Entries in the database have both (i) structural evidence demonstrating the structural multiplicity or dynamic disorder of the ID region(s) in the partner bound form of the protein and (ii) in vitro or in vivo biological evidence that indicates the significance of the fuzzy region(s) in the formation, function or regulation of the assembly. Unlike the other intrinsically disordered or unfolded protein databases, FuzDB focuses on ID regions within a biological context, including higher-order assemblies and presents a detailed analysis of the structural and functional data. FuzDB also provides interpretation of experimental results to elucidate the molecular mechanisms by which fuzzy regions—classified on the basis of topology and mechanism—interfere with the structural ensembles and activity of protein assemblies. Regulatory sites generated by alternative splicing (AS) or post-translational modifications (PTMs) are also collected. By assembling all this information, FuzDB could be utilized to develop stochastic structure–function relationships for proteins and could contribute to the emergence of a new paradigm.FuzDB ( Intrinsically disordered proteins (IDPs) or protein regions (IDRs) are abundant in eukaryotic organisms and are known to serve versatile biological roles ,2. IDPs/Experimental characterization of structurally heterogeneous protein complexes faces various difficulties. Fuzzy regions tend to be sticky and often result in amorphous aggregates, presenting a bottleneck for purification and impairing crystallization. The solution spectra of fuzzy complexes resembles to that found in the unbound state, often masking the actual binding sites . As compIntrinsically disordered protein databases  aims to facilitate the identification and characterization of fuzzy assemblies. Second, by detailed analysis of experimental data, FuzDB relates structural heterogeneity to its biological consequences. It provides molecular insights into how fuzziness impacts the conformational ensemble and thereby its biological activity. A unique feature of FuzDB is to present the molecular scenarios and functional significance of structural multiplicity or dynamic disorder in an easily accessible format for scientists with a wide-range of expertise. In this manner, FuzDB contributes importantly to the development of a novel, stochastic structure–function paradigm , transcriptional, proteolytic or enzymatic assays as well as microscopic techniques. Sequence manipulations, such as site-directed mutagenesis, truncation or scrambling could also provide valuable functional information. Truncating fuzzy regions often results in gradual activity changes. Certain fuzzy complexes, such as yeast prions , acidic transcriptional activators  or histone tails [FC0027] tolerate a great degree of sequence modifications referred to as ‘sequence independent binding’, as long as their amino acid composition remains to be conserved.The fuzziness of a protein assembly is evaluated on the basis of two conditions, which both have to be fulfilled.Methods module of the database.All structural and functional techniques are listed in the Fuzzy protein complexes can be classified on the basis of topology and mechanism. The different categories have been extensively discussed elsewhere ,10–12; hflank (F) or link—clamp (C)—globular binding segments. In random (R) complexes, short linear motifs in the fuzzy region interact with the partner in a variable manner. Assignment of topology is based on structural analysis.Topological classes are defined based on the arrangement of the fuzzy region with reference to the globular regions or binding domains. There are two main categories: polymorphic (P), where the ID region folds into alternative structures upon binding to the same partner, and dynamic, where it interconverts between various different conformations in the complex. In dynamic complexes fuzzy regions can conformational selection) [FC0015 (flexibility modulation) [FC0047 (tethering) [FC0060 (competitive binding) [FC0052   if the structure is available. Inconsistencies in sequence and organism are sorted out. Chimera sequences are marked and stored. Domain information is added based on Pfam, functional regions are indicated based on Uniprot, protein interaction databases and the literature. Post-translational modifications (PTMs), isoforms and medical relevance are collected from Uniprot and the literature. References are linked to PubMed.Verifying the fuzziness of the complex and defining the fuzzy region by residues. Data in the key references are corroborated by the analysis of the structure/conformational ensemble and functional data. If needed, an additional literature survey is performed, and authors may be contacted again. If the data are still incomplete, the working entry is transferred to the Buffer database. Entries can be freely exchanged between the Working and the Buffer databases. Entries may also be excluded from the Working database in the case of conflicting data.Classification of the fuzzy region based on topology and mechanism.Detailed structural and functional analysis. Information is extracted from the references and is compiled in a concise manner. The goal is to highlight the type of structural information that is indicative of fuzziness and also its functional relevance in a very clear, possibly quantitative, way. Two descriptive sections are formulated and added to the working entry.Deriving structure-function relationships. If data are available, the mechanism by which the fuzzy region impacts the conformation ensemble and thereby the biological activity of the assembly is detailed. The significance of fuzziness is highlighted.Data are derived from two sources: either from a literature search or uploaded via the FuzDB website  Figure . FuzDB h) Figure .ComplemTable of Entries and in the corresponding Expanded Entry page, as will be detailed below.The completed working entry is transferred to the publicly accessible FuzDB. The minimum information needed for a public FuzDB entry is the following: the name of the protein and its partner, cross-reference to Uniprot, protein function, both structural and functional data supporting fuzziness, detection method, annotation and classification of the fuzzy region, significance of fuzziness and key reference(s). Additional data can be incorporated at later stage via the Working database. During the updating process, the original public FuzDB information remains to be displayed. The public FuzDB entry is presented in two parts, in the Home module introduces fuzziness and the criteria to define fuzziness based on the cited reviews. The Browse module displays the Table of Entries of 108 fuzzy complexes grouped into 10 complexes per page. This contains the core data, and each complex also has an associated Expanded Entry page, where the detailed analysis and derived information is presented. The Search module enables a simple text or advanced search across the database. Specific terms related to derived information, such as the mechanism category, can also be used. The Methods module collects the experimental techniques that have been applied to study fuzzy assemblies. Methods is linked to the Detection method column of the Table of Entries in the Browse module and can also appear as a pop-up window. The Classes module describes the topological and mechanistic categories of fuzzy complexes. This is linked to the Class column of the Table of Entries in the Browse module and can also appear as a pop-up window. The Upload module is for submission of new entries to the database. This module is highly protected, requiring a safety key to upload data, which can be obtained by email. The process of submission is described in detail step-by-step and the data will be validated (see above). The Analysis and Help modules provide information on how to use the resource. The Analysis module details examples on how to interpret data for individual systems and how to perform comparative analysis of fuzzy complexes. The Help module describes the modules as well as the columns in the Table of Entries and the fields in the Expanded Entry page. The FAQs module discusses questions related to conceptual issues and biological significance of fuzzy complexes.FuzDB has a modular architecture. The Browse module . All fuzzy complexes have an identifier in the format of FCxxxx, and the same protein can be involved in multiple entries. The protein name also includes domain specifications and abbreviations. The Uniprot identifier is defined for the ID protein and links to the sequence in fasta format. In the case of chimeras, the sequence is displayed in the Expanded Entry page. The organism of the protein is also reported in the Table of Entries. If the structure is available, a cross-reference to the Protein Data Bank and/or the Biological Magnetic Resonance Bank is provided. The Detection method(s) column lists the experimental techniques and is linked to the Methods module. Fuzzy regions are annotated by defining the start and terminating amino acids with reference to the Uniprot sequence; multiple regions are separated by colons. The topological classification of the fuzzy region is displayed in the Class column, which is linked to the Classes module. PubMed identifier(s) of the key reference(s) is/are given in the last column. Detailed information on each complex can be found in the corresponding Expanded Entry page by clicking either the FCxxxx identifier or the protein name.The core of FuzDB is the e Figure . Data inExpanded Entry page provides detailed insights into the experimental characterization of fuzzy complexes and higher-order assemblies, and can be used to develop structure-function relationships. If coordinates are available, the structure is displayed on the left side with an option to view the full, higher-resolution image .In the advanced search is recommended when looking for terms in specific sections, such as protein or partner name, organism, the PDB or BMRB code, Uniprot or PubMed ID. The user may also search for fuzzy complexes that have been determined by a given experimental technique, as listed in the Methods module. It is possible to use a combination of specific fields, such as topology and mechanism. For example, applying ‘flanking’ (topology) and ‘tethering’ (mechanism) filters will select those fuzzy regions that flank ordered binding sites and serve to anchor them to their partners [FC0064  and either the structural or the biochemical evidence, although for a new FuzDB entry both types of data will be required. The user is advised to provide a reference, such as a PubMed or a structure identifier. Specifying the sequence of the protein is recommended, but is not obligatory. An automatic email is generated upon submission to notify the FuzDB team. In most cases, the authors are contacted for missing or ambiguous information and an extensive literature search is performed to complement data (see Data Processing). Obviously, email inquiries regarding fuzzy complex candidates are also considered. The contact name and email of the submitter is displayed in the bottom of the Expanded Entry page.Information submitted by users is a valuable source to expand the database. At present only new entries can be submitted; additional information to existing entries should be sent by email and not via the website. The submission process is described step-by-step in the front page of the advanced search of the Search module. Fuzzy complexes, which have been studied by the given experimental approach, will be selected and their corresponding Expanded Entry pages detail the structural evidence indicating dynamic disorder or polymorphism in the bound state. For functional analysis, proteins with related activities can be selected in the simple text search of the Search module, and their corresponding Expanded Entry pages discuss the in vitro or in vivo biochemical data supporting fuzziness. Once both criteria are fulfilled and the fuzziness of the assembly is confirmed, it is advisable to classify the complex based on topology. Then the topology class filter in the advanced search of the Search module can be applied to relate the complex to other assemblies of the same class.FuzDB can be used to perform comparative analysis or to interpret experimental data for the system of interest, as exemplified in the Analysis module. In the case of a fuzzy complex candidate, a technique-specific filter can be applied in the Search module on data either in the Table of Entries or the Expanded Entry page, by organism, partner, function, topology or mechanism. Individual or combined categories can be used, e.g. comparing different topology classes in the same organism or analyzing mechanisms derived from the same experimental approach. The data and cross-links can be subjected to bioinformatics analysis using more advanced computational tools, e.g. to quantify the propensity of fuzzy region(s) within a protein or to compare the lengths of fuzzy regions within an organism.A comparative analysis can be performed via the Expanded Entry pages. These can be related to the observations on the system of interest, or can be related to other assemblies, either from the same organism, function or topological class, or those studied using the same experimental approach.One of the goals of FuzDB is to provide not only data, but also derived information on the relationships between structural ensembles and biological activities. The molecular scenarios by which fuzzy regions affect conformational ensembles and influence functions are discussed in the post-translational modifications and the isoforms, context-dependence sections of the Expanded Entry page. Context-dependence refers to environment-dependent functional features of the different isoform, which are modulated by the PTM or AS of the fuzzy region. This information can complement and be combined with the molecular mechanism that is described in the Expanded Entry page, and can help to interpret the structural and functional properties of the system of interest under different conditions.Regulation of protein assemblies often involves fuzzy regions ,13 that Structural biology is engraved in a deterministic relationship between protein sequence, structure and function. Proteins without a well-defined three dimensional conformation in their native state contradict this paradigm and transform our thinking on the structural pre-requisites of biological functions. Upon interacting with their partners, however, ID proteins were assumed to adopt a folded state and still follow the classic principles. In contrast, the concept of fuzzy complexes establishes a direct relationship between the structural heterogeneity and biological function of proteins within their physiological context. Conformational freedom is not only preserved in fuzzy assemblies, but also contributes to their interactions, activities or regulation.FuzDB collects fuzzy assemblies of proteins and provides annotations for fuzzy protein regions. It contains proteins with a wide variety of functions, ranging from gene expression regulation, signaling, endocytosis, aggregation, chaperoning, cytoskeleton organization, as well as enzymes , which mAll entries in FuzDB are based on experimental data. An ongoing effort is to develop a computational approach to predict fuzzy regions that tend to remain unfolded even when bound. This will be an efficient engine to identify further candidates, which will be validated on the basis of experimental results. Preliminary studies indicate ∼5000 long fuzzy regions (>30 AA) in the human proteome . The prediction algorithm for fuzzy regions will be incorporated in a web-based server and will be linked to FuzDB. Predicting fuzzy regions facilitates pre-screening of new candidates and promotes analysis of biologically important complexes.In summary, FuzDB facilitates the identification of fuzzy protein assemblies and establishes a direct connection between the conformational ensembles and functions of proteins. This is the basis of a new, stochastic paradigm, leading to a more holistic view on how proteins fulfill their biological roles.


Chagas cardiomyopathy (CCM) is traditionally considered a disease restricted to areas of endemicity. However, an estimated 300,000 people living in the United States today have CCM, of which its majority is undiagnosed. We present a case of CCM acquired in an endemic area and detected in its early stage. A 42-year-old El Salvadoran woman presented with recurrent chest pain and syncopal episodes. Significant family history includes a sister in El Salvador who also began suffering similar episodes. Physical exam and ancillary studies were only remarkable for sinus bradycardia. The patient was diagnosed with symptomatic sinus bradycardia and a pacemaker was placed. During her hospital course, Chagas serology was ordered given the epidemiological context from which she came. With no other identifiable cause, CCM was the suspected etiology. This case highlights the underrecognized presence of Chagas in the United States and the economic and public health importance of its consideration in the etiological differential diagnosis of electrocardiographic changes among Latin American immigrants. While the United States is not considered an endemic area for Chagas disease, the influx of Latin American immigrants has created a new challenge to identify at-risk populations, diagnose suspected cases, and provide adequate treatment for this disease.  Trypanosoma cruzi [Chagas cardiomyopathy is a vector-borne parasitic infection caused by the protozoama cruzi . With thma cruzi , 3. The ma cruzi . T. cruzi positive patients had a significantly higher prevalence of arrhythmias, severe heart failure, and mortality with respect to their seronegative peers [The infection is most commonly transmitted by insects of the Reduviidae family at blood meal bite sites but may also be transmitted vertically from mother to child, through blood transfusions, organ donation , and aneve peers . Life exve peers .While Chagas has traditionally been considered a disease restricted to areas of endemicity, the ever-increasing mobility of populations across regions and borders has changed disease distribution dynamics . There aIn this paper, we present a case of Chagas cardiomyopathy acquired in an endemic area, detected in its early stages of chronic manifestation and before the classic late-stage morphologic changes to the myocardium. This case is representative of the underappreciated prevalence of the disease and its consequences in an area with a large immigrant population of Latin American origin.A 42-year-old El Salvadoran woman presented to the ED after a syncopal episode. The patient's symptoms began with left sided chest pain radiating to her left arm followed by dizziness. She had loss of consciousness for 5 minutes and had suffered multiple syncopal episodes with increasing frequency over the past year. She was advised to see a cardiologist for slow heart rate but did not follow up. She denied palpitations, vision or hearing changes, or seizure activity.The patient grew up in rural El Salvador and immigrated to the United States 10 years ago. Of note, her sister of 40 years of age, who currently lives in El Salvador, also recently began suffering similar episodes over the past 2 years.On examination, her blood pressure was 105/66 mmHg and was found to be bradycardic; her heart rate fluctuated between 30 and 42 beats per minute. The patient's lungs were clear. There was no lymphadenopathy or peripheral edema. Investigations showed negative serial troponin levels and normal hematologic and blood chemistry tests. The patient's electrocardiogram was remarkable for sinus bradycardia, with conserved axis and segment intervals and no other conduction abnormalities . Chest XDuring her initial hospital course, a first Chagas serology test was ordered given the fact that the patient had no other identifiable causes or risk factors for cardiomyopathy beyond the epidemiological context from which she came. This result came back positive after discharge, and the patient was followed up as an outpatient for a confirmatory second serology, which was also positive. Without any other clear etiology, it is suspected that Chagas was the cause of the cardiomyopathy.It was agreed upon by the medical team that, given the seropositive status of the patient with only electrocardiographic evidence of disease and no appreciable cardiomegaly or congestive changes, antiparasitic treatment should be offered. This issue was discussed at length in follow-up visits with the patient. While she initially expressed interest in receiving pharmacological treatment, she has been persistently hesitant to commence therapy. This remains an active discussion with the patient.This case highlights several key points as a result of the underrecognized prevalence of Chagas in the United States and the importance of considering Chagas in the etiological differential diagnosis of electrocardiographic changes among Latin American immigrants. Two clear issues that must be addressed from the clinical and public healthcare stewardship standpoint are the fact that the patient has 4 children who could potentially be seropositive and in the indeterminate phase and must therefore be screened and the fact that the patient has no macroevidence of CCM, and the question remains of whether or not to offer her treatment that could change the course of disease.One key aspect of this case is recognizing the heterogeneity of the endemic nature of Chagas. In the case of El Salvador, the country of origin of this patient, the countrywide prevalence is 3.37% , third iAdditionally, there is an inherent obligation from the part of the medical profession in the United States to maintain an index of suspicion for Chagas, as it is frequently underappreciated by the immigrant population as well, as was observed in a study in Latin American immigrants living in Los Angeles . T. cruzi, are suspected to play an important part in the more commonly recognized morphologic changes of the myocardium resulting in dilated cardiomyopathy and congestive heart failure [Chagas disease follows a biphasic progression in which the acute phase typically culminates in a 4- to 8-week mild febrile illness and skin welt at the site of infection. This gives way to an indeterminate phase, of which 20–30% of infected persons will evolve to become symptomatic in 15–25 years with CCM . The earWhile there is clear indication that treatment of Chagas disease in the acute phase has proven efficacy , there iThe only large scale trial to date showed that while treatment in patients with established CCM reduced serum parasite detection, a reduction in cardiac clinical deterioration was not appreciated over the following 5 years of follow-up . HoweverIn consideration of the side-effect profile of antitrypanosomal treatment, a Cochrane review found that severe side effects occurred in 2.4% of patients to benznidazole, with mild skin reactions, severe GI intolerance, lymphadenopathy, neuritis, and fever, while nifurtimox carried a 6.7% incidence and included neuritis and hepatitis . TherefoRegarding prevention of sudden cardiac death in patients with CCM presenting with symptomatic bradyarrhythmias, placement of a pacemaker, as was done in this patient, follows best practices guidelines established in areas in which Chagas is endemic . In the Vertical transmission of Chagas disease from a seropositive mother to offspring carries a risk of 5% , 29. As An economic analysis of the cost-effectiveness of screening versus no screening done in Spain, a nonendemic area with Latin American immigrants like the United States, showed that antenatal screening of Latin American mothers, their offspring in positive cases, and consequent treatment resulted to be more cost-effective than not screening when projecting the long-term healthcare costs a seropositive person would place upon the system . This isWhile the United States is not considered an endemic area for Chagas disease, the significant influx of immigrant populations from Latin America has created a new challenge to identify susceptible populations, diagnose suspected cases, and provide adequate treatment for this disease. The opportunity to carry out appropriate population based screening and offer secondary and tertiary prevention of disease progression is emblemized by this case and is an area that offers the opportunity to improve population-directed patient care and cost-effectiveness of healthcare delivery.Although there is inconclusive evidence regarding the etiologic treatment of the indeterminate form of Chagas disease, small studies have reported a decrease in disease advancement after treatment. This is an area that should continue to be investigated in the future. With respect to prevention of sudden cardiac death, current guidelines for pacemaker placement prove to be beneficial.Finally, there is substantial evidence to suggest that the realization of screening to suspected cases, especially during the indeterminate form, as in the children of the index patient in this case, would lead to a substantial savings for the healthcare system.


The operational management of healthcare services is expected to directly touch patient experiences. Iranian Ministry of Health and Medical Education (MoHME) for the first time, as such, has sought to improve the operational management of healthcare delivery within a reform agenda by setting benchmarks for ‘number of visit per hour’ and waiting time in outpatient clinics of about 700 affiliated hospitals. As a new initiative, it has faced with mixed reactions and various doubts have been cast on its successful implementation. This manuscript aims to shed some light on the operational challenges of the initiative and the requirements of its successful implementation. In this regard, patient planning and scheduling tools and techniques have been used to facilitate the smooth flow of patients through health settings; to reduce waiting time; to minimize the idle time of healthcare resources, and ultimately to boost the efficient use of expensive personnel and medical equipment.3A key concerning issue commonplace in most developing countries, as highlighted by the World Health Organization (WHO), is not just the small share of their gross domestic product (GDP) pouring into their health sector, but, most importantly, is the poor operational management linked with this spending.5 Long waits and delays have been approached by the most recent initiative, Health Sector Transformation Plan (HTP) which strives to improve the operational management of health service delivery in Iran. Instruction 4, out of HTP’s eight policy instructions, seeks to specifically promote the service quality of visits in terms of both waiting and consultation time in clinics and other outpatient settings.6 It rightfully addresses the long waits and delays in healthcare settings; however, since launched it has been the subject of various scrutiny7 both in regard to the quality of an evidence-basis upon which the instruction is built and to the inadequate considerations to implementation issues. In this manuscript we intend to review the instruction from a HOM perspective and to discuss the implementation issues related to this instruction.As a less expensive alternative, the speciality care at outpatient clinics has become an essential structure for service delivery in Iran. However, the quality of services at such clinics has been criticized by professionals, researchers, and laymen due to the long waits, delays, and the short consultation time in those settings.status quo of patient planning in most public healthcare settings of the country including outpatient clinics has been rather chaotic. Such chaos has always existed with the public healthcare settings given the high load of their patients. It has even exacerbated with the introduction of the HTP which has made healthcare services more accessible and affordable than ever. Despite this, there exist extremely few appointment scheduling systems in operation, thus patients have to usually wait for several hours in the clinics or outpatient departments. In most cases, with very few exceptions, the providers admit their patients on a walk-in basis requiring the patients to attend in the service centres in an ad-hoc and random way. At most, the clinics may occasionally set the day of the visit ahead of time. However, neither does the patient know the time of visit, nor has s/he an estimate of the expected waiting time. In busier clinics, patients may wait for several hours. Consequently, the clinics become extremely overcrowded and medical specialists undergo a tremendous pressure to see a larger number of patients within a specific time range.8 This is expected to make patients extremely frustrated and ultimately dissatisfied with the services.The 9 Prior to the HTP, in its very first initiative, MoHME had once embarked on total quality management (TQM) tools to institutionalize the culture of quality and to support the providers to become effective in addressing all types of quality deficits such as waiting time.11 This initiative was of very limited achievements in Iranian healthcare organizations, mainly because of the inadequate attentions paid to its implementation eg, in planning and management commitment to the initiative.12Iranian Ministry of Health and Medical Education (MoHME) has sought to somehow improve the operations management of health services in the country.13 The HTP is the most recent initiative to improve operational management of health services delivery in Iran, which has started to be effective in about 700 hospitals affiliated with the MoHME in the whole country since May 2014. It was originally initiated due to the highly increased out-of-pocket, around 57% at its highest level in 2013, and catastrophic payments.14 As a multi-purpose plan addressing quality, fair financial contribution by all individuals, equity and access, the initiative is of a comprehensive perspective covering a wide range of activities such as health, treatment and medical education. Policy instruction 4 of the HTP specifically seeks to promote the service quality of visits in clinics and other ambulatory care settings.6 A part of instruction 4, among other things, sets a benchmark and requires all public hospitals to constrain the number of visits per hour. At the first year of the program, 8 visits per hour of a specialist was planned (except for psychiatrists that should see 6 patients only), which might be reduced in the next year. By this instruction the decision makers in the MoHME were keen to close a yawning gap between consumer expectations and perceptions of service quality and to provide services on a par with international standards and thus to, in a way, improve the responsiveness.15 However, after 2 years since the instruction has been introduced, waiting time in the specialized outpatient clinics is still markedly high, fluctuating between 2 to 4 hours,16 which is way above acceptable benchmarks. This signals that the instruction 4 has not been effectively implemented to reduce waiting time in the outpatient clinics. Despite this, consultation time in the specialized outpatient clinics has slightly increased by the instruction 4.16Long waiting and short consultation time have never been addressed in a reform agenda as specific as in the HTP. This was designed in line with the fifth 5-year health development national strategies (2011-2016) and mainly with the new President’s manifest in order to achieve the universal health coverage, within which a large amount of money was injected into the health sector early 2014.17 the instruction 4 de facto seems to be challenging and less likely to be easily implemented. A set of prerequisites are expected to determine the degree to which the instruction is successfully implemented. These include ‘quality of evidence underpinning the instruction,’ ‘capacity of hospitals’ including qualifications of people involved in the implementation, ‘the process of implementation,’ and ‘rules and regulations’ surrounding the instruction.From the Implementation Science perspective17 Though this instruction was suggested at the right time, it looks fairly simplistic and builds on a rather weak form of evidence. Adaptability to the local conditions is neither a small concern. This instruction only sets a very general, ‘one-size-fits-all benchmark’ for all various specialities and patients, while not every patient is similar in terms of their demographic conditions, nor alike in terms of the severity of their illness. The least expectation is to develop speciality-specific benchmarks. Furthermore, the demand for services usually fluctuates daily, weekly, and seasonally. The teaching tasks of the university hospitals could also add to this complexity. Lastly, the cost of instruction deployment in terms of supply and equipment required is also unclear.Instruction 4, whilst being an overly effective step towards quality improvement, is fairly debatable with respect to its evidence base supporting it. The strength and quality of evidence, stakeholders’ perception of the relative value of the instruction, the adaptability of the instruction to the local needs and its operational costs are questioned.19 These issues if not addressed thoroughly may pose serious challenges in moving toward the use of patient planning systems. The current planning systems are by and large computer-based in the mainstream of scheduling systems.The compliance with the instruction is incentivised by payments; however, the implementation guide of the instruction must consider several factors related to the internal capacity of hospitals implementing the instruction. In other words, the use of planning systems as an effective tool for improving patient throughput are closely linked to the operational and implementation issues inherent to planning systems. These issues are availability of computerized scheduling systems, perception of professionals regarding patient planning, facility layout, and context of clinics eg, culture of quality.20This instruction motivates the hospitals to apply appointment scheduling systems to accomplish the benchmark set. The operational guide of this instruction emphasizes that the hospitals must design appropriate computer-based patient planning and scheduling systems for their clinics and outpatient settings. Currently, few hospitals wherein the HTP are implemented make use of computer-based scheduling systems. Much fewer have even designated a call centre or designed a website to allow patients make an appointment. Considering the first phase of HTP which involved 584 clinics enrolling 9500 medical specialists from different disciplines, the amount of work to be done could be guessed. On the other hand, the use of scheduling in patient planning requires a technical and management capability to understand the demand fluctuation and resource allocation.21 Furthermore, such a system should access to information on the available resources in terms of rooms, equipment, specialists, and support staff to meet the demand, whilst there is an uncertainty in the availability of resources, which makes patient planning very difficult. As such, scheduling systems take a great deal of efforts and a long time to develop. Although the knowledge and expertise of scheduling is available, its application in Iranian health settings is rather limited. To the best of our knowledge there are few studies demonstrating the application of scheduling in Iran.8A workable scheduling system needs information on the extent and characteristics of demand; that is, on the type of patients and their socio-economic status and its fluctuation. However, the demand information is either non-existent or is dispersed.22 Implementation needs a plan describing its processes.12 It encompasses steps-by-step phases to plan, assimilate and engage people, act and evaluate results.17 It is important to engage medical specialists as this, on the one hand, might improve the quality of their clinical services and, on the other, they may perceive patient planning systems as a burden to their work planning, hindering their successful implementation. Overall, the execution can succeed provided that the above prerequisites are in place to support its process.The benchmark or any other target for visit time either general or disease-specific can hardly be operationalized by a top-down hierarchical order only.External factors such as rules and regulations that surround target organizations of the instruction could influence the implementation process. Since the most target hospitals are public entities they are subject to the state rules and regulations. Spending money for a patient planning system mainly seems a non-core service and thus is constrained by the initiatives intended to cut costs of support services. Therefore, outpatient clinics managers have been prohibited to spend more resources including staff to run and maintain the appointment planning systems.19So far it has been debated that instruction 4 has not been effectively implemented due to several hindering factors, particularly a contentious evidence basis and overlooked technical aspects of appointment systems. Nevertheless, no evidence-based standards of acceptable waiting time for outpatient visits either routine primary or speciality care is existing. Even in the United States, two accepted benchmarks; ie, ‘third next available’ (TNA) appointment and ‘office visit cycle time’ have resulted in controversial results.22 Most Western European countries such as the United Kingdom and the Netherlands have to a large extent resolved long waits and delays using operational models.24 Even in China, Singapore, and India this problem has been partially solved.25 We provide a generic operational model that could be used to adjust the appointment schedules to specific conditions of clinics. This model first considers the context of outpatient care and then proposes the design of a replicable appointment system.Because of the argumentative evidences on the performance of benchmarks we fall short of being able to recommend feasible benchmarks for number of visit per hour and patient waiting time for outpatient clinics in Iran. Instead we recommend outpatient clinics to use health operational models to tailor benchmarks to the conditions of a clinic. Operational models, rooted in the HOM, provide a basis for analysis and building solutions for scheduling problems. These tools are built on simulation, queuing theory, value stream mapping, and so forth.16 There is also still some inclination mostly with these people because of their experience of the current system, thus they tend not to trust the internet-based and tele-scheduling systems and prefer attending physically in the outpatient settings hours before their due time. Furthermore, the cross-flow of patients between specialties is important given a high prevalence of chronic patients who are in need of different specialties to accomplish a diagnosis or a treatment path.26 As demand fluctuates, resource allocation should be also flexible.20 Conversely, the resource allocation in Iranian outpatient clinics is currently based on group allocation and fixed rather than to be responsive to the actual demands. As a result, while demands fluctuate daily, weekly, and seasonally, the resources capacities remain constant, which in turn could lead to overcrowding in peak demand and idle resources in low demand. These challenges should be taken into consideration in the design of an appointment system.The context of care in Iranian outpatient clinics is perplexing and poses daunting challenges; including the shortage of qualified specialists in remote areas, a high fluctuation of demands and consequently the large variability in the length of appointments and out-of-town patients.27A priority step in designing an effective appointment system is to meet some basic preconditions. Iranian health providers have to learn to consider patient and family as a primary factor in designing an appointment system. This will result in an appropriate set of choices, attention to patient preference, and shorter waiting times. The providers also need to develop different solutions to accommodate demand fluctuations caused by out of town patients and cross-flow between specialities. Furthermore, they need to redesign and simplify care models with fewer steps, standard processes, pre-visit tasks by non-physician staff, and internet-based visits.20 A daily scheduling improves capacity utilization by letting providers to fit appointment of varying length in a daily schedule, by which providers can take care of deviations from planned clinic time and scheduled appointments. It facilitates servicing walk-in, out-of-town, and cross-flow patients.Some studies prescribe advance booking coupled with daily scheduling to accommodate current demand characteristics.28 An appointment rule is a basic decision which determines the number of patients in each block and length of interval between appointments. Appointment rules determine the amount of reserved capacity for types of demand ie, appointments and for unanticipated urgent calls. Appointment rules also reserve capacity for patients who live out-of-town eg, rural areas who might attend in a clinic in an ad hoc manner. Furthermore, appointment rules can accommodate the cross-flow of patients between specialties.29 However, no single appointment rule creates desired outcomes in all clinics, thus appointment rule should be adjusted to specific condition of each clinic. Cayirli et al propose a procedure to tailor rules for the clinics with different environments characterized by the probability of no-show, variability of service time, number of patients per session, and the relative value of doctor’s time to patient’s time. This procedure as claimed by Cayirli et al has proved more effective in minimizing adverse effects of no-shows and walk-ins than traditional appointment rules in literature .28 By providing adjustment procedures and an open-source decision-support tool this appointment rule can be used to develop appointment schedules for clinics.Well-designed scheduling system involves three types of decisions: appointment rule, patient classification, and adjust for no-show and walk-in.The initiative to tackle waiting time is highly expected to improve the responsiveness of Iranian health system. However, operationalizing this policy to minimize waits with the current capabilities of public healthcare settings seems to be not an easy job. We encourage decision-makers in MoHME to eradicate barriers hindering the successful implementation of the HTP’s instruction 4. We furthermore advocate the use of modern tools and techniques to measure and enhance the patient planning in outpatient clinics. In this vein, we encourage operations management researchers to address patient planning issues and employ the demonstration projects showing the value and use of appointment systems in better quality of services.We are grateful to Professor Jan Vissers for sharing his insights and comments on the earlier versions of the manuscript.Not applicable.Authors declare that they have no competing interests.MM had the first idea that was later developed into the current form jointly by EJP. Both MP and SG provided their constructive comments on the final version.1Tehran University of Medical Science, Tehran, Iran. 2Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Science, Tehran, Iran. 3Department of Health Management & Economics, School of Public Health, Tehran University of Medical Science, Tehran, Iran. 4Social Security Research Institute, Tehran, Iran.


Green Toxicology refers to the application of predictive toxicology in the sustainable development and production of new less harmful materials and chemicals, subsequently reducing waste and exposure. Built upon the foundation of “Green Chemistry” and “Green Engineering”, “Green Toxicology” aims to shape future manufacturing processes and safe synthesis of chemicals in terms of environmental and human health impacts. Being an integral part of Green Chemistry, the principles of Green Toxicology amplify the role of health-related aspects for the benefit of consumers and the environment, in addition to being economical for manufacturing companies. Due to the costly development and preparation of new materials and chemicals for market entry, it is no longer practical to ignore the safety and environmental status of new products during product development stages. However, this is only possible if toxicologists and chemists work together early on in the development of materials and chemicals to utilize safe design strategies and innovative in vitro and in silico tools. This paper discusses some of the most relevant aspects, advances and limitations of the emergence of Green Toxicology from the perspective of different industry and research groups. The integration of new testing methods and strategies in product development, testing and regulation stages are presented with examples of the application of in silico, omics and in vitro methods. Other tools for Green Toxicology, including the reduction of animal testing, alternative test methods, and read-across approaches are also discussed. Over the past two decades, the movement of Green Chemistry has become a new standard embraced for the development of less harmful materials and chemicals that are safer for both the environment and consumers , 2. The ®; , the use and applicability of which may lead to more successful Predictive Toxicology. QSARs are a predictive tool that provide insight into the potential toxicity of chemicals based on patterns of structure–function relationships from similar chemicals whose activities have already been assessed. Successful use of QSARs requires a sufficient amount of input data in order to support structure–toxicity relationships for risk assessment. A number of computerized models have been developed and improved to accommodate large datasets from high-throughput screening efforts . AsAdditional challenges for the use of in silico methods are that toxicological studies, to a large extent, are done by the industrial sector, and thus are used for registration as proprietary data and are not typically published or publicly available. This is especially true for the high-quality, standardized test data produced according to international test guidelines under quality assurance schemes such as Good Laboratory Practice (GLP). There was a pivotal change in the sharing and availability of such data, when for the first time, the European REACH legislation mandated that dossier summaries be published, making data readily available on a new scale. While this, together with the enormous testing demands of REACH , 28, stiFurthermore, the grouping of substances and read-across approaches were developed for filling data gaps in registrations of chemicals. Read-across approaches utilize weight-of-evidence approaches to make use of shared properties of an untested compound to a known compound. While sharing many characteristics of a QSAR, read-across approaches do not seek a mathematical formula for larger parts of the chemical universe but instead are based on “local” similarity and shared properties of chemicals , 37. In In the initial stages of chemical development, the identification of the sequential processes and perturbations of biological pathways at a molecular level  through to the cellular or organ level leading to an adverse outcome provides insight into the SAR and can allow for the grouping of similar mechanisms of biological response . These a50 values in enzyme or receptor assays) could be used to develop predictive in silico tools. With the availability of such computational methods, chemists can test and screen their new structures for warnings, and learn to avoid the synthesis of toxic compounds at earlier stages of development. As such, the development of in vitro methods complement and may eventually reduce the essential use of in vivo studies, which directly supports the principles of Green Toxicology. Thus, with the addition of any new in vitro method for (eco)toxicity testing we will get one step closer towards an integrated process of gaining early toxicological information and adapting substance synthesis, leading to the efficient development of green chemicals and products with reduce toxicity.AOPs and PoTs will have major implications for the advancement of predictive toxicology, an important tool which would improve Green Toxicology approaches in developing and designing safer chemicals. An understanding of the molecular structure, functionality and adverse outcomes associated with chemicals is essential in aiding in the benign-by-design concept. In vitro assays are typically rapid, with high-throughput and large data output that can be reliably reproducible and cost effective. These qualities are particularly important for the safety and hazard assessment of new products, which can result in substantial costs for failures and problems detected late in the development and regulatory testing phase. In addition, many in vitro methods produce mechanism-specific data, which is another important aspect that can aid with the design of alternative compounds with lesser toxicity. With newer and more innovative in vitro tests, the focus moves more towards chronic exposures at low concentrations on cells and organ systems rather than the traditional adverse effects observed in in vivo animal tests that are often conducted at high doses. Furthermore, the results of in vitro testing . Regulatory rationales were also an additional driver of alternative test methods, particularly those that identified compounds with alerts for “cut-off” hazards, such as mutagenicity and endocrine disruption. Therefore, the currently validated in vitro assays particularly apply to the aforementioned endpoints , 46.In the following paragraphs, a brief overview is provided concerning the 3Rs method currently employed at BASF, which are used within a screening context to avoid the development of compounds with an unfavourable hazard profile (i.e. concept of Green Toxicology).epidermis (RhE) that closely mimics the biochemical and physiological properties of the upper parts of the human skin using human derived non-transformed keratinocytes as cell sources. The indication of corrosive and irritant test substances is determined by their ability to decease cell viability (cytotoxicity) below defined threshold levels as measured via the MTT-assay.The traditional in vivo Draize irritation test for skin and eyes, in which a restrained, conscious animal is exposed  to a test substance for a set amount of time to determine toxicological effects, has long since been criticized for the limitations in species differences, subjective scoring, and experimental variability. The replacement of the Draize test for skin irritation was historically one of the first steps towards the full replacement of animal testing. BASF, and similar chemical companies, use two methods suitable to provide data for classification as corrosive (Epiderm™ skin corrosion test) or irritant (Epiderm™ skin irritation test) to the skin. These tests are employed within the context of a simple testing strategy described elsewhere –50. In bFor eye irritation, the situation is essentially quite similar. The replacement of the Draize test for eye irritation again was achieved by two methods. The ex vivo Bovine Corneal Opacity and Permeability BCOP; , 52) eye eye52]) Skin sensitization is a process more complex than skin or eye irritation, and includes several key events such as (1) dermal penetration, (2) protein reactivity, (3) inducing stress responses in keratinocytes, (4) activation of immune cells (dendritic cells) in the skin, and (5) their translocation to the lymph nodes. Given this complexity, it is difficult to imagine one single test that would be able to incorporate all of these steps . TherefoThe endpoint of systemic toxicity has not been of major interest to chemical companies and regulatory bodies. With good intention, it was proposed to use the results of cytotoxicity testing to determine the starting dose for acute oral toxicity testing. However, it should be noted here that good intention is not always a good guidance. With years of experience following this principle, a post validation study demonstrated that dose selection based on expert knowledge provided better results than following the cytotoxicity guidance, which was not significantly better than a fixed starting dose of 300 mg/kg b.w. . Such exTo screen for compounds with endocrine effects, two in vitro systems are often used that address the most common causes for endocrine activity: (1) agonist or antagonist effects on the androgen receptor (AR) or estrogen receptor (ER) and (2) interference with steroid synthesis. There are a variety of in vitro, wildlife and mammalian screen tests available to screen for endocrine disruptor activity, with details on each provided elsewhere . In partAnother important aspect of systemic toxicity, with respect to avoidance of chemicals, with a problematic hazard profile is neurotoxicity. For screening purposes, the “neurons on a chip” assay is utilized , 73. In The last, and possibly the most important endpoint in toxicology, which has been investigated in screening strategies, is the toxic effects on development. It should be noted here that relatively little is unfortunately known about the modes of action involved in developmental toxicity, in comparison with many endpoints in systemic toxicity such as neurotoxicity, endocrine effects or carcinogenicity. Therefore, mode of action-based screens are not readily available. Two more holistic approaches are used to assess developmental toxicity: (1) the chick embryotoxicity screening test (CHEST)  and (2) With new omics technologies becoming more readily available, we are now at a point where there is a chance to tackle complex toxicological concerns, such as systemic toxicity. Following the successful development of a metabolomics based approach to predict systemic toxicity from a single drop of blood from short-term toxicity studies , the potFinally, with the availability of sufficient data on, for example, compound–receptor interactions, it is possible to create mathematical models which can assign a likelihood that a particular chemical structure will interact with a biological target. The process of developing such models is not necessarily fast and depends very much on the quality of the input data. However, the availability of such models will help chemists to design new compounds that perform the desired task and have a higher likelihood of a favourable toxicological profile. Safety-by-design will result in a win–win situation, with less animal testing and intrinsically safer products.In contrast to household and consumer chemicals, where the optimization process of the properties during product development is often independent of the safety assessment, the drug development process can be seen as a series of iterative steps to optimize efficacy and simultaneously lower the safety as early as possible. Therefore, the early assessment of toxicity before the first application to man  plays a pivotal role in this process. Compounds for which the preclinical toxicological assessment identifies an adverse effect profile that exceeds the expected benefit for the patient will be excluded from progression in the development pipeline. Preclinical toxicology is hereby facing two challenges: on the one hand, the predictivity of the applied toxicological assays should be improved on a continuous basis to avoid false predictions , while on the other hand, the predictions should be made as early as possible during the process of drug candidate selection. This early assessment causes a shift from in vivo to in vitro to in silico methods. Maertens et al.  possible carcinogenic to humans (Group 2B) based on sufficient evidence in experimental animals and inadequate evidence from epidemiological studies” [2-particles and the predicted outcomes. Comparisons of the materials used for the critical animal studies with that produced for the sunscreens should allow for the determination of the similarities in the materials and if the benign-by-design principle should be considered more thoroughly for future development of sunscreens.Cosmetics, especially sunscreens, should protect us from ultraviolet (UV)-light induced sunburn and skin cancer. This protection has been achieved by a multitude of chemicals with different structures, some of which are under suspicion of being endocrine disruptors or of having other effects in environmental organism in receiving aquatic environments. Over the last two decades, nanoparticles consisting of ZnO or TiOd rivers , the usestudies” . This exAnother critical nanomaterial is carbon nanotubes (CNTs), a lightweight but very strong material with a multitude of different possible applications in its various modifications . This material is described to have a strong similarity to asbestos with respect to the adverse health effects caused via inhalation. The properties of CNTs, such as the biopersistent, long fibre-like structure, and induction of oxidative stress, lead to the same biological consequences in lung tissue as asbestos, and thus, the use of CNTs is still under debate regarding the carcinogenic effect. However, following the important principle “benign-by-design”, recent studies have demonstrated that CNTs are biodegradable under specific circumstances, which would dramatically reduce their carcinogenic potency , 102. InAs mentioned above, more and more products that consist of or contain nanomaterials will soon enter the market or are already in use. An adequate risk assessment of environment and health is seemingly not possible because of the tremendous need for biological experiments, animal testing and laboratory capacity. The usefulness and applicability of in vitro methods must be demonstrated on a case-by-case basis, but they represent enabling technologies to address these demands , 105. ThIn addition to hazard-oriented key events, properties such as stability or bioaccumulation can serve as further building blocks for testing strategies . Bio-perThe cases shown above for chemical and pharmaceutical companies, as well as nanotechnology development, clearly demonstrate that Green Chemistry, together with the principles of Green Toxicology more specifically related to the environmental and health effects of compounds or materials, may achieve a sustainable and safe production scenario of new chemicals. However, in the case of pharmaceutical compounds, there may also be limitations with regard to achieving safe and efficacious drugs that are at the same time environmentally friendly. As the examples from the European Environmental Agency (EEA) demonstrate , it is nLast but not least, such measures are not free of charge, hence, all stakeholders should be convinced to use these principles and the consumer has to accept the higher costs for such products. As a very important step in producing chemicals and materials for future applications regarding environmental- and health-safety issues, the goals of Green Toxicology have to be accepted by all societal groups.


We read with interest the recent study by Jones et al.  who founP < 0.05) less than the improvement observed with deceptive feedback. Hence, we propose the presence of an avatar alone is only partially responsible for the magnitude of the performance improvement observed in our study.First, the final trial of our study followed a deceptive feedback trial, and it is possible the inclusion of an avatar on the computer screen contributed in part to the enhanced performance observed in this trial by an implicit competition effect. The presence of an avatar could be perceived as either competition or support . To date, studies investigating the performance influence of a second athlete have been equivocal . Flyger,  did not  Flyger,  and 4 km Flyger,  TTs. TheFinally, overall performance time or mean power output does not imply the use of identical pacing strategies across trials. Indeed, subjects can achieve identical mean power outputs despite purposefully different pacing strategies, which can impact fatigue development and performance  of a 4 km time trial (Stone et al., In summary, differentiating between the gain in performance from competition alone, and the additive gain from deception is important. Differences in cycling speed, pacing strategy, and time trial distance can each influence energy reserves, the magnitude of the “end-spurt,” and overall performance, ultimately determining an individual's response to deception. The variable of deception remains a useful tool in pacing research and it may ultimately become a viable training tool for improving cycling performance.R-JS, EA, JR, KT, and TM all contributed substantially to the conception or design of the work, the drafting the work and revising it critically for important intellectual content, and the final approval of the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Camino Verde (the Green Way) is an evidence-based community mobilisation tool for prevention of dengue and other mosquito-borne viral diseases. Its effectiveness was demonstrated in a cluster-randomised controlled trial conducted in 2010–2013 in Nicaragua and Mexico. The common approach that brought functional consistency to the Camino Verde intervention in both Mexico and Nicaragua is Socialisation of Evidence for Participatory Action (SEPA).In this article, we explain the SEPA concept and its theoretical origins, giving examples of its previous application in different countries and contexts. We describe how the approach was used in the Camino Verde intervention, with details that show commonalities and differences in the application of the approach in Mexico and Nicaragua. We discuss issues of cost, replicability and sustainability, and comment on which components of the intervention were most important to its success. In complex interventions, multiple components act in synergy to produce change. Among key factors in the success of Camino Verde were the use of community volunteers called brigadistas, the house-to-house visits they conducted, the use of evidence derived from the communities themselves, and community ownership of the undertaking.Communities received the intervention by random assignment; dengue was not necessarily their greatest concern. The very nature of the dengue threat dictated many of the actions that needed to be taken at household and neighbourhood levels to control it. But within these parameters, communities exercised a large degree of control over the intervention and displayed considerable ingenuity in the process.ISRCTN27581154. In a 2011–13 cluster-randomised controlled trial in Managua, Nicaragua, and three coastal regions of Guerrero, Mexico, a pesticide-free, evidence-based approach to community mobilisation, called Camino Verde (Green Way), reduced dengue virus infection among children, self-reported dengue illness at all ages, and all dengue vector entomological indices [Vertically organized and insecticide-based vector control efforts have important limitations, and there is a need to develop and test community-based strategies that include environmental management . Up to tAedes aegypti. Actions reported from trials and other studies include education of households through their children [These community-based strategies, used in various countries, all involve some form of community participation, and they are all aimed at controlling the mosquitoes that carry the virus, children , 5, 7, achildren , communichildren , provisichildren . Severalchildren –7.Most community-based efforts have included, or were conducted in parallel with, the use of chemical larvicides and/or insecticide spraying, but others, called eco-friendly interventions, used only bio-control agents such as small fish and crustaceans that consume larvae in water receptacles or “ovitraps” that use non-chemical toxins to kill mosquito eggs –11.Although all these interventions were community-based, many appear to have been driven for the most part from above. However, in four randomised controlled trials that were successful in reducing entomological indicators, two studies from Cuba and one each from Fortaleza, Brazil, and Chennai, India, there was serious community participation that helped tailor the interventions to local realities –6.In this article, we describe how the Camino Verde trial incorporated several of the actions mentioned above, along with others, within a broader approach called SEPA . We describe the elements of the SEPA approach using the TIDieR reporting guidelines for describing interventions , discusshttp://www.ciet.org) – has developed an approach to the production and use of evidence for health promotion and community development, which we call “socialising evidence for participatory action” (SEPA).Over 30 years, CIET – a non-governmental research organisation that grew out of the Centro de Investigación de Enfermedades Tropicales (CIET) at the Autonomous University of Guerrero in Mexico  trained them in the life cycle and habits of the dengue virus-transmitting The facilitators’ role was to (1) make initial contact with the community and facilitate a brigadista recruitment process, (2) present evidence from the baseline survey, (3) provide training, and (4) support the community in its assuming of responsibility for the intervention. In Nicaragua, the facilitators were former brigadistas active in the 2004–2008 feasibility study on the same subject. During this pilot experience, the research team came to appreciate the sense of solidarity that makes daily life possible in the neighbourhoods of Managua and to understand that its role was to reinforce values of respect for individual differences and collective responsibility already present in the community and pass this appreciation on to the first generation of brigadistas in the trial. Facilitators in Mexico, mostly recent graduates from the University of Guerrero where CIET is located, received more formal ethical training in which a Mexican communications expert and a member of the Nicaraguan field team participated. In both countries, facilitators sought to move as quickly as possible from leadership roles to supporting roles.Aedes aegypti mosquito at the household level was indispensable to the entire effort. All consenting households in the research clusters participated in the intervention, and all members of each household were invited to join in the effort. While the clusters used to measure the effects of the intervention were limited to approximately 140 households, the intervention often reached households in the surrounding neighbourhood as well.Environmental control of the The Nicaraguan trial was entirely concentrated in the capital city, Managua, where neighbourhoods typically have recognized, active leadership closely allied with the Sandinista government. The SEPA strategy there was to work with these leaders and deliberately avoid creating parallel structures, while striving to maintain the brigade’s autonomy and political neutrality. Several brigadistas were also community leaders. The Mexican trial covered the entire coastal area of the state of Guerrero. In Guerrero’s rural areas, the strategy was similar to that in Nicaragua, especially where the communities are primarily indigenous and more organised. In urban areas, mainly in the city of Acapulco, identifiable community leadership tended to be less unified and less effective for our purposes. The organisation of many urban communities has been disrupted by drug-related violence, and the Camino Verde brigades in some cases helped to restore community organization.Children of both sexes were very active in the intervention – in their own households, in the schools, and in collective neighbourhood activities. Some brigades in Nicaragua were made up predominantly of children. In Mexico, two student brigadistas were selected to coordinate activities in each of the schools. Once made aware of what mosquito larvae and pupae look like up-close, children can become fascinated with them and become dedicated foot-soldiers in the struggle to eliminate them.Numerous national and regional organisations in both countries, while usually not rooted in any individual community, are active at the grassroots level. These organisations had diverse main agendas, but the threat of dengue and the need for mosquito control was a common concern. The SEPA programme partnered with as many of these organisations as possible in its mobilisation activities.the use of community volunteers, called brigadistas;visitas de acompañamiento;house-to-house visits, called simple mosquito control tools accessible to every household;collective elimination of breeding sites not under the control of individual households;engaging schools, churches, shops, clubs and other organizations in the effort;a wide variety of media used to educate and motivate.A feasibility study in Nicaragua between 2004 and 2008  developebrigadistas were allowed inside the dwelling, the dialogue about dengue was engaged and reinforced by joint inspection of water receptacles and discussion about how to prevent infestation with mosquito larvae and pupae.The first three of these activities went together in the household visit. The initial interaction with residents leading to an invitation to enter the home was a critical moment in the relationship between brigades and community members, as it raised key issues of consent, trust, and confidentiality. Once Since, however, dengue is not just a household problem but also a community one, brigadistas were called upon to encourage collective action to eliminate the vector from abandoned properties, public spaces such as cemeteries, parks, playing fields, bus stations, and central squares, and private commercial spaces. School children, churchgoers and club members were often willing participants in these collective actions and they were also active in their own homes reinforcing the lessons imparted by the household visits. Media for educating and mobilising community members included graffiti, murals, banners, street theatre, parades, pamphlets, piñatas, festivals and songs.To launch the intervention, facilitators convened and ran intervention design groups to discuss survey results, cost implications, and specific prevention strategies in each community. In Nicaragua, these groups were composed of community leaders and were focused on evidence about costs of dengue illness and money spent on personal protection against mosquitoes. Each participant was given a sheet of paper with four questions, each question accompanied by the evidence from the baseline survey specific to her/his neighbourhood. Each question was discussed by the group and the group formulated group responses or commentaries . In Mexihttp://caminoverde.ciet.org/en/nicaragua/activities/andhttp://caminoverde.ciet.org/en/mexico/activities/Details, including photographs of these activities can be found at: Aedes aegypti mosquito. The entomological evidence was of two kinds: a) aggregate numbers and percentages of mosquito larvae and pupae derived from systematic inspections of household and community water receptacles and b) visual demonstration of the presence of these larvae and pupae to householders on their own premises. The epidemiological evidence came from the baseline study of the Camino Verde trial in the same communities and concerned risks from failure to protect against dengue and likelihoods of protection from various actions that households and communities could take to minimize those risks. The economic evidence was a) cost data on dengue and dengue control gathered in the baseline surveys and b) reflection by each household on the costs they incur from seeking treatment for dengue illness and from purchases of anti-mosquito chemicals and devices.The principal material used in Camino Verde was evidence. The evidence was biological, entomological, epidemiological, and economic. The biological evidence concerns the development cycle of the immature One piece of evidence was lurking in each household’s own water containers. During the household visits, brigadistas would accompany the household head, and often the children, on an inspection of the various water receptacles on their property. Finding mosquito larvae and/or pupae right on the premises offered a unique opportunity for making people aware of their presence and discussing ways of preventing them from maturing and/or from ever being there in the first place Fig. . Rather At the household level, brigadistas carried a water jar or plastic bag containing live mosquito larvae and simple tools  for collecting larvae in the household. A particularly important tool was a laminated graphic showing the mosquito vector’s life cycle on one side, with alternative control measures and the advantages and disadvantages of each option on the other. This graphic was also displayed on the t-shirts worn by many of the brigadistas, who used it while explaining to households how to interrupt the cycle before the mosquito matures. The graphic was also projected or otherwise displayed during collective events and included in murals and posters in public places Fig. .Fig. 2Cohttp://caminoverde.ciet.org/en/nicaragua/activities/ and http://caminoverde.ciet.org/en/mexico/activities/and also at http://caminoverde.ciet.org/en/galleries/photos/.At the neighbourhood level, brigadistas helped organize various collective events – both awareness-raising events and events to control mosquito breeding in public spaces. Materials used for awareness-raising events included leaflets, posters, parades, songs, games, murals, graffiti, t-shirts and piñatas. Examples of these events and the materials used can be found at http://sepa-nic.blogspot.com) where community brigadistas, residents and leaders could give testimonies of their own experiences, share anecdotes, enjoy photos, access their own data and work tools, monitor the product of their own work, and ask, comment and learn about what was happening in other neighbourhoods [At the household level, brigadistas delivered the intervention face to face. At the community level, brigadistas and facilitators delivered the intervention both in small groups and at large public events. The only electronic tool used was a blog created in Nicaragua  in order to measure recent dengue virus infection. The baseline also gathered demographic and socio-economic data, data on dengue illness and its costs to the household, on knowledge about dengue and its vectors, attitudes and practices related to dengue control, and expenditures on anti-mosquito products. Results from the saliva samples were returned to the individual households in both intervention and control communities. Meanwhile, we contacted local health authorities, local NGOs, and community leaders to obtain their consent and cooperation. Finally, before launching the intervention, we held a series of community discussions of baseline results. In Nicaragua, discussions with community leaders focused on evidence about costs to households from dengue illness and money spent on personal protection against mosquitoes . In MexiHawe and colleagues have argued that, in complex interventions, the function and process of the intervention should be standardized, rather than the components themselves, thus allowing the form to be tailored to local conditions . We desiThis difference between function and form is apparent at different levels. At the inter-country level, it can be seen in the difference between Mexico and Nicaragua in the way that brigadistas were recruited, trained and rewarded. In Nicaragua, all the brigadistas were volunteers who responded to an invitation from community leaders. There was a good deal of turnover among the brigadistas, but as some left the brigades, there was a steady stream of replacements who were trained by those remaining. Some brigadistas eventually took on broader roles, joining the leadership of their already-existing neighbourhood structures but with an identity that was free of past conflicts . In MexiAnother difference between countries was introduction of biological control using fish, practiced in Mexico but not in Nicaragua. Brigadistas and facilitators in Mexico learned that in some intervention communities, residents kept fish in water containers, using them to prevent the development of mosquito larvae. They then encouraged other communities to do the same. Secondary analysis of the Camino Verde results in Mexico provides evidence that fish in water containers can reduce the risk of dengue virus infection and dengue illness .At the neighbourhood level, some examples of how communities interfaced with public services show how useless it would have been to arrive with preconceived solutions. In one Mexican community, members decided to conduct a street cleaning campaign. They first had to obtain the cooperation of the sanitation services. Having had success in doing this they went further and arranged for monthly clean-ups with the cooperation of that department plus those of water supply and public works. In Nicaragua, what citizens learned from accompanying the entomological inspections in their own homes led to the realization that full control of the mosquito problem required community-wide solutions. This led to collective action to put pressure on small businesses and repair shops to control infestation on their premises, and this in turn gave them confidence to deal with public institutions such as the local health centre and the municipal authorities responsible for refuse collection and the repair and maintenance of streets and drains.In another Mexican community, the lack of regular garbage collection had led one resident to dump her garbage in empty lots and even sometimes on property belonging to other residents. This became the cause of chronic tension among the residents, who requested help from the brigades in resolving it. They first visited the woman and with her consent arranged a meeting between her and her neighbours, the upshot of which was a petition to the municipal sanitation services for increased garbage collection in the whole community.Appendix 2 to the main Camino Verde report presents lists of commonalities and differences in the way that SEPA was implemented in Mexico and Nicaragua. There were aspects common to both countries by reason of the trial’s content and objectives, aspects common to both countries by reason of the experience gained in the feasibility study conducted in Nicaragua before the trial, and aspects that differed by country and/or community .Fidelity to the SEPA approach was assured by supervision and by exchanges among communities. Both countries made use of peer monitoring. Brigadistas from one community visited another community under supervision of a different facilitator without intervention on the part of researchers. They applied a brief questionnaire and conducted entomological inspections in consenting households. Afterwards, the visiting brigadistas presented their findings to the host brigadistas and together they discussed possible adjustments to the approaches being used. Meetings among facilitators and brigadistas from different communities also helped to assure commonality of purpose and process.We now know that the Camino Verde trial was successful in reducing dengue virus infection among children, self-reported dengue illness at all ages, and all dengue vector entomological indices in Managua, Nicaragua, and three coastal regions of Guerrero, Mexico. SEPA is not a recipe. It is an approach that serves a common function of assisting communities – always with respect for their knowledge and customs – to identify ways of reaching common objectives. Nevertheless, if we want the Camino Verde approach to be scaled-up and replicated elsewhere, we need to identify some practical aspects that appear to be essential to success.Aedes aegypti control [Studies of community-based dengue control strategies have been criticized for failing to distinguish which specific components of the intervention have the greatest impact on  control . Guidanc control . An apprThe two most consistent strategic elements in the Camino Verde trial were the use of community volunteers (brigadistas) and repeated house-to-house visits (visitas de acompañamiento).Brigadistas, a variety of community health workers , were thWhat appears to be a key aspect of this process is “learning by doing” and the “ownership effect” (efecto de lo propio) … in which learning about the mosquito’s reproductive cycle, the recognition of larvae, pupae and eggs, and learning what to do about it  is accompanied by inspection and sometimes by actual cleaning….The repetition of the visits makes possible a transition from distrust to confidence and also reinforces what people have learned. Posters and street theatre then act as complementary reinforcement of what has been learned.The attitude of the brigadistas and their motivation also encourages residents to maintain their surroundings properly.The house-to-house visits were the brigadistas’ most regular activity, weekly in principle but frequent at least. The method most consistently used was the demonstration to householders of the presence of mosquito larvae and pupae on their own premises. In Mexico, an anthropologist from the University of Guerrero analysed householders’ narratives of change collected during the trial and summarized his findings in these terms:--From a 2011 internal report by Prof. Joan Muela.Thus, the brigadistas  and the household visits appear to be the most “active ingredients” responsible for the trial’s success, but underlying the brigadistas’ role and their household visits was also the climate of community trust and community ownership of the process.Randomised controlled trials in Fortaleza, Brazil  and ChenAs long ago as 1951, Lewin argued that the process of ‘unfreezing’ existing behaviour patterns needs to take place in a group environment and to involve open and supportive communication among those involved in negotiating the change . And, inFrom this perspective, it is unlikely that the presence of brigadistas and their household visits alone account for the success of Camino Verde. Although there is no direct proof for it, we believe that community ownership of the undertaking was also a key factor and that attempts to replicate the Camino Verde trial’s success while discounting the importance of community autonomy and community control of the social change process are unlikely to succeed . The WHOSecondary results of the trial show behavioural differences between households in the intervention and control communities. In the trial’s follow-up survey, the household respondents in intervention communities reported higher levels of collective self-efficacy: agreeing that communities themselves can do something to control mosquitoes in their environment .While sustainability ultimately depends on the will and capacity of community members to control their own environment, some external stimulus and guidance will be needed. During the trial, the external actors were the facilitators whose work came to an end once the trial ended. In Nicaragua, where communities and government work together in many endeavours, we have reason to expect that government will step in and make the Camino Verde process its own, at least on a trial basis. In some parts of Mexico, where community trust in the government is weaker, non-governmental solutions may be required.Creating favourable environments and fostering community ownership of the SEPA process are time-consuming activities and are not inexpensive. We have collaborated with health economists on an as yet unpublished economic analysis of the trial outcomes. Preliminary results show that the annualized cost of the intervention per member of the intervention community in Mexico was USD 16.72 while that for Nicaragua was USD 7.47. Using the perspective of the World Health Organization’s “Choosing Interventions that are Cost-Effective” project , the intThis cost-effectiveness analysis excludes two significant economic aspects of the Camino Verde intervention. One is the savings from dengue cases averted, including the number of work and/or school days that patients and caregivers might have lost but were saved thanks to the intervention . The othAedes aegypti mosquito is a vector not only for dengue but also for chikungunya, Zika, yellow fever and other diseases. We do not have proof of Camino Verde’s impact on these diseases, but it is likely to be comparable with the impact on dengue. For example, the prevention of Aedes aegypti proliferation could make Camino Verde cost-effective for its effect on Zika alone. Given the risk of microcephaly from Zika, the resulting benefits would be substantial [Nevertheless, our efforts going forward will include achieving more favourable cost-effectiveness, which could be done in three ways: by achieving the same result at a lower cost; by improving the result for the same cost; and/or by achieving more than one objective at a comparable cost. First, as we had no precedent for measuring the impact of community mobilisation on dengue virus infection incidence and reported dengue illness, we were less concerned about costs than ensuring a favourable result, and we spent the entire budget provided by the sponsor to achieve it. We believe that all three ways of improving cost-effectiveness will apply here. First, with the experience now acquired, we believe that economies of scale and other measures could make the intervention more efficient. Secondly, we have evidence to suggest that the intervention could be made more effective for a similar cost. Analysis of the trial’s intermediate outcomes from a gender perspective, presented in a companion article, shows that what the trial achieved was due almost entirely to the women in the intervention communities . We needstantial –67.brigadistas to deal with other health conditions would not only respond to community priorities, but would also be another way to achieve additional objectives for comparable costs. The house-to-house visits and community events that were part of Camino Verde could well become the occasions for educating and encouraging households and communities toward other preventive actions such as immunisation, screening for other infectious and chronic diseases, and healthier behaviours.We also know that the communities themselves did not always consider dengue control to be their highest priority , 50. BroFor these reasons, therefore, we believe that the SEPA approach can be judged cost-effective, building on lessons learned from the Camino Verde trial.Aedes aegypti threat has been permanently embedded in any of the communities that participated in the Camino Verde trial. The real gains achieved in this trial need to be consolidated and extended until a societal tipping point is reached where it will be normal for all households to monitor water receptacles on their own properties and cooperate with their neighbours and public and private institutions in a shared responsibility for thorough and consistent control of their environment to make it free of dengue, chikungunya, Zika, and other arboviruses. We should not exaggerate the degree of autonomy exercised by the communities in this trial. They did not choose dengue as the subject they most wanted to mobilise around and they received the intervention by random assignment. The very nature of the dengue threat dictated many of the actions that needed to be taken to control it.It would be foolish to pretend, nonetheless, that a self-sustaining culture of vigilance and cooperation in response to the brigadistas. Community leaders invited neighbours to join the brigades, and many leaders themselves joined the brigades. And in both countries, communities were empowered by evidence to interact with public authorities in new and creative ways.However, within these limitations, communities exercised a large degree of control and displayed considerable interest and ingenuity in the process. None of the investigators in Mexico, for example, had thought of biological control before it was suggested by community members. In Nicaragua, the researchers played no direct role in the selection of Aedes aegypti mosquito and is applicable in different community and country conditions.The SEPA approach is an appropriate one for mobilising communities to combat diseases transmitted by the