Abelson (ABL1) gene. Despite BCR-ABL1 being one of the most studied oncogenic proteins, some molecular mechanisms remain enigmatic, and several of the proteins, acting either as positive or negative BCR-ABL1 regulators, are still unknown. The Drosophila melanogaster represents a powerful tool for genetic investigations and a promising model to study the BCR-ABL1 signaling pathway. To identify new components involved in BCR-ABL1 transforming activity, we conducted an extensive genetic screening using different Drosophila mutant strains carrying specific small deletions within the chromosomes 2 and 3 and the gmrGal4,UAS-BCR-ABL1 4M/TM3 transgenic Drosophila as the background. From the screening, we identified several putative candidate genes that may be involved either in sustaining chronic myeloid leukemia (CML) or in its progression. We also identified, for the first time, a tight connection between the BCR-ABL1 protein and Rab family members, and this correlation was also validated in CML patients. In conclusion, our data identified many genes that, by interacting with BCR-ABL1, regulate several important biological pathways and could promote disease onset and progression.Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome that originates from the reciprocal translocation t and encodes for the constitutively active tyrosine kinase protein BCR-ABL1 from the  Breakpoint Cluster Region (BCR) sequence and the Abelson (ABL1) gene, which encodes for the constitutively active tyrosine kinase protein BCR-ABL1 [Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome (Ph), which originates from the reciprocal translocation t9;22)q34;q11.2). This translocation results in a fusion oncogene between the 1.2. This2q34;q11.BCR-ABL1 ,6,7,8,9.Drosophila melanogaster, represents a powerful tool for genetic investigations and a promising model to study the BCR-ABL1 signaling pathway through the identification of genes encoding for proteins that, to a different extent, contribute to the BCR-ABL1 phenotype. The simplicity of genetics and ease of manipulation in Drosophila present it as an attractive model. In addition, Drosophila hematopoiesis is comparable to that of mammals, and the high homology levels between human and Drosophila genes are important advantages of using Drosophila to understand human diseases [Drosophila model for t was used to successfully identify new modulators of the AML1-ETO fusion protein [Drosophila model of CML suitable to perform genetic screening to fish out genes potentially involved in CML onset and/or progression [Drosophila carrying specific small deletions for chromosomes 2 and 3 and our gmrGal4,UAS-BCR-ABL1 4M/TM3 transgenic Drosophila as “bait”. By observing the changes in the phenotype, it was possible to identify several candidate genes in different pathways that may be involved in CML sustaining and progression.The fruit fly, diseases . This aldiseases ,12. Inde protein ,14. In ogression . In our Drosophila looking for positive and negative regulators of the BCR-ABL1 oncogenic protein that may be involved in the sustaining and progression of CML. Using a recombinant fly carrying both the gmrGal4 and the UAS-BCR-ABL1 transgenes in cis on the third chromosome , we were able to perform a genetic screening of most of the Drosophila genome (autosomes 2 and 3). This was performed by crossing the BCR-ABL1 recombinant fly with more than 200 stocks of deletions  belonging to class +2 and 25 (12%) belonging to class −2. Furthermore, we found 90 minor modifiers, 43 (20%) classified as +1 and 47 (22%) as −1. A total of 63 stocks were classified as nonmodifiers  . In partgmrGal4,UAS-BCR-ABL1 4M/TM3 showed a particularly severe worsening of the BCR-ABL1 phenotype , one overexpressing the wild-type isoform (UAS Rab5 wt), and one encoding a constitutively active form (UAS Rab5.Q88L). The eyes of the flies co-expressing BCR-ABL1 and the dominant-negative (DN) of Rab5 showed a marked worsening of the BCR-ABL1 phenotype, affecting the normal round morphology and pigmentation and showing areas of necrosis  and bone marrow (BM) samples from patients at different stages of the disease: at diagnosis, during the cytogenetic or molecular response, and during the secondary resistance to tyrosine kinase inhibitors (TKI). In addition, another 10 specimens were collected from patients with a primary resistance to TKI. The results were presented in RAB5A in CML patients at diagnosis , during secondary resistance , and in primary resistant subjects  compared to healthy controls (mean +1.04) . This pattern was also observed when we stratified specimens by sample types (BM or PB). Our data suggested an inverse correlation between RAB5A expression and the presence of a BCR-ABL1 transcript, highlighted by their opposite behavior  and molecular remission (<0.1%). As indicated by RAB5A in the presence of the Philadelphia chromosome . This data was consistent with what we observed in Drosophila, meaning RAB5A and/or its family could potentially be new targets involved in BCR-ABL1 pathological signaling.Concerning all GO observed in henotype . Since tbehavior B. To invDrosophila melanogaster, represents a powerful tool for a genome-wide genetic analysis and screens, given the functional conservation and sequence homology between human and Drosophila genes. In this work, we conducted a genome-wide genetic screening using commercial gene deletion stocks for autosomal chromosomes 2 and 3 and our previously generated and validated CML Drosophila model, gmrGal4,UAS-BCR-ABL1 4M/TM3, as “bait” [patched gene (FBgn0003892), an ortholog to human PTCH1 (patched 1) and PTCH2 (patched 2). Protein patched homolog 1 (PTCH1) encodes for a cell-surface receptor and is a member of the Hedgehog (Hh) signaling pathway. The PTCH1 protein exerts its function by negatively regulating the activity of the frizzled family receptor smoothened (SMO) [PTCH1 overexpression and mutations in CML patients are associated with poor prognosis and primary or secondary resistance to tyrosine kinase inhibitors [patched gene generates a better phenotype, suggesting that BCR-ABL1 and patched, when present together, cooperate for the disease etiology. In addition, among genes of “eye-antennal disc morphogenesis”, we found babo (FBgn0011300), a gene activated by patched and SMO signaling in Drosophila [babo is the ortholog of TGFBR1 (best score), a representative of the Transforming Growth Factor-β Receptor family (TGF-βR). It is known that the TGF-βR signaling pathway plays an important role in CML, leading to cell growth inhibition, differentiation, and apoptosis [patched and babo were simultaneously deleted, as in stock #201, we observed a better phenotype compared to #198, where only patched was suppressed. This result suggests the powerful cooperation of these genes with BCR-ABL1, further highlighting that Drosophila is a powerful model to identify positive and negative BCR-ABL1 partners. Additionally, in the worst phenotype classes, we identified some genes that could cooperate with BCR-ABL1 in CML. One of these is frizzled (FBgn0001085), a seven-pass transmembrane domain receptor, ortholog of human FZD7, involved in the Wnt signaling pathway, a gene probably involved in CML cell protection mediated by bone marrow-derived mesenchymal stem cells [aPKC (FBgn0261854) and four-jointed (FBgn0000658). Furthermore, interactions with the cadherins pathway (dachsous (FBgn0284247), Cadherin-N (FBgn0015609), and adherens junction protein p120 (FBgn0260799)) are also supported by data of the literature. Indeed, Zang et al. reported that N-cadherin expression contributes to increased resistance to farnesyltransferase inhibitor SCH66336 in BCR-ABL1p190-positive acute lymphoblastic leukemia [CDH13mRNA in CML patients is lower than in healthy subjects, showing a negative correlation with the BCR-ABL1 fusion gene that may contribute to CML development [Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome. Despite BCR-ABL1 representing one of the most studied oncogenic proteins, some molecular mechanisms leading to cellular transformation are still partially unknown, and its positive or negative regulators are not completely identified. The fruit fly, s “bait” . Althouged (SMO) . There ied (SMO) . It has ed (SMO) . Furtherhibitors ,20,21,22osophila . Intrigupoptosis . Interesem cells . Recentlem cells . BCR-ABLleukemia . Furtherous FBgn04247, CadSaccharomyces cerevisiae and are the master regulators of all stages of the intracellular trafficking processes in eukaryotic cells evolutionarily conserved from fly to vertebrates. The RAB5 gene, which encodes for a monomeric small GTPase, is a key member of the Rab family, and RAB5A is its most important subtype, with well-identified functions and mechanisms. RAB5A affects the internalization and intracellular transport of receptors, such as receptor tyrosine kinases, GPCRs, and antigen-recognition receptors, by recruiting Rab5 effectors. The signals mediated by RAB5A range between gene transcription, cell morphology, growth, differentiation, apoptosis, and disease development [RAB5A transcript was significantly reduced in leukemic cells while returned to normal levels during the remission phase. In addition, we identified a significant opposite regulation of BCR-ABL1 and RAB5A mRNA. The BCR-ABL1 transcript, when present, seems to downmodulate RAB5A expression, probably in order to acquire some molecular advantages, such as the reduction of receptor turnover and the increase of the proliferative stimuli. In support of this hypothesis, we previously identified that the downregulation of Rab interactor 1 (RIN1) and Bridging integrator 1 (BIN1), two proteins directly involved with Rab-mediated receptor tyrosine kinase intracellular trafficking, caused aberrant and constitutive receptor signaling and are often observed deregulated in CML-resistant patients [Taken together, these results suggest the usefulness of our screening system and, therefore, give us confidence in the goodness of the data we have obtained for new possible modulators that have never been described before. In particular, in our study, we identified for the first time a tight connection between the BCR-ABL1 protein and Rab family members (Rab5 (FBgn0014010), Rab6 (FBgn0015797), Rab7 (FBgn0015795), Rab11 (FBgn0015790), and Rab23 (FBgn0037364)) and between BCR-ABL1 and the class of microtubule-based kinesin and cytoplasmic dynein motor complexes that are the main Rab effectors (Dhc64C (FBgn0261797) and DCTN1-p150 (FBgn0001108)). The Rab GTPase proteins were first studied in yeast elopment . In our patients . A plauspatients . Successpatients . In a coWe are aware of the limitations of our artificial system that could include different “non-gene-specific” deletions on chromosomes 2 and 3, the interactions between proteins of different species, and eye-limited protein expression. Some of these were anyway shared with other drosophila models, such as AML-ETO1, for which different modulators were successfully identified ,14. In ahttps://flybase.org/). BCR-ABL1 transgenic flies were generated as previously described [gmrGal4 and the UAS-BCR-ABL1 transgenes in cis on the 3rd chromosome  were crossed with lines carrying different deletions on the second (DK2L and DK2R) and third chromosomes (DK3L and DK3R). This resulted in an eye phenotype that can be easily and quickly scrutinized. To evaluate the phenotype, 15–30 first-generation (F1) flies, from three independent crosses, were classified into five arbitrary phenotypic classes. Class 0 represents the average BCR-ABL1 phenotype: the eye was smaller, bar-shaped, and misplaced extra sensory bristles appeared in the dorsal region. In addition, ommatidia were no longer distinguishable. Class +1 represents a weaker phenotype, characterized by an improvement of either dimension or differentiation. Some disorganized ommatidia could be present. Class +2 represents the weakest phenotype, with an improvement in both dimension and differentiation, with at least a partial rescue of the phenotype. Indeed, large regions of organized ommatidia were observed, although the eye was not completely developed. Class −1 represents a stronger phenotype, characterized by a worsening of either dimension or differentiation. Some lack of pigmentation and sporadic very small necrosis areas were observed in the eye. Class −2 represents the strongest phenotype, characterized by a worsening of pigmentation, dimension, and differentiation. This phenotype was characterized by the presence of large necrosis areas and more misplaced extrasensory bristles. In addition, we analyzed also the following BDSC Rab5 stocks: Rab5.S43N (#9771), UAS Rab5 wild type (#24616), and UAS Rab5.Q88L (#9773). All flies were cultured in standard medium and grown at 25 °C, if not otherwise specified. To examine the eye phenotypes, images of adult eyes were captured using a Leica stereomicroscope 5.7× equipped with a photo-camera , Italy).Fly stocks of drivers and deletions were obtained from the Bloomington Drosophila Stock Center (BDSC) , and they are described at FlyBase  using specific primers and probes assays  according to the published methods [Eighty-seven bone marrow aspirate (BM) and peripheral blood (PB) samples from 56 CML patients and 16 healthy subjects were collected. The study was approved by the San Luigi Hospital Institutional Review Board . All the patients included in the study signed written informed consent. Thirty-one samples were collected at diagnosis, 23 during cytogenetic or molecular remission, and 7 during secondary resistance to tyrosine kinase inhibitors (TKI). An additional 10 specimens were collected from patients with a primary resistance to TKI. White blood cells were separated by buffy coat, and total RNA was extracted using standard procedures. Expression levels of  methods .Drosophila stock were reannotated by a homemade Perl script using updated information from the Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/) and FlyBase (https://flybase.org/). Each stock was annotated in the appropriate phenotypic class . To highlight the genes implied in the phenotype changes, we filtered with the following criteria: For each stock, we removed from the list of deleted genes those that (a) were concomitantly present in class 0, because they were plausibly not altering the phenotype and (b) those that were found concurrently in opposed phenotypic classes . Subsequently, the remaining genes were annotated and sorted based on their frequency of the gene ontology “biological process” in ameliorative (+1 and +2 class) or pejorative phenotypes (−1 and −2 class)  .RAB5AmRNA cycle threshold values (CtT) were calculated by Bio-Rad CFX manager 3.1 software and normalized against the internal control GUSB (CtR) by subtraction (CtT-CtR). ΔCt values generated were then standardized, with ΔCt obtained by universal reference RNA (Stratagene), evaluated in the same run. Differential RAB5A transcript expression between −ΔCt values of healthy controls and samples derived from each different clinical subgroup or between groups were evaluated using the t-test. RAB5AmRNA expression levels were dichotomized into two groups of “high” and “low” expression using the median value as the threshold cut-off. The BCR-ABL1 transcript presence, evaluated as the percentage of BCR-ABL1/ABL1, was dichotomized into groups using two diagnostic criteria: molecular remission (<0.1%) and cytogenetic remission (<1%). The association between the RAB5A and BCR-ABL1 transcripts was evaluated using the Fisher exact test. Statistical analysis was performed using R statistical software .The Drosophila model identified still unknown pathways, thus providing the basis for the development of new potential therapeutic strategies for the treatment of CML patients in line with the findings recently published by Outa et al. [Drosophila appeared to be a powerful tool to dissect the BCR-ABL1 etiology.The power and the novelty of our model was the possibility to highlight modulators that do not physically interact with BCR-ABL1, and we identified several new putative pathways affecting the BCR-ABL1 phenotype. These molecules regulate several important biological pathways and could promote CML onset and/or progression. Among these, we showed, for the first time, that several members of the RAB family were associated with BCR-ABL1 signaling. Our model also identified several pathways already known for their role in TKI resistance or in CML blast crisis, and this reinforces the strength of our findings. In conclusion, the a et al. . Althoug


The diagnosis of pulmonary embolism (PE) needs clinical manifestations and radiological findings. CT angiography (CTA) of pulmonary vessels is the gold standard of diagnosis of PE. However, endobronchial ultrasound (EBUS) can be a reliable and accurate alternative method of diagnosis in patients who are not candidates for CTA. Invasiveness and high cost are still the major limitations for EBUS, however, they should be considered in the appropriate population in future practice. We present a case of a 62-year-old asymptomatic male diagnosed with PE during EBUS for mediastinal lymph node assessment and biopsy. Pulmonary embolism is a common thromboembolic disease that usually presents with highly variable clinical manifestations -3 and isIt is important to mention that this case was presented as an abstract in the 50th Critical Care Congress of the Society of Critical Care Medicine held in January - February 2021.A 62-year-old male, former smoker, with a past medical history of large exudative pericardial effusion, epilepsy, and hypothyroidism presented for outpatient EBUS for follow-up of positron emission tomography (PET)-avid mediastinal and hilar lymphadenopathy that were found during the workup of his pericardial effusion. The patient was physically active walking daily without chest pain or dyspnea. He reported having a non-productive cough. On examination, the patient was alert, sitting comfortably, without lower extremities edema, and his vitals were stable. Chest auscultation revealed loud S2 in the pulmonary area. All of his blood tests, including troponin I and pro-B-type natriuretic peptide (pro-BNP) were completely normal. His electrocardiogram (EKG) showed normal sinus rhythm.During EBUS, an abnormal floating object was visualized in a vascular structure of left pulmonary vessels as shown in Figure A pulmonary embolism is a life-threatening pulmonary vascular thromboembolic disease that affects around 900,000 people  each year in the United States, with no sex predilection, and it usually results in between 60,000 and 100,000 deaths per year according to the Centers for Disease Control and Prevention (CDC) -8. It haA pulmonary embolism is a critical condition that requires rapid diagnosis and treatment for favorable outcomes. CT pulmonary angiography is considered the diagnostic imaging modality of choice due to its accuracy, non-invasive nature, its availability, and its ability to identify other lung pathologies ,11. AlthEBUS is usually performed as an adjunct to transbronchial needle aspiration (TBNA) of mediastinal and hilar lymph nodes for the assessment and evaluation of different lymph node pathologies and the staging of lung cancer. The anatomical proximity of the bronchial airways and the pulmonary vasculature allows the EBUS theoretically to evaluate the pulmonary arteries for any abnormalities or pulmonary embolism . SeveralMajor pulmonary vessels can be viewed during EBUS, and thus pulmonologists should be aware that EBUS can be a good alternative modality to diagnose PE with high sensitivity and specificity in patients who are not candidates for CT pulmonary angiography. More studies are needed for the further evaluation of the diagnostic value of EBUS for PE.


Popliteal artery entrapment syndrome (PAES) is a rare cause of limb-threatening vascular disease. Usually, it arises from aberrant embryological development or acquired dysfunctionality of the popliteal artery and its surrounding musculotendinous structures in the popliteal fossa. Here, we present a case report of a young woman with relatively sudden-onset short-distance claudication and paraesthesia affecting her right leg primarily. She had no recent traumatic history and no atherosclerotic risk factors and was otherwise previously very active. She had a feeble right popliteal artery pulse and no foot pulses. Nerve conduction studies demonstrated no electrophysiological abnormalities. Following computed tomography angiography and magnetic resonance imaging, it was determined she had type 2 PAES. Subsequently, the patient underwent surgical division of a lateralised head of her medial gastrocnemius, resection of her fibrosed popliteal artery, and repair with a reversed long saphenous vein interposition graft. Following surgery, her symptoms resolved, and she remains on aspirin and ultrasound surveillance. A 28-year-old woman was referred by her local general practitioner to the vascular surgical department with intermittent claudication, predominantly affecting her right leg. The claudication had gradually worsened over a 9-month period, now limiting her to a walking distance of 30 metres—relieved by 10-15 minutes of rest. Though denying rest pain, she also described some numbness and pins and needles in her right foot and occasionally had similar sensations in her left. Ultimately, her symptoms were impacting her ability to work, where as a teacher, she found it difficult to walk between classrooms. She denied any previous lower limb trauma, though previously regularly exercised by running. She had no relevant personal or family past medical history, with no significant risk factors for atherosclerotic disease given she was a nonsmoker, nondiabetic, and without hypertension or hypercholesterolemia.On physical examination, she appeared generally well. A pulse examination demonstrated that she had a feebly palpable right popliteal artery pulse (in comparison to her left) and no palpable pedal pulses. Despite this, she had a normal capillary return, with no lower limb ulceration.Initially, the patient was referred for Doppler ultrasound, which demonstrated a normal right popliteal vein; however, it did demonstrate a short popliteal artery occlusion with notable large collateral vessels. For completion, blood tests were collected including a vasculitic screen and a thrombophilia screen, both of which were negative. Nerve conduction studies were also then performed, which demonstrated no electrophysiological abnormalities.To quantify her disease further, computed tomography angiography (CTA) was conducted confirming in-fact bilateral short-segment popliteal artery disease, with a total occlusion on the right, a high-grade stenosis on the left, and three vessel run-offs bilaterally . MagnetiFollowing an anaesthetic evaluation, the patient was taken to theatre. Under general anaesthetic, a left leg long saphenous vein harvest was performed, followed by repositioning the patient prone. A lazy-S incision was made over the right popliteal fossa, with appreciation and preservation of the tibial nerve and popliteal vein. The popliteal artery was dissected free, noting a tough muscular band of the medial head of the gastrocnemius travelling laterally over the popliteal artery causing extrinsic compression . This baPopliteal artery entrapment syndrome (PAES) is a rare cause of potentially limb-threatening vascular disease. Typically, it arises from aberrant embryological development or acquired traumatic dysfunctionality of the popliteal artery secondary to surrounding musculotendinous structures in the popliteal fossa . UsuallyWhether acquired or embryological in nature, given that PAES is generally complex in its development, the potential anatomical variations have been described in a multitude of classification systems—the Whelan and Love system (see ), as menA high suspicion for PAES should be held when assessing young patients presenting with intermittent claudication, particularly in the absence of other atherosclerotic risk factors. In conjunction with a thorough history and examination, a targeted imaging assessment should be conducted to assist in narrowing the differential diagnosis, as well as prompt for early operative salvage from potential limb-threatening complications.