Different methodological approaches for implementation research in global health focusing on how interventions are developed, implemented and evaluated are needed. In this paper, we detail the approach developed and implemented in the COmmunity HEalth System InnovatiON (COHESION) Project, a global health project aimed at strengthening health systems in Mozambique, Nepal and Peru. This project developed innovative formative research at policy, health system and community levels to gain a comprehensive understanding of the barriers, enablers, needs and lessons for the management of chronic disease using non-communicable and neglected tropical diseases as tracer conditions. After formative research, COHESION adopted a co-creation approach in the planning of interventions. The approach included two interactions with each type of stakeholder at policy, health system and community level in each country which aimed to develop interventions to improve the delivery of care of the tracer conditions. Diverse tools and methods were used in order to prioritise interventions based on support, resources and impact. Additionally, a COHESION score that assessed feasibility, sustainability and scaling up was used to select three potential interventions. Next steps for the COHESION Project are to further detail and develop the interventions propositioned through this process. Besides providing some useful tools and methods, this work also highlights the challenges and lessons learned from such an approach. There have been calls for improved methodological approaches to implementation research in global health.Co-creation approaches allow the development of interventions with local stakeholders playing an active role and are gaining traction in implementation research, but few practical approaches have been described in the global health literature.Prior to co-creation, formative research with a policy analysis, health systems assessment and community perception study using non-communicable diseases and neglected tropical diseases was completed to assess barriers, enablers and lessons for the management of these tracer conditions.The tools and methods used for co-creation need to be adapted according to the characteristics of the stakeholders and each setting.The principal challenges for researchers using this approach is the investment in time and resources in working closely with partners and not knowing which interventions may result.The WHO highlights that ‘research is fundamental to generate knowledge and information for formulating evidence-informed policies and practices in support of global public health and health equity’One element at the design stage that is often overlooked is the initial development of interventions, as these are often planned and implemented in an ad-hoc manner.Although guidance on implementation research exists,The COmmunity HEalth System InnovatiON (COHESION)The COHESION project includes formative research at policy, health system and community levels using non-communicable diseases (NCD) and neglected tropical diseases (NTD) as tracer conditions for policy, health system and community responses10.1136/bmjgh-2018-001183.supp1Supplementary dataIn each country two sites, a rural and an urban or periurban area were selected as being ‘representative’ by local stakeholders. The research protocols were submitted and approved by the following ethics committees:Commission Cantonale d'éthique de la recherche, Genève, Switzerland.Comité Institucional de Bioética em Saúde da Faculdade de Medicina/Hospital Central de Maputo, Maputo, Mozambique.Nepal Health Research Council, Kathmandu, Nepal.Institutional Review Board of the Universidad Peruana Cayetano Heredia, Lima, Peru.et al,At global and national levels, a range of activities, for example, the inclusion of NCDs and NTDs in the Sustainable Development Goals, have taken place in recent years and resulted in a stream of documentation and policy pathways. As part of the formative research, these were mapped and enabled an understanding of how NCDs and NTDs are addressed in both global and national political agendas. As discussed by Walt COHESION’s formative research also included a health systems assessment building off expertise in developingKnowledge of community capacities is necessary to be able to improve healthcare in line with local needs and resources.The COHESION Project, used the WHO’s definition of intervention: ‘a health intervention is an act performed for, with or on behalf of a person or population whose purpose is to assess, improve, maintain, promote or modify health, functioning or health conditions.’COHESION’s approach to co-creationOnce the COHESION team ‘filtered’ the interventions, the remaining options were presented to stakeholders again and then they were asked to prioritise and evaluate their feasibility. For the health system and policy level stakeholders, a structured approach was taken using a tool for prioritisation of interventions during the second meeting . For theThis section describes the implementation of the co-creation process which took approximately 2 months and the adaptations made in each context.In order to gain insight into possible interventions from policy level stakeholders, the Peru and Nepal teams used the opportunity provided by the interviews for the health system assessment, to ask additional questions. This was done to capitalise on the opportunity of interviewing people who usually have little ‘time to spare’ at the policy level in both contexts. The additional questions revolved around suggesting policies or projects that could be implemented to improve PHC for managing the target diseases.In addition to these questions, a meeting in Kathmandu Nepal was organised with community medicine physicians and public healthcare workers to obtain the perspective of health system stakeholders. During the meeting, researchers presented the findings of the health system assessment and community perceptions study and then asked for suggestions of interventions. Additionally, input for interventions was collected during a meeting with the Nepal Health Research Council, where preliminary results were presented and participants were asked for suggestions for interventions. A similar meeting was organised in Peru for stakeholders at health system level.In Peru, at the community level, a meeting was organised with the local population to provide information on selected diseases followed by group discussions to propose possible interventions. In addition, a meeting with healthcare workers at PHC level was held explaining the co-creation process and asking them to provide ideas for possible interventions. In Nepal, a joint meeting between the community and PHC workers was held. This meeting took a similar approach to that in Peru by providing information on selected diseases, including the findings of the health systems assessment with an emphasis on the problems from the community and followed by group discussion asking for any suggestions.In Mozambique, the results were presented at meetings that were divided into policy, health system and community levels. PHC workers were included in the health system meeting. After the presentation, participants were asked for suggestions to improve the situation of people affected by NCDs or NTDs.In the first consultation, different numbers of participants provided their suggestions in each country. Across actors, the number of suggestions ranged from 113 to 231 in Nepal, 10 to 84 in Peru and 6 to 11 in Mozambique. In both Nepal and Peru, the largest number of inputs came from the policy level stakeholders.Between the first and second consultation, filters based on prioritisation by each stakeholder and feasibility were applied by the research teams in each country. In Peru, the process consisted of: systematisation of proposals according to implementation level, elimination of duplicates and a meeting with experts in the project’s region. In Mozambique, the team first eliminated duplicates and reviewed pros and cons of the interventions to select the most feasible ones. Nepal grouped similar interventions and removed duplicates from the list. This resulted in 6 interventions in Mozambique, 17 in Nepal and 7 in Peru.During the second consultation, a prioritisation process was conducted with the interventions selected after the first consultation. Policy and health system stakeholders were asked to use specific evaluation criteria  to assess interventions. Community level participants were asked to prioritise the interventions according to the importance for their community. In Peru, in order to prioritise interventions at the policy level, an email was sent to the participants of the first consultation. They were provided with the tool shown in In Mozambique, only interventions that passed the internal filtering process were presented. In both Mozambique and Nepal, a more qualitative approach was taken with communities by explaining the potential interventions and asking them what they thought and felt about each of them. The Nepal Team presented all interventions listed by order of priority to policy level stakeholders. At the policy and health system levels, the potential interventions were presented and described and then the tool with evaluation criteria was used , similarAfter the second consultation, every country had a different number of proposals. In Nepal, after the prioritisation, the top potential interventions were selected and  was usedhttp://www.cohesionproject.info.All potential interventions were synthesised into three potential areas for intervention Next steps are to further detail and develop the final interventions co-created through this process using the United Kingdom Medical Research Council guidelines.The formative research approach is innovative in that it covers all levels of the health system from policy to the community enabling an overall view of the context in which interventions will be implemented. In addition, the use of tracers allows for an overall view of different elements of the health system. As the process of intervention development is integrated into the formative research this allowed the researchers to better understand the local context, for example, how a disease is understood and perceived by local communities. Using NCDs and NTDs as tracer conditions aims to ensure that responses developed do not increase competing demands on the health system, but rather enable NCD and NTD interventions to benefit the health system as a whole.Involving local stakeholders early identified opportunities and challenges in the environments where the interventions will be implemented. In this process, stakeholders are first key informants, then active participants in intervention development and finally possible users of the interventions. Others have argued that this process allows for better ‘buy-in’ of the interventions and might impact their integration and sustainability in practice.It is important to reflect that knowledge is not the same for the stakeholders and their capacities to provide proposals are unequal. For these reasons, adaptation of messages was required by researchers when presenting the results to communities, individuals within the health system and those at policy level. This was facilitated in the COHESION project, as the team included health communication scholars and the funding allowed for substantial investments in communication processes. The funding mechanism also allowed for conducting formative research, intervention development and implementation without having these interventions predefined. This highlights the importance given for research to identify solutions and options for overcoming implementation obstacles taking into account the local context.Co-creation has become more widely used as it allows an alignment of research and intervention development to better address the needs of the targeted populations.Besides providing some useful tools and methods, this work also highlights the challenges and lessons learnt from such an approach. Researchers need to think about the methodological aspects of the research and creation of interventions based on the best available evidence and also communication and engagement activities in order to foster partnership between stakeholders on the ground. In so doing, this project rejected a paternalistic view of defining research needs and priorities and rather generated and guaranteed spaces to gather, organise, prioritise and jointly define areas for intervention with all key stakeholder groups. This approach provides researchers with both challenges and opportunities. The challenges relate to investment in time and resources in working closely with partners and not knowing which interventions may result. On the flipside, this offers a unique opportunity to develop interventions together with local stakeholders and ensuring their buy-in from the start of the process.


Implications for the future of male and female representation in orchestral personnel are then considered.This study examines the representation of male and female musicians in world-class symphony orchestras. Personnel of 40 orchestras of three regions, the UK, Europe, and the USA, and distributions of men and women across the four orchestral departments, strings, woodwind, brass, and percussion, are compared. Significant differences in representation between orchestras of the three regions are reported. Practices adopted by orchestras when appointing musicians to vacant positions are reviewed and numbers of males and females appointed to rank-and-file and Section Principals are compared. Career patterns of male and female musicians are also compared. Increases in numbers of women appointed to orchestral posts in the last three decades are compared with increases in the proportion of women in the general workforce. The data of orchestral membership are then compared with the numbers of young people receiving tuition on orchestral instruments retrieved from a large national database ( During the eighteenth and nineteenth centuries, acquisition of musical skills by women was applauded, but social conventions prevailing in Europe and America approved their display in private but not in public. Except for the piano and the voice, women were severely limited in their access to musical training, witness the difficulties suffered by the English composer Ethel Smyth (1858–1944) and by other women , p. 188.,As a consequence of these social attitudes, women were excluded from professional music-making, and until the second decade of the twentieth century, membership of professional orchestras was restricted to male musicians . The firIt was not until 1930 that the first woman was appointed to a tenured fully professional post in an American orchestra, when Edna Phillips joined the Philadelphia Orchestra as its harpistPrior to Wood’s Queen’s Hall appointments, the only opportunities for women to play professionally had been with women-only orchestras, for example, that formed in Berlin in 1898, directed by Mary Wurm, but these were few in number . Later e.Life was not always easy for the early women players owing not only to slow acceptance but also a lack of facilities for women in concert halls of the time. Archives of the Cleveland Orchestra include a photographSome orchestras have been markedly slow to admit women to playing positions: the Berlin Philharmonic did not do so until 1982, and the Vienna Philharmonic as late as 1997; prior to that date, women might be engaged regularly  but were not publicly listed as orchestral members or awarded tenured posts.An international survey of professional orchestras by The progressive entry of women musicians into the once male community of the symphony orchestra has generated a sizeable literature. A number of studies have been concerned with sociological effects of these changes on the orchestral community, discussing issues such as inter-player relations, conditions of employment, membership stability, opportunities for personal growth, facilities, and financial resources . Few stuSome published commentary has been more characteristic of journalistic polemic than of evidenced reportage, with headlines such as “Sexism is rife in classical music” ; “Women In the following sections, we present an objective analysis of the current representation of male and female musicians holding tenured positions in orchestras across the world, and the instruments on which they perform.Data of the relative presence of male and female musicians in each orchestral section and for each instrument were collected from the current websites of 40 major orchestras in the UK, North America, and Europe representing a total of 3,420 musicians. Criteria for inclusion in the sample were that an orchestra should be fully professional, recognized as a having world-class status, have made published recordings under established labels, and have accessible data of participant musicians. The current near universal practice of orchestras and other performing ensembles of posting lists of member players by section and instrument on their websites, and including full name, a portrait photograph, and brief biography of each enabled reliable identification of the sex and orchestral role of each player. Vacancies or pending appointments listed on websites were not includedp < 0.0001), but there were highly significant regional differences in male-female proportions.Modern symphony orchestras list an average of 100–120 musicians; though regional orchestras tend to be smaller than those based in major or capital cities. Overall, across all instruments, a significant majority of the musicians of the orchestras reviewed were male , but assignments of men and women to 1st and 2nd violin positions were not significantly different .Violin: women were numerically predominant in the violin sections (p < 0.021).Flute: A majority of flautists were female . A number of orchestras do not list contracted harpists, engaging players whenever repertoire necessitates.Almost all harpists were female (p < 0.0001). Where women were present, they were typically found among French horns. In two orchestras, principals in the trombone section were women. In one orchestra, the principal tuba was a woman.Males predominated in all brass sections (p < 0.0001) of other percussionists were female.No tympanists were female, and only 12.5% , but the increased representation of women was confined to higher-pitched upper strings, woodwind, and harp; brass and percussion positions of all orchestras remained almost exclusively male territory .When the same test was applied to orchestras in the UK; however, no differences linked to orchestra location were evident: orchestras in the capital did not include a greater proportion of males than did orchestras in the UK provinces , an own-choice work, previously notified extracts from orchestral works, and to read at sight passages from orchestral works. An audition will typically take around 45 min. Juries for auditions normally comprise the Principal and other members of the relevant section  plus a representative from related sections of the orchestra. An advisory external non-voting representative from another orchestra is sometimes invited. The orchestra’s principal conductor usually has the right to attend, but this is not often exercisedIt is common practice in orchestras in the USA for the successful candidate to be hired solely on the evidence of the audition: the player judged to be the best performer gets the post. Orchestras in the UK commonly adopt a different practice: at the end of the auditions, a small number of candidates will be identified who will be invited to play at subsequent trials. At each trial, the candidate will attend scheduled rehearsals with the orchestra and then take the vacant chair at the orchestra’s relevant public concerts. This “audition-plus-trials” procedure allows a more comprehensive assessment of a candidate’s sight-reading competence, goodness of fit of tone, and style with that established by the collective orchestra and its director, ability to operate under the stress of a concert and personal adjustment to section members. Its disadvantages are that the sex of the candidate will be known to the jury members. As the selected candidates may each be offered as many as six trials, the process of filling an orchestral chair can become protracted: 2 years would not be exceptional. Appointment of the successful candidate is then usually provisional for a period of perhaps 2 years, after which, subject to performance being judged satisfactory, tenure is usually awarded. Until recently, this “extended trial” procedure has been unique to British orchestras but is now being adopted more widely.Orchestras do not restrict their considerations to respondents to their advertisements but commonly invite other experienced players whom they have reason to believe may be interested, and these persons are likely to be invited to trials without being subject to audition.Few instrumentalists achieve positions in elite orchestras end-on to their conservatoire training, though those who are shown to have served for 45–50 years must be presumed to have done so. The majority gain experience in other orchestras from which starting point they later gain promotion, and this is evident from the personal career profiles published on the orchestras’ websites. Each section of a modern orchestra—cellos, flutes, horns, etc.—will have a designated Principal or Section-leader . Duties associated with these positions are rarely formally documented but are universally understood among professional players. The appointee to each section will be a musician of exceptional performance abilities, with extended experience of the orchestral repertoire and the workings of the orchestral world. They must be competent to advise on stylistic or instrumental issues such as bowing, articulation, and phrasing. The section Principal will normally take any solo passages demanded in the score and will act as agent of the conductor in securing cohesion and unanimity among section members in all aspects of their collective performance . A co/su“End-of-the-line” players  also commonly have Principal status, as do players of single-instrument sections such as tuba, despite the absence of second players of those instruments over whom leadership would be exercised. Management policies differ as to the number of musicians that are identified as Principals within an orchestra, ranging in our sample between a minimum of 12 and a maximum of 29. Some orchestral managements give the appearance of regarding Principal ranking as a reward for long service.χ2 = 41.29, df = 1, p < 0.000), but proportions did not differ significantly across the three geographical regions. Some European orchestras, however, have appointed very few women to Principal chairs: the Berlin Philharmonic currently lists only one , Vienna Philharmonic one , the Bavarian Radio OrchestraAcross the 40 orchestras reviewed, 83.2% of persons occupying Principal chairs were male, and 16.8% were women; 64.82% of co-Principals were male, and 35.18% were female  . Twelve male players are shown to have served between 40 and 51 years, suggesting that musicians do not lose their skills early, but only one long-serviced woman is reported to have spent as long as 40 years with her orchestra. These extended career patterns are consonant with those reported from Four of the 40 orchestras reviewed  include the dates of appointment of their musicians to the orchestra in the website biographies. From analysis of these, it is possible to map the typical career patterns of their collective 403 member musicians. The mean length of service of Principals and assistant/co-Principals with present orchestra was 17.15 compared with the mean duration for all players of 17.5 years.Kulturorchestern), ranging from local to metropolitan, reporting an age range of 20–69 years with a mean age of 45.25 years.Current ages of members of the four orchestras are not published on their respective websites, but It is evident from Because of these typically extended lengths of tenure of both sexes, few positions become vacant in an orchestra any year: from It is evident from the linear trend at Over the past 30 years, an extensive literature has accrued reporting differential associations of musical instruments, some instruments perceived to have feminine connections, others masculine. Judgments of these associations have proved to show high reliability . This haDespite the considerable number of related studies and commentaries, a definitive ordering of perceived gendering of instruments—most “feminine” to most “masculine”—has been difficult to determine owing to inconsistency among researches in the instruments that have been considered, differences in the musical genres with which they have been associated, and in the target populations addressed. A study by n = 58; violin n = 75,763), no instruments had a total n that was sufficiently small for their associated probability value to be considered unreliable. Instruments for which the number of girl students significantly exceeded the number of boys were listed as feminine type, those for which the number of boys significantly exceed girls were listed as masculine type. Instruments for which the boy-girl binomial probability proved to be statistically not significant were considered to be gender-neutral. From these values, it was possible to construct an hierarchy of instruments by sex type, most feminine to most masculine , the order is expressed as percentages at By comparing the numbers of male and female students receiving tuition on each of the orchestral instruments listed in the Hallam et al.’s study, a binomial probability could be calculated for the gender distribution of students for each instrument, and by ranking the probability levels according to magnitude, an order of gendering could be determined. Although the numbers of students relating to individual instruments inevitably showed wide variance , and conversely, a greater percentage of men achieve orchestral positions than would be predicted by numbers of boys receiving tuition .By comparing the Hallam et al.’s dataset with our data for male-female orchestral representation above, it was also possible to measure the level of correspondence between the numbers of male and female students receiving tuition on each instrument with the numbers of professional artists in post in the orchestras sampled. The levels of correlation were high and highly significant for both sexes. Despite these high correlations, however, the analysis revealed a marked and consistent discrepancy between the proportions of male and female students who achieved orchestral positions. The comparisons at It is evident from the plots for both the students and the in-post professionals that both groups adhere to characteristic sex-typed instrument choices illustrated in Preference for quality of sound/timbre ;appearance of an instrument; its manner of playing ;approximation of pitch range of instruments to player’s vocal range ;personality factors ;educational and training opportunities, attitudes of teachers and music directors, and lack of female role models in secondary and higher education ;socio-economic factors ;gender stereotyping , confirming the proposition of Hallam et al. and others that females show a preference for smaller, lighter instruments, whereas heavier and larger instruments are preferred by boys.Similarly, instrument weights were ascertainedCorrelations carry no implications of causality, but these results indicate that the sounding pitch, size, and weight of instruments are factors significantly associated with instrument choices of both sexes.prima facie evidence, our review of 40 world-class orchestras shows that overall male musicians have a greater numerical presence, significantly outnumbering females by a ratio of 60% males: 40% females. Within these simple proportions of imbalance, however, other significant contrasts are present: between orchestras of different geographical regions and their contextual employment and promotion structures, between individual orchestras, the respective career patterns of male and female musicians, their ages and lengths of tenure of positions, and the instruments they play.The comparative representation of men and women in professional orchestras is an issue of considerable complexity in which many disparate variables intersect. On p < 0.0001), though there were some notable exceptions.Our review show that balance of sexes in orchestras varies significantly according to region. Small differences in the presence of women between those in the UK (44%) and the USA (40%) were not statistically significant, but orchestras in Europe showed a significantly lower female presence  unequal overall representation of men and women and (2) skewed distribution of males and females across instrumental sections associated with the proclivity of women for smaller, lighter instruments of higher pitch, and men for larger, heavier, instruments of lower pitch, as prominently evidenced in the data of Reasons for the numerical disbalance are primarily historical, having their origins in a male-dominated labor force of post-Victorian society and its pervasive influence on attitudes to the respective social roles of men and women, especially those concerning child bearing and family responsibilities. The intensive work schedules of an orchestra are not easily accommodated with family life, though there are many examples to show that it is not impossible. The conspicuous discrepancy between the proportions of male-versus-female students receiving instruction on orchestral instruments and the representation of their respective sexes in orchestral membership raises complex questions. It is beyond the scope or purpose of the present paper to give them adequate consideration, but it is probable that the causes are associated with sociological and psychological issues such as personality, self-imagery, career expectancies, and choices, preferred lifestyles and education.What factors influence young people when they choose the instrument they will learn? Reasons are likely to be complex, involving both socio-cultural and practical issues, and therefore difficult to identify with accuracy. Apart from the small-scale investigation by Since their first introduction in the 1970s, blind auditions for orchestral appointments have generated much commentary particularly in the press. The impression is given that they have now been adopted universally and have brought about a revolution in the male-to-female representations and rid the orchestral world of its supposedly endemic sexism. The headline of Rice’s feature of 2013 “How blind auditions help orchestras to eliminate gender bias” is one of the more moderate contributions.The phenomenon we call music begins with the composer who assembles sounds to create sequences of musical gestures that enact his inner musical and emotional experiences: these are encoded in notational form in the musical score. The information encoded in the score sets out only the material structures of the composer’s ideas: it cannot encapsulate the emotional experiences that engendered the composition—its “feelingfulness”—nor encode quality of tone, rhythmic, or dynamic articulation or other nuances of performance. These qualities are realized from the score by the performer. The sounds perceived by a listener are therefore a synthesis of the sounds first envisioned in the composer’s imagination overlaid by new qualities that are products musical imaginings, emotions, and experiences of the performer. In the case of an orchestral performance, this new realization depends on the powers of re-envisioning of the musical director engaging the artists of the orchestra in a collective re-enactment. This is achieved through communication in rehearsal sessions and by the subtlety of the director’s gesture and the collective artistry of the orchestra’s musicians. An orchestra is therefore a dynamic unit whose purpose is to realize the ideas of the composer in actualities of sound, re-imagined through the orchestral director, and realized in sonic acts by the orchestral players.Unanimity of style and intention in this endeavor are the quintessential qualities of a fine orchestral ensemble. Technical competencies at the instrument are only basic necessities expected of a musician at audition. Compatibility of sound production with developed qualities of the ensemble: quality, attack and release of tone, extent and rate of vibrato, articulation of bowing, awareness and sensitivity to subtleties nuance empathy to nuance, and rhythmic articulation are among critical qualities. Principal players are soloists in their own right and their performance qualities and style are critical to an orchestra’s stylistic personality. It is therefore not surprising that orchestras exercise considerable care in their recruitment procedures in their efforts to secure appropriate appointees.Advertisements for positions in major orchestras will commonly draw around a hundred applications: this number will be reduced at the paper level to some 50 or 60 who will be invited for audition. From this large pool, five or six will be selected for trials. Sometimes, after the trials have taken place, none of those selected are found to be satisfactorily matched to the orchestral ensemble, and the advertisement and application process may be repeated, sometimes more than once. Additionally selection panels do not regard themselves as restricted to those musicians who respond to the advertisements but may invite players in post with other orchestras to apply. The appointment process may therefore take many months before a universally approved player is found.From the forgoing, it will be understood that calls, such as Engagements with world-class orchestras are typically long-tenured and owing to the tendency to late retirement ages, especially of male musicians, vacancies for new recruits occur relatively infrequently—an example of unintended consequences of legislations. This has caused change in the balance of sexes in the personnel of orchestras to take place rather slowly. Nevertheless, many orchestras are approaching numerical equality: British orchestras have already achieved that state, and those of North America are close to it. A number of those in Europe are still some distance away, however. If screened auditions, where they are appropriate, do protect women from possible bias at auditions, equality may be achieved, but if women continue to favor higher pitched instruments, given the number of brass and lower string players in the composition of a symphony orchestra, overall equality may prove elusive.The datasets generated for this study are available on request to the corresponding author.DS and EH claim authorship of this manuscript and also the copyright of the research work that this manuscript reports.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Measles is a highly infectious viral disease that continues to be a challenge for many countries worldwide. Although significant improvements have been observed since the introduction of vaccines, measles remains endemic in Romania. Contributing factors include vaccine hesitancy, difficulties in delivering doses to the population, and even the lack of sufficient vaccine supplies. These problems are further exacerbated by an inadequate implementation of public health measures, ranging from inefficient communication programs to the absence of a legislative framework concerning immunization. Moreover, many of the recent outbreaks were associated with chains of transmission in other countries, thus making the control of measles in Romania relevant at an international level. As many difficulties exist, understanding the key factors that limit the success of public health programs may provide guidance in shaping future strategies. Because similar issues are being faced in various other countries, the management of measles in Romania offers valuable lessons for researchers and policy-makers alike. Measles is a highly infectious viral disease which can result in severe complications and even death , 2. To tIn Romania, the first dose of the monovalent measles-containing vaccine (MCV1) was introduced in 1979 for children aged 9–11 months, and the second dose MCV2) was implemented in 1994 for children 6–7 years of age  (2). The was implAlthough these public health strategies have achieved and maintained a high vaccination coverage, outbreaks of variable severity have still been taking place, sometimes exhibiting elevated levels of mortality, especially in young children . MoreoveIn this paper, a synthesis of the available information about measles in Romania is provided. Recent outbreaks, some of their underlying factors, and specific control measures are presented in order to illustrate the successes and failures of national public health strategies. At the same time, possible solutions are assessed in terms of their potential to limit the impact of future epidemics.After the introduction of the measles-containing vaccine in 1979, the dynamics of the disease changed, exhibiting a decreased annual incidence and longer inter-epidemic periods see  10–17).–17.10–17Although the disease burden caused by measles had decreased substantially in Romania by the beginning of the current millennium, the epidemics of 2004–2007 and 2011–2013 deserve attention, as well as the one that began in 2016 . GeneralThe measles outbreak of 2016 resulted in more than 15,500 cases with a total of 59 deaths by the end of 2018  29). Th. Th29). As no specific treatment for measles exists, vaccination remains the only effective measure in combating this disease , 2. In RIn response to measles epidemics, additional vaccination campaigns have been conducted over the years to supplement the national program , 12, 13.An additional problem is represented by the low vaccine uptake in vulnerable communities, and more targeted measures would be needed to achieve good immunization coverage in these populations , 21, 35.The declining trends in vaccination coverage in the context of the recent measles epidemics have also constituted the basis for recurrent discussions about mandatory immunization –38. AlthMedical practitioners are offered regular training sessions by the county branches of the Romanian National Institute of Public Health (INSP), which are aimed at providing them with skills in vaccine delivery and in communicating with patients . FurtherOverall, the majority of the public (83%) believes that immunizing children as part of the national vaccination strategy is beneficial, according to a social study carried out in 2017 . Furtherspiritual and medical assistance” to the public, including access to and information about vaccines (Religious arguments have also been used against vaccination on many occasions. According to a 2011 census, more than 85% of the population identified as Orthodox Christian, and this factor is likely to influence the views of the public concerning immunization . The offvaccines –49. Howevaccines , 50, 51.The global anti-vaccine movement has also become more prevalent in Romania in recent years , 38. TheThere are a multitude of factors contributing to the negative outcomes of the prevention and control of measles in Romania, such as difficulties in vaccine delivery, the public's views on immunization, and even the absence of a legislative framework regarding vaccination. Consequently, although an elaborate methodology exists, most of the outbreaks that have occurred in recent years often failed to be contained in an appropriate way. With this in mind, the outcomes of public health measures in Romania will strictly depend on the way in which current and future policies will be implemented. The results will be contingent on thorough evaluations of existing methodologies, efficient public awareness campaigns, adequate intervention strategies, and more targeted measures toward vulnerable subsets of the population. Regarding this latter factor, conducting community-level serosurveys might have the potential to identify individuals that are at high risk of infection with measles. At the same time, understanding why people are hesitant about, or actively opposed to vaccination may provide invaluable insights for designing tactics to address these problems. Currently, it remains to be seen how Romanian politicians, public health authorities, and members of the civil society will address these issues in the upcoming years. Because similar problems are being faced on an international scale, the lessons learned from the measles epidemics in Romania may provide guidance in shaping future strategies not only for the Romanian authorities, but also for other countries that are challenged by this disease.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background and purpose: The recent advances in stroke therapy have placed focus on delivering care within the first hour after stroke onset (golden hour), principally through the use of Mobile Stroke Unit (MSU) to bring the hospital to the patient. The aim of this project is to search the location of MSU hub in Sydney, Australia, optimizing for catchment, transport to nearest thrombolysis and endovascular clot retrieval (ECR)/thrombectomy capable hospital and population at risk.Methods: Traveling time was performed using ggmap package in R to interface with Google Maps application program interface (API). This analysis estimates the travel time from the centroids of each suburbs to five potential MSU hubs  and eight thrombolysis capable hospitals. It is proposed that the MSU should be deployed at ECR hub to cover the suburbs, not well-covered by thrombolysis and ECR capable hospitals. This step was performed by assigning membership to hospitals within 30 min traveling time to the ECR hub. The base hub of the MSU was proposed as the closest hub (providing ECR) to the least well-served suburbs. The population serviceable by MSU was estimated using stroke incidence studies in Melbourne and Adelaide.Results: The largest population, serviceable by MSU within 30 min , 45 min radius , and 60 min , was Royal North Shore followed by Royal Prince Alfred, Liverpool, Westmead, and Prince of Wales hospitals. Prince of Wales hospital has the smallest catchment within 30 min , 45 min , and 60 min . Suburbs at the edge of metropolitan Sydney such as the Northern Suburbs are less well-served by thrombolysis and ECR capable hospitals. There are 10 suburbs within 30 min travel of one hospital. The remainders are within 30 min of two or more hospitals.Conclusions: Any of the five endovascular clot retrieval capable hospitals are capable of serving as a hub for MSU. We provide a method to identify the hub based on location of suburbs less well-served by other hospital. The recent advances in stroke therapy have placed focus on delivering care within the first hour after stroke onset (golden hour) . A poten2  with tiles from OpenStreetMap   .We formulated the problem as a variant of the maximum coverage . In a trIn addition, we provide a sensitivity analysis by estimating the population at risk of stroke serviced by MSU. This analysis was done by extending the base from 30 to 60 min .The map  illustraThe largest population, serviceable by MSU within 30 min , 45 min radius , and 60 min , was by Royal North Shore hospital followed by Royal Prince Alfred, Liverpool, Westmead, and Prince of Wales hospitals . Prince In this study, we have used data driven method to map location of MSU base in Sydney using Google Maps API estimate of travel time from the ECR and thrombolysis capable hospitals. This issue arises as there are five ECR capable hospitals in Sydney; each has their merits of operating as a base for MSU. Using the idea of equity, an ECR hub was identified as the one that was closest to the suburbs, least served by ECR and thrombolysis capable hospitals. Our methodological approach is of use when planning to invest in MSU.ggmap or other tools discussed in this special issue of Frontiers in Neurology (Stroke). In the Republic of China (Taiwan), Google Maps API is available for travel time estimation.The use of geographical information systems for health service design is evolving since 2017 , 26–30. The approach we have taken for evaluating MSU base in Sydney is different from that in Melbourne . The MSUIt is possible that the issue of deciding between thrombolysis and ECR capable hospitals and MSU in a well-service catchment has not been explored in detail, as some of the centers operating MSU have only one ECR hub and have a small catchment population. Sites such as Lucas County serves a population of 433,689 ; and theSimilar to the Houston and Melbourne model of basing the MSU hub in a major public teaching hospital , the ratProjection of patients at risk of stroke is based on stroke incidence studies from Melbourne  and Adelaide  , 19. TheA limitation of this study is that it is based on simulations and not observed cases of stroke. We had circumvented this by simulating stroke cases in the centroid of each suburb. This approach provides an average of the trip time. Another limitation in the proposal for MSU is the issue of cost effectiveness, which has not yet been done. Current studies are underway on cost effectiveness of analyses of MSU , 38. A cDepending on the configuration of MSU, the cost has been estimated to be around US$750,000 to US$1,400,000. The running cost of operating MSU during office hour is estimated to be around US$1,000,000 . RecentlThis study was not designed to address the issue of cost effectiveness or propose the purchase of MSU but rather to evaluate the location for deployment of MSU. In the process of performing these analyses, there were issues on clustering or proximity of hospitals with thrombolysis and ECR capability. There may be a need to allocate resources equitably from inner Sydney to outer Sydney while planning the location of MSU hub. This is an issue that is not unique to Australia but is also relevant in Europe  and CanaIn summary, any of the ECR-capable hospital can serve as a hub for MSU. We provide a method to identify the hub based on location of suburbs less well-served by other hospital.The datasets generated for this study are available on request to the corresponding author.TP: design, analysis, and writing the manuscript. RB, VS, and HM: writing the manuscript.TP is on the Advisory Board of Genzyme on Fabry Disease and has received payment for lectures including service on speakers' bureaus for Bayer, Boehringer Ingelheim, Pfizer, and Genzyme. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brucellosis, also known as “undulant fever” or “Malta fever”, is a zoonotic infection caused by microorganisms belonging to Brucella, a genus of gram-negative coccobacilli that behave as facultative intracellular pathogens of ruminants, swine and other animals. Brucellosis is a threat to public health, hence identifying the optimal way of preventing disease spread is important. Under certain circumstances, integrated, multidisciplinary “One Health” (OH) initiatives provide added value compared to unidisciplinary or conventional health initiatives. Conceptualizing and conducting evaluations of OH approaches may help facilitate decisions on resource allocation. This article historically describes and compares Malta's 1995–1997 with Serbia's 2004–2006 brucellosis control programmes and quantitatively assesses the extent to which they were compliant with a OH approach. For both case studies, we describe the OH initiative and the system within which it operates. Characteristic OH operations  and supporting infrastructures  were evaluated. We scored the different aspects of these programmes, with values ranging from zero to one (1 = strong integration of OH). Malta demonstrated a higher OH index (0.54) and ratio (1.37) than Serbia (0.49 and 1.14 respectively). We conclude that context and timing are key to determining how, when and why a One Health approach should be applied. The adoption of a true OH approach that involved systemic organization, leadership clarity and transdisciplinary communication, collaboration, and co-ordination was essential to Malta's successful eradication of brucellosis after several failed attempts. In contrast, contextual factors in Serbia permitted the successful adoption of a primarily sectorial approach for short term control of brucellosis. However, while a fully-fledged transdisciplinary OH approach was not initially required, it is likely to be key to maintenance of brucellosis control in the medium and long term. Through these two case studies, we demonstrate that One Health initiatives should be applied at the right place, at the right time, with the right people and using the appropriate conditions/infrastructure. Lastly, OH evaluations should include economic assessments to identify optimal of resources in these situations, thereby justifying funding and political support required. Brucella, a genus of gram-negative coccobacilli that behave as facultative intracellular pathogens of ruminants, swine and other animals , and situations where farm animals are kept in close proximity to humans . The epiwww.onehealthinitiative.com), “One Health” is an umbrella term referring to the commonalities between people, animals, plants and the environment. It recommends integrative approaches to health by expanding interdisciplinary collaboration across these highly interlinked components , had long been endemic in Malta, to the point where it was known as “Malta fever”  and milk. Policy makers were highly engaged in the process and introduced effective legislation that required the registration of all herds in Malta. In 1987, the Veterinary Services Department (VSD) launched the “Test and Slaughter” scheme  across aBrucella antibody titer of > 1 in 320 dilution or a positive culture of B. melitensis, or who had a member of their household with a Brucella antibody titer > 1 in 320 dilution (with or without symptoms), was classified as a case. The Disease Surveillance branch of the Department of Public Health extensively sampled and tested cheeselets sold in shops, street vendors and supermarkets across the Maltese islands. The Department of Agriculture was subsequently notified regarding suspect herds, which were examined further and blood testing carried out  was set up. During this period, a “test and slaughter” programme similar in scope to that described for Malta was established. Overall, the veterinary services destroyed 1,497 animals  in the northern part of Serbia after screening 1,485,702 animals. No data is available for the southern part of Serbia. The number of infected humans and animals significantly decreased in northern Serbia (Vojvodina province) after 2006, and in southern Serbia after 2009, and overall Brucellosis incidence now shows a declining trend   in an integrated manner, namely through the holistic assessment of these aspects. This analysis includes a historical account intended to offer insight into the geopolitical context of Brucellosis outbreaks and to identify and delimit the systems within which the OH initiatives were developed to differing extents. The TOC  . The NEObal.net) . This co The TOC  underlie The TOC , 7.Within a OH approach, the TOC defines the objectives of the initiative, as well as the changes required to achieve these goals . Therefo* AND Malta OR Serbia. Furthermore, the bibliography of articles emerging from the search were reviewed to identify additional potentially relevant literature.A non-systematic literature search around brucellosis was conducted between January 2016 and April 2016 using Google Scholar, PubMed and EMBASE to identify scientific articles and gray literature that would inform the background of this study. The following search terms and key words were used: BrucellUsing a case study approach, the authors obtained information primarily through 15 documented interviews carried out in both countries. In Malta, these interviews were recorded and transcribed by John Rizzo Naudi prior to 2005  and invoWe therefore retrospectively identified and discussed the various steps and systemic changes that needed to take place for brucellosis eradication or control to be achieved. Assessment of the following aspects: Thinking, Planning, Working, Sharing, Learning and Systemic organization—was conducted. The dimensions within each aspect—where each “dimension” refers to an entity that can be captured by the same metric or concept, such as geographical space or time—were scored in increments of 0.2  by SB for Malta and SS for Serbia. Information on the scales for the different dimensions can be found in the appendices. Scoring of the NEOH evaluation tool  was thenLastly, a conceptual essay for economic evaluation that assesses the flow of cost and benefits is provided. This offers a basis for further evaluation aiming at assessing the advantages of the OH approach in comparison with traditional approaches toward disease control and eradication.The Public Health Department  was the main actor for Malta, whereas the Directorate of Veterinary Medicine was the main actor for Serbia. Public health services in both countries included reference laboratories for human diagnostics; physicians, and hospitals. The Public Health Department assumed responsibility for these services in Malta, whereas Local Health Authorities were responsible for public health services in Serbia. Food safety/consumer health was only relevant to the Maltese scenario and involved the Superintendence of Public Health and Department of Consumer Affairs. Other major stakeholders included health education/promotion and policy makers from the Health, Agriculture, Justice and Internal Affairs Ministries in the case of Malta; and policy makers from the Ministry of Agriculture and Ministry of Internal Affairs in the case of Serbia.set of elements or parts that is coherently organized and interconnected in a pattern or structure that produces a characteristic set of behaviors, often classified as its ‘function’ or ‘purpose.”’ . Social practices and behaviors, as well as the general state of knowledge about mechanisms of disease spread, are of relevance to the wider socio-cultural sub-system which determines the insurgence and the persistence of the disease in the society .Meadows and Wright define a system as a “rpose.”’ . The apprpose.”’  the presOther relevant elements of the system could not be shown in Figure Br. problem, their organization and strategies . The institutional framework can be considered to be a sub-system permeating and affecting the functioning of the basic system outlined in Figures –The institutional framework, i.e., the institutions charged with solving the Br., the accumulation of scientific knowledge and of what was effective  and the increase in social awareness, finally leading to the implementation of the OH-like model of intervention.–The evolution of the institutional framework over time. Together with geographic space, time is a relevant dimension of the system, particularly because of the sequence of repeated attempts to control In the case of Serbia Figure , the samLocal breeding systems reliant on the use of pastureland by different herds moving across the countryside (which promotes the dissemination of the disease within farms or family production units), characterized by prolonged close contact between animals and humans;The health system, in particular the lack of effective counter-measures and governance to address brucellosis;Limited knowledge regarding the risks related to brucellosis among the general population, which in turn determines local practices and behaviors in production, processing and consumption. In the case of Malta, this explained the social mis-representation of dairy product safety and led to the persistence of traditional processing and consumption practices, despite health measures implemented several times over a number of decades.Based on the system identification process outlined above, we developed a comparative approach between the two cases. General similarities between the two countries include the basic contextual units of the system, such as the general epidemiological features of the disease and—assuming that the geopolitical territory of Malta and Serbia respectively represents the spatial limits of each system—its dissemination to human beings via direct contact and through the food supply chain . Additional similar elements include:The differences in the political context The differences at institutional or organizational levels The greater relevance of traditional consumption habits in Malta, in comparison to Serbia, which resulted in a greater emphasis on the food supply chain for OH initiatives in that countryLast but not least, Malta had a pioneering role in the discovery of brucellosis epidemiology. This probably contributed to the differences in timing and method of intervention strategies between the two countries.On the other hand, some features uniquely characteristic to each country may explain, at least in part, differences in the timeline and key characteristics of brucellosis development in the Maltese and Serbian systems. In particular, the unique geographical characteristics of the two countries resulted in different patterns of disease emergence and resilience to eradication and control, particularly in combination with:A further step in system identification concerns the institutional and governance aspects of the health measures adopted in 1995–1997 and 2004–2006 in Malta and Serbia respectively. While Serbia's strong Veterinary Services played a leading role in the control of brucellosis, Malta's veterinary services were not developed to an equivalent extent and hence could not lead the control and eradication programme. Instead, Malta relied on a historically powerful public health sector, which was in a position to take on a leadership role in the most recent outbreak. These have been amply described in this paper.Table The rationale of the eradication and control processes in both countries was to address the infectious disease, with efforts primarily focused on systemic organization, leadership clarity and transdisciplinary communication, collaboration, and co-ordination. The following drivers spurred the intensity of efforts to control brucellosis in the two countries:Economical: high health care cost of treating human brucellosis; costs of surveillance; costs of government subsidies to farmers whose animals are eliminated because of the diseaseEmotional/Psychological: suffering of patients  affected by brucellosis; suffering of family and friends, particularly in fatal cases. Human cases of brucellosis were more prominent in Malta than in Serbia, where the disease seemed to have caused substantial emotional and psychological distressGeographical: Malta's island status contributed to the recognition of disease vectors and also helped to contain and maintain eradication of the disease. This driver was more of a challenge in Serbia, since the importation of infected animals was facilitated by porous land borders. Hence geographical location is a crucial consideration—Serbia depended on the actions of neighboring countries to manage its Brucellosis control process, whereas this was not the case for MaltaSocial: Malta's sister island—Gozo seemed to be less receptive to public health warnings regarding brucellosis, as manifested by the lingering belief that aseptic (clean) farming and retail environments were sufficient to ensure food safety of milk/products. The social driver in Serbia was primarily related to the country's post-war relations with neighboring countries and the lack of communication and trust between people from different (Former Yugoslavia) regions.This section of the results deals with the quantitative evaluation of the “One-Health Index.” Each of the six assessments outlined below is represented by a spoke in the spider diagrams for Malta and Serbia Figure  where thThinking refers to the way actors and stakeholders think within and about the system and the One Health initiative. This includes an assessment of how the dimensions and scales under consideration  may support or limit the outcomes and impacts of the initiative. The overall scores are 0.80 for Malta and 0.60 for Serbia. A major strength for both countries was the integrated health approach adopted during the eradication process. The lower overall score for Serbia is attributed to the lower sub-scores in the different dimensions' coverage and balance, and to sustainability and socio-ecological considerations. The focus in Serbia was timely control of the disease in animals before it spread to humans, hence fewer dimensions were covered and less importance was given to sustainability once there was successful control and effective law enforcement by the Veterinary Service. Although Malta's score for thinking is higher, this was mainly due to Malta's previous failed attempts at eradicating the disease, and reflects the fact that more stakeholders needed to be involved in order to finally achieve success.One Health planning requires that aims, problem formulation, responsibilities, resource allocation and financing of the initiative are systematically organized. It also requires clarity in establishing roles, tasks, responsibilities, and competencies of participants . In thisThis assessment explores the extent to which engagement in the OH initiative was interdisciplinary and participatory  . TransdiSharing refers to the information and data-sharing infrastructures in One Health initiatives . ElementThe learning infrastructure within the One Health initiative comprises the learning style  and setting . It also encompasses the type of environment: namely the stakeholders involved (“direct” environment), and the cultural, economic, and political situation surrounding the OH initiative . Our assessment considered whether these learning styles and environments supported a OH approach. The overall scores are: 0.60 for Malta and 0.60 for Serbia. Malta showed slightly greater emphasis on adaptive and generative individual, team and organizational learning, as a result of the crisis that ensued following the emergence of Brucellosis in humans.This assessment probes whether implementation of the OH initiative was facilitated by change-oriented leadership and effective teamwork, and therefore is closely related to and influenced by OH Planning. The overall scores are 0.80 for both Malta and Serbia. Despite differences in the methods leading to control and eradication, both countries manifested a rather strong sense of systemic organization as reflected by social and leadership structures and skills, team structures, competence and focus on innovation.Table Following the 1995 outbreak in Malta and subsequent efforts to eradicate the disease, there have been no cases of brucellosis in humans recorded since 1997. The control of the process, including monitoring and pasteurization of milk and cheese production and the enforcement of labeling and packaging is now co-ordinated by four collaborating departments: The Veterinary Services Department, the Public Health Directorate, the Agricultural Department and the Department for Consumer Affairs. Sharing and linking of information in inter/trans-disciplinary groups was well established in the 1995–1997 outbreak, which ultimately led to successful eradication. The information was successfully shared by representatives of disciplines on the outbreak committee and also to the non-scientific communities through information packages released by Ministries of Health and Agriculture.Seminars and education activities are organized in order to increase the knowledge on the disease and involve stakeholders in surveillance activities, e.g., seminars and courses targeting official veterinarians and practitioners, medical doctors in hospitals and family doctors, and educational outreach campaigns targeting the general public.Sharing and linking of information between the Ministry of Health and Ministry of Agriculture was not carried out officially. While annual reports on zoonotic disease cases in humans (including brucellosis) are publicly available online, annual reports on animal screening from the Directorate of Veterinary Medicine are not publicly available. The yearly prevalence of brucellosis in animals can be found on the web site of the World Organization for Animal Health. Therefore, in contrast to Malta's isolation as an island state, which facilitated control of Brucellosis, Serbia remains susceptible to importation of the disease unless strict, vigilant border control is continuously maintained.This section focuses on the economic outcomes of brucellosis. It is likely that political support is more forthcoming when the adoption of an approach like One Health translates into economic gains. Although this study involves a conceptual, rather than an empirical, economic evaluation, potential economic impact of brucellosis may be identified through the schematic models in Figures Figure Public costs: arrows commencing from these boxes list the type of public costs, mainly incurred by the public health system ; and Disease containment – Private costs: similarly, arrows staring from these boxes list the types of private costs ; Loss of revenue due to temporary or permanent disability, which translates into costs for individuals, families and society, depending on the social relevance of the disease; Adoption of good practices in milk/dairy product preparation and consumption; and Psychological suffering due to uncertainty around health status).– As shown in Figures The positive and negative economic impacts outlined in Figures These two case studies, despite their common goal of eradicating or controlling Brucellosis, have quite diverse backgrounds and show different degrees of “One Health” thinking in their respective approaches. In both countries, control was only possible due to constant reminders to farmers and animal owners that the disease could easily spread to humans, together with strict enforcement of legislation. The Malta case study spans a century of failed measures and setbacks, and demonstrates a paradigm shift in the approach to brucellosis eradication over time. The measures implemented ranged from initially relatively isolated actions such as processing of milk through pasteurization (introduced prior to the second World War), to a more sectorial approach adopted in the 1980's. However, it was only upon the adoption of a true “One Health” transdisciplinary approach in the mid-1990's that Brucellosis eradication was successfully achieved and maintained. Enforcement of existing and newly implemented legislation was crucial to this success, and required collaboration by all stakeholders involved including public health, veterinary health, policy-makers and legislators, as well as farmers and consumers' representatives.The Serbian context differed from the Maltese scenario. Serbia did not have a long history of brucellosis, which was largely sporadic before the mid-1990's and became endemic only as a result of the non-regulated importation of infected cattle and sheep from neighboring countries. There was no cultural resistance to the destruction of potentially infected herds of cattle and sheep, and a greater willingness to accept the scientific rationale for culling within the general population. Additionally, Serbia was able to capitalize on the experience and knowledge of OH thinking adopted in other countries, which explains the somewhat predominantly sectorial approach adopted by the Veterinary Directorate. This proved effective, so that escalation to a fully-fledged transdisciplinary OH approach was not required. It should be noted that the geopolitical conflict in Serbia that ultimately led to “United Nations Security Council resolution 1244”  meant thWhile brucellosis control was the primary concern in the short term, surveillance and ongoing monitoring remain important medium and long term concerns. This is also reflected in the timing and extent of adoption of OH thinking in the two countries: in Serbia a true OH approach was not required for control, however it is likely to be key to its maintenance in the medium and long term. In Malta, the OH approach was critical in the short term in order to eradicate brucellosis, and together with strict enforcement of legislation remains key to ensuring that the disease does not return. The strength of the OH approach has been tested in recent years. In 2012 a “phantom” herd of unregistered  sheep was identified in Gozo, leading the Veterinary Services Directorate to commence testing and culling of 216 potentially infected sheep. The farmer launched a court case to prevent the remainder of the herd from being depopulated, and a series of appeals and counter-appeals followed with the farmer, the Attorney General, the Police Commissioner and the Director General of Veterinary Services as the main protagonists .There are several lessons to be learnt. In these two case studies, we hope to showcase that One Health initiatives should be applied at the right place, at the right time, with the right people and using the appropriate conditions/infrastructure. One should not adopt a OH approach purely for its own sake or rather wait for all the disciplines to be involved before concrete action is taken. In other words, the One Health transdisciplinary action should not replace but should reinforce the unidisciplinary initiatives taken at the stages of problem identification and action. For example, on the one hand, in the case of Malta, because of the failed attempts at eradication due to fragmented unidisciplinary efforts, only when the Maltese rigorously adopted the OH approach in a transdisciplinary manner, namely by also actively involving the non-scientific community, did they achieve success. The Serbian case study on the other hand showed that the health and agricultural authorities could rely on the aggressive action taken by the Directorate of Veterinary Medicine before moving onto the One Health approach mainly for surveillance prevention. It was the case because most of the infection appeared in animals and number of infected humans was not as high as in Malta. The disease mostly developed in cattle and sheep leaving most of the consequences in economic losses in animal breeding.The timing of the OH approach is particularly important: in Malta, the right conditions took decades to develop and lessons were painfully learnt over a long period of time, whereas in Serbia the OH approach followed the drastic intervention and leadership of the veterinary department. Sustaining the processes that prevent the re-emergence of brucellosis, however, are likely to require a OH approach.There are a number of limitations to the method used in this study, including potential bias in the selection of interviewees. In both countries, the fact that the interviewers were “insiders”—members of the outbreak teams—might have influenced the purposive sampling approach used in this study . FurtherThis comparative case study shows that context and timing are key to determining how, when and why a One Health approach should be applied. We conclude that one need not wait for the start of a fully-fledged One Health approach to address a potential health crisis. Instead, each relevant discipline should be on the alert and perform its key responsibilities at an early stage, before scaling up to a transdisciplinary level becomes necessary. Nevertheless, as evident in this article, adopting a OH approach has provided added value not only during the periods of crisis but also in the medium and long term, particularly in the areas of disease prevention and control, surveillance, health promotion and health education. Adopting a OH approach may also translate into cost savings. We therefore propose that OH evaluations should include economic assessments, in order to be able to better understand the optimal use of resources in these situations, thereby justifying funding and political support required.SB and SS were responsible for data collection and providing the first framework of the paper. SB, SS, DC, and EL were responsible for preparing the first draft. MC and MA were responsible for the system dimensions, boundaries, elements, relationships, and functioning, as well as the theoretical perspective regarding an economic evaluation and providing advice in the early stages of formulating the case study. All authors contributed in the preparation of the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Yeast and filamentous fungi have been successfully harnessed to produce fine and high value-added chemical products. In this review, we present some of the most promising advances from recent years in the use of fungi for this purpose, focusing on the manipulation of fungal strains using systems and synthetic biology tools to improve metabolic flow and the flow of secondary metabolites by pathway redesign. We also review the roles of bioinformatics analysis and predictions in synthetic circuits, highlighting  Systems biology has risen as a relevant approach to encompass the phenomena that occur concomitantly in determined environments. Since its advent, research was modeled after a global perception of biological systems operation. Systems biology presents scientific possibilities to understand processes as a whole and to integrate new data and new analytical tools that are intended to solve biological problems by integrating parts and modules. Specifically, systems biology offers us the possibility to study the combination of individual parts which can play different roles in the behavior of the biological system depending on the context.On the order hand, synthetic biology refers to the use of rational approaches that provide us with the possibility to study a unique part from a module or a system in a minimalist manner to understand how the creation of nonexistent parts could enable us to engineer biological circuits, for the engineering of microbes as cell factories. These approaches seek to address the growing industrial demand for biotechnological processes that manufacture products in the required scale in an economically efficient manner. Many efforts in the genetics and metabolic engineering fields have explored the use of viable fungal cells to generate fine chemicals.Synthetic biology plays a central role in these efforts by means of innovating the use of the components of biological circuits, pathway, or processes and ultimately of systematically harnessing these modules to achieve the goals of industrial scale production. The advent of the bioinformatics era has allowed the redesigning of living information in the cell environment. This has significantly contributed to the development of new strategies based on systems biology approaches, mainly computational and rational ones. Since all these advances in single parts of a system and integrative modules direct efforts toward a common goal, systems and synthetic biology permit the deeper exploration and elucidation of cellular mechanisms, mainly the genetic and metabolic engineering of fungal cells in an integrative way.Here, we review and discuss the most promising recent advances in this field, focusing on the use of single module, genetic and metabolic designs to engineer filamentous fungi and yeasts for industrial biotechnological processes. We describe some of the most promising industrially relevant tools for this purpose. Also, a list of significant studies that have used synthetic biology approaches to engineer fungi is provided in Table (G3PDH) promoter in Pichia pastoris, a well-known methylotrophic yeast model. The study adopted a robust strategy of simultaneous deletions and duplications of transcription factor binding sites (TFBSs) to control expression of transcription factor (TF) genes to understand the transcriptional dynamics in these cells. This is a promising approach to unravel the mechanisms of transcriptional dynamics that could be potentially expanded to further investigations of the transcription logic in yeast, with the ultimate goal of robust and improved strain engineering.Transcriptional control redesign is a general term encompassing processes used to engineer fungi that have been the most intensively studied ones. Many single modules that are involved are pivotal to drive heterologous protein expression. Advances in synthetic promoter design have significantly contributed to the creation of engineered strains. In this sense, Ata et al.  recentlyThis strategy allows the creation of promoter variants with different strengths in the presence or in the absence of critical regulators. It relies on the premise that these dynamics directly influence the transcriptional response and the regulation logic. Approaches like these are the most common and robust tools for understanding the dynamics of regulation in yeast cells. The design of a set of promoters that rely on different architectural compositions is crucial for the systemic understanding of regulation dynamics and is a convenient application for more detailed studies to engineer strains for industrial purposes Figure . AlthougAshbya gossypii translation elongation factor (TEF) promoter architecture  are elegant ways to engineer promoters, as are combinatorial fusions of regulatory elements creating novel, promoter architectures. Both approaches could optimize transcription in fungal cells. One of the greatest benefits of this approach is the possibility to supply the industrial demand for metabolites or fine chemicals, which is currently limited by extreme chemical conditions, such as low pH. A systematic and expansive molecular strategy to overcome the limits of transcriptional regulation is needed to increase the level of production of high-value added products. To this end, a construct based on the core architecture of Trichoderma reesei promoters, the most utilized fungus for the production of cellulases  of intronic sequences in the promoter of genes encoding 40S ribosomal proteins (RPs) increased the strength of the promoter. The luciferase activity was 17-fold higher when an intronic promoter was used to drive expression when compared with the strong wild type TDH3 promoter in S. cerevisiae  terminator to increased protein expression was described as a process mediated by NAB6p and PAP1p trans-acting RNA-binding proteins. This relationship was demonstrated by the analysis of mutations of the DIT1 terminator sequence. The mutations that were introduced improved the terminator activity by 500% when compared with an internal terminator used as the control  tools for the engineering of microbes as cell factories are an efficient and reliable strategy in synthetic biology. Fungal cells, even filamentous species, have already been modified for their successful use in industrial bioprocesses. The CRISPR/CRISPR-associated protein 9 (Cas9) technique, basically, consists of using a Cas9 endonuclease-guided RNA to target a DNA sequence, which allows the precisely targeted disruption of this sequence and the activation of repair mechanisms in cells. This strategy fits a chimeric single guide RNA (sgRNA) and a Cas9 into a unique and easily manipulatable plasmid vector. Since the appearance of CRISPR/Cas9, numerous efforts have aimed to engineer fungal cells for industrial purposes Figure . Recent A major advantage of using the CRISPR/Cas9 system to edit genomes is the system's plasticity for repairing double-strand DNA breaks. Led by sgRNA, Cas9 cleaves the target DNA, which activates a repair mechanism in eukaryotic cells. Therefore, the nonhomologous end joining system of repair is considered an error-prone method for fixing broken sequences and may generate disruptions in these sequences by deleting or incorporating small DNA molecules to the site of damage. This type of repair system is a suitable alternative to generate knock down strains or to disrupt an open reading frame (ORF) of the gene of interest. Additionally, the repair promoted by homologous recombination provides an error-free system through the incorporation of a faithful donor DNA sequence, which is a valuable strategy when the goal is to replace a sequence of interest.P. pastoris in a precise and efficient manner  that naturally produce higher rates of this molecule. Therefore, strategies to manipulate both species have been reported  lipid.Metabolic engineering for the production of oleaginous compounds is focuse on the central carbon metabolism, with the aim of increasing the molecular levels of acetyl-CoA, malonyl-CoA, and acyl-CoA. This generates precursors for the production of fatty acids that are used for the manufacture of detergents, lubricants, biodiesel, plastics, and coatings  and a protein related to the general stress response (Whi2) increased yeast performance and ethanol productivity, even in highly acidic media.Another problem during the biomass fermentation is the acidification of the growth medium due to the acidic pretreatment of the biomass  library, and they identified genes that could be related to this acetic acid resistance phenotype. The authors also observed that the glucose consumption rates and ethanol productivity in media containing acetic acid were increased in the mutants when compared with the wild type. In another study, Rajkumar et al.  than when the same enzyme was expressed in the cytosol, demonstrating that the mitochondrial matrix can be a desirable environment to redirect metabolic pathways. Furthermore, a pathway for isobutanol production was also improved by localizing biosynthetic enzymes to the mitochondria of S. cerevisiae. Park et al. , polyketides (for antibiotics), and terpenoids, such as ß-carotene and amorphadiene  responsible for the incorporation of acyl-CoA onto the diacylglycerol backbone to synthesize TAGs. This, along with a deletion from a lipase regulator, increased the lipid content to 77%. Both the studies reported the efficiency of using synthetic pathways to increase the production of fatty acids by Y. lipolytica, which later can be converted to oleochemicals that are important for industrial applications, including biodiesel, detergents, and bioplastics, among others  and amorphadiene synthase (ads). Remarkably, the production of these chemicals increased when the organism grew using lignocellulose hydrolysates when compared with to purified substrates. A brief overview about the systems and synthetic biology strategies used for rewiring and dynamic control of metabolism in fungi are summarized in Figure i et al.  reportedEven after considering the aforementioned accomplishments, it is important to note that there is still a huge difference between static and dynamic metabolism. Biological systems are much more complex than a physical, steady system. Results are hard to reproduce and, thus, hard to predict. This is especially true if we only consider a limited part of the system, such as enzymes in a metabolic pathway  in S. cerevisiae and reported the highest PHDA yield ever achieved in yeast. Still, in this sense, ON-OFF genetic circuits can also be achieved by adding responses to temperature or other inducers, such as isopropyl β-D-1-thiogalactopyranoside . An example of a study that took advantage of genetic circuits was performed by Williams et al. . The autContinuous genetic circuits can also be constructed for the dynamic control of target metabolic pathways, coupling gene expression to the sensing of a specific metabolite. Metabolite sensors allow the circuit to respond in accordance with the cellular environment so that they become sensitive to variations. As an example, Xu et al.  developefaa1 under the control of different promoters to prevent the accumulation of free fatty acids in industrial mutant strains, thus avoiding the loss of precursors to the extracellular medium. This approach enhanced the production of fatty alcohols and expanded the knowledge regarding the control of metabolite flux of this pathway. This is an excellent example of the use of dynamic control to increase industrial production. Venayak et al. , can be utilized for industrial purposes. The lipids extracted from T. cacao beans are the basic component of cocoa butter, a valuable ingredient that is becoming increasingly scarce in the market. In this sense, Wei et al.  product.The bioproduction of lipid molecules in fungi can provide novel renewable and sustainable material for the production of food ingredients independent of plant cultivation, climate changes, and seasonal availability. Lipids extracted from plants, such as the cocoa tree  to cancer (vinblastine and vincristine). Owing to their structural complexity, most of those important chemicals are produced through extraction or semi-synthesis from plant species. Addressing problems like seasonal variations in crop yields and batch variation of e et al.  developed Smolke  for the S. cerevisiae. Although the yield obtained by Galanie et al. (Cannabis constituent (tetrahydrocannabinol (THC)) . pcbAB and npgA are responsible for the synthesis of the benzylpenicillin (PEN) precursor, amino-adipyl-cysteine-valine (ACV), while the products of pcbC, pclA, and penDE are responsible for the conversion of ACV to PEN.The production of nonribosomal peptides (NRP) is of interest for their use in synthetic biology approaches, since the modularity of NRP synthetases (NRPSs) allows enhanced compound production or the synthesis of entirely new structures. A milestone in the application of synthetic biology to produce NRP using n et al. . They maS. cerevisiae and of PEN in Hansenula polymorpha were previously reported by  variation can change TF nucleotide sequence preferences, as reviewed in Inukai et al.  and SiggS. cerevisiae have a primary and secondary binding motif, which may even perform distinct regulatory functions. These findings indicate a property that could be further explored in motif search algorithms that focus on yeasts. Even with many experimental approaches that generate high-throughput data, it is impossible to test all the environmental conditions that a natural regulatory network is able to respond to. Despite this limitation, several methods and approaches have been used to identify motifs in high-throughput data. In this regard, computational motif discovery tools that deal with large data  that are currently being used include HMS (Hu et al., Studies using high-throughput data regarding TF binding specifity are another source of valuable information on parameters affecting gene regulators. In this sense, Gordân et al.  suggesteBioinformatics research is being allied with synthetic biology for the discovery of new modules and their interactions. Yeast promoters have motifs/sequences that are necessary for promoter function and for assembly of the preinitiation complex, which leads to the recruitment of the entire cellular transcription machinery (Thomas and Chiang, cis-regulatory elements (Pedersen et al., Promoter prediction is a hurdle. Transcriptional initiation is the first step in gene expression and, so, is an important control point. Despite its importance, eukaryotic promoter prediction is not simple because of the structural complexity of natural cis-regulatory elements. This was followed by the use of a neural network with back propagation to create a promoter classifier, thus, allowing an improvement in accuracy. Another notable approach to solve the prediction bottleneck was described by Umarov and Solovyev (Owing to the complexity of promoter prediction in general, we presents prediction below some useful tools that have been applied in fungi/yeast or that could be adapted to these organisms. For example, Shahmuradov et al.  attainedSolovyev . The autSchizosaccharomyces pombe and A. nidulans (Sibthorp et al., In addition to the aforementioned prediction methods that deal with pre existing data, several studies have used transcriptomics to find putative TSSs. With specific RNA-seq protocols to obtain 5′ regions (Gowda et al., Considering current efforts in analyzing large amounts of data, Yu et al.  proposedWhile these tools may help in studies to overcome limitations in motif discovery, working with high-throughput data remains a bottleneck. To deal with this, Guzman and D'Orso  developeSystems and synthetic biology are playing a pivotal role in the development of tools for engineering yeast and filamentous fungi. Studies based on them have shed light on how single modules can influence an entire and robust system. Besides, the particularity of each tool presented in this review, the approaches to engineer fungi as cell factories for biotechnological industrial processes, can be successfully combined to guarantee a viable and cost-efficient strategy. The tools described here can cover a wide area of applications, substantially improving the already existing methodologies to engineer these organisms. Still, despite the great promises of synthetic biology, the genetic manipulation of filamentous fungi remains to be one of the major challenges for industrial applications. The difficulty of manipulating their genomes still hinders the use of metabolic engineering for filamentous species. However, synthetic biology approaches used for yeast are significantly contributing to the spread of methodologies and tools for engineering microbes to produce high value-added products at an achievable cost and to benefit equipoise. In general, synthetic biology approaches have presented successful examples of management and the acquisition of mutant strains that meet industrial demand. Finally, we anticipate that novel computational tools, especially for the investigation and design of regulatory elements, will play a pivotal role in future engineering attempts of these remarkable organisms.LM-S and RS-R conceived the work. LM-S, LN, AS-M, GL, MC, and RS-R assembled the first draft of the manuscript. LM-S and RS-R revised the final version of the work. All the authors have read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer EB and handling Editor declared their shared affiliation.