The legalisation of cannabis for medicinal use is a contentious space both politically and in the medical community. In 2014, the Medical Innovation Bill introduced by Mario Oriani-Ambrosini MP, aimed to shift the political and legal positions of cannabis as an illegal substance to one available for research and medical use. To date, progress on this has been slow. Cannabis and cannabinoid products are currently available for medicinal use in several countries, including the Netherlands and 29 states in the United States. Locally, anecdotal reports suggest that many of our patients with chronic medical conditions are using cannabis and cannabis-derived or cannabinoid products for symptom alleviation.2 The review identified moderate quality evidence to support the use of medicinal cannabis for chronic pain, chemotherapy-induced nausea, vomiting, and multiple sclerosis,2 but the many formulations, dosages and routes of administration limit recommendations.In January 2016, the Alcohol, Tobacco and Other Drug Research Unit (ATODRU) of the South African Medical Research Council (SAMRC) produced a policy brief evaluating the findings of a published systematic review of current evidence to inform the discussion.Subsequently, the SAMRC brought key stakeholders representing all nine faculties of health sciences at South African universities together on 08 February 2017 at the SAMRC Medicina Campus, Cape Town, to explore opportunities for conducting local research and clinical trials of medicinal cannabis and cannabinoids to inform local policy-making. The workshop identified three major research priorities:Conduct a national, multisite clinical trial of cannabinoids following identification of the optimal formulations, dosage and relevant clinical indications from the current evidence base to inform trial protocol development.Support exploratory research to quantify the prevalence and qualify the current use of extracts (e.g. oils) in the community to alleviate pain and other symptoms; methods to include are as follows, (1) community- or clinic-based cross-sectional surveys or online survey of general public, (2) cross-sectional surveys of practising general practitioners and/or specialists in pain clinics with respect to their knowledge of patient use of cannabis extracts through patient disclosure.Conduct qualitative evaluation(s) of possible barriers and facilitators to medical practitioners prescribing cannabis or cannabinoids for medicinal purposes, should it be legalised in the future.3 Only three studies, all using a fixed dose combination of tetrahydrocannabinol (THC) and cannabidiol (CBD) delivered via oromucosal spray were identified, and the results of the GRADE4 assessment are shown in Further to this meeting, an SAMRC-supported team of researchers evaluated the evidence for a trial of a cannabinoid for the management of pain related to HIV associated sensory neuropathy (HIV-SN). The team selected painful HIV-SN as the condition based on: (1) the high burden of HIV and HIV-SN in the South African population, despite availability of improved antiretroviral drug regimens, and (2) the absence of empirical evidence supporting the efficacy of pharmacological agents typically recommended for the management of neuropathic pain in this population group. They limited their assessment to high-quality trials of greater than 12 weeks duration, included participants with at least moderate intensity pain at baseline and who met current recommendations for trial design in the field of chronic pain.5 exists for a fixed dose THC:CBD oromucosal cannabinoid spray trial for the management of painful HIV-SN. This was based on the GRADE assessment, the absence of demonstrably effective alternative therapies for painful HIV-SN and the clinical need in people living with HIV. The team is currently working on a study protocol for a multisite trial that conforms with the current highest standards for trials of analgesic agents for chronic pain.5 The SAMRC will be releasing a Request for Applications (RFA) from groups interested in participating in trials in due course.The team concluded that clinical equipoiseIn medical practice, there is a balance between early adoption of new innovation and a slower, more considered approach prioritising evidence and safety over potential benefit. Though there are some clear areas where evidence supports the use of medicinal cannabis (as reviewed in the SAMRC policy brief), this should not imply that cannabis is a panacea for all ails or that it should be freely available without due control of production quality, disease indications and specific dosing recommendations. Research is urgently needed and regulatory changes will promote and allow for high priority, locally relevant research using regionally sourced products.It is imperative that the medical and scientific community – both practitioners and researchers – are involved in informing and developing evolving cannabis policies. Much confusion exists between medical versus recreational usage, the legal statutes of medical and recreational possession and usage, and the scientific evidence compared to impassioned anecdotal reports of its efficacy in addressing a host of medical conditions. We owe it to our patients to offer the best relief of suffering with all available therapies based on the rational assessment of benefit, cost and potential harms of cannabis and cannabinoid products for medicinal use.


To maximize the impact of Zika prevention programming efforts, a prioritization process for social and behavior change programming was developed based on a combination of research evidence and programmatic experience. Prioritized behaviors were: application of mosquito repellent, use of condoms, removing unintentional standing water, covering and scrubbing walls of water storage containers, seeking prenatal care, and seeking counseling on family planning if not planning to get pregnant. To maximize the impact of Zika prevention programming efforts, a prioritization process for social and behavior change programming was developed based on a combination of research evidence and programmatic experience. Prioritized behaviors were: application of mosquito repellent, use of condoms, removing unintentional standing water, covering and scrubbing walls of water storage containers, seeking prenatal care, and seeking counseling on family planning if not planning to get pregnant. Aedes-borne diseases between 2012 and 2018, review of seminal papers, and review of gray literature. We examined articles to determine each behavior's potential effectiveness in preventing Zika transmission or reducing the Aedes aegypti population. We also developed assessment criteria to delineate the ease with which the target population could adopt each behavior, including: (1) required frequency; (2) feasibility of the behavior; and (3) accessibility and cost of the necessary materials in the setting. These behaviors were refined through a consensus-building process with USAID's Zika implementing partners, considering contextual factors. The resulting 7 evidence-based preventive behaviors have high potential to strengthen SBC programming's impact in USAID's Zika response: (1) apply mosquito repellent, (2) use condoms during pregnancy, (3) remove standing water, (4) cover water storage containers, (5) clean/remove mosquito eggs from water containers, (6) seek antenatal care, and (7) seek family planning counseling. This case study documents a flexible process that can be adapted to inform the prioritization of behaviors when there is limited evidence available, as during many emergency responses.Since the 2015 Zika outbreak in Latin America and the Caribbean, a plethora of behavior change messages have been promoted to reduce Zika transmission. One year after the United States Agency for International Development (USAID) initiated its Zika response, more than 30 variants of preventive behaviors were being promoted. This situation challenged social and behavior change (SBC) programming efforts that require a coordinated response and agreed upon set of focus behaviors to be effective. To support USAID implementing partners in harmonizing prevention efforts to reduce Zika infection, we developed an evidence-based process to identify behaviors with the highest potential to reduce Zika infection and transmission. We compiled a full list of behaviors and selected the most promising for a full evidence review. The review included systematic keyword searches on Google Scholar, extraction of all relevant published articles on  Résumé en español al final del artículo.Aedes aegypti mosquito, a vector that also transmits other arboviruses including dengue, chikungunya, West Nile virus, and yellow fever. The first outbreak of Zika detected in the Americas occurred in 2015, with a spike in suspected congenital malformations and other neurological complications such as Guillain-Barré syndrome.Zika virus is a communicable disease primarily transmitted by the The outbreak in LAC demanded a concerted regional response, given the wide distribution of the mosquito vector, the lack of population-level immunity, the absence of a vaccine or rapid diagnostic test, uneven access to water due to low quality water and sanitation infrastructure, water shortages, lack of information about the disease, and inadequate health systems to respond to the health impacts.–USAID's SBC programming was comprised mainly of mass and social media, community engagement, and interpersonal communication, with the goal to “raise awareness, reduce misinformation, and address the barriers that prevent individuals, families, and communities from practicing lifesaving behaviors to improve health outcomes.”Aedes aegypti mosquito) and practices to reduce sexual transmission had already been identified in the literature and could be assessed for Zika. Understanding the transmission dynamics was critical to identifying behaviors to consider for prevention. The exercise, referred to as the “behavior prioritization process,” focused on a range of individual- and household-level behaviors to reduce the risk of Zika acquisition and transmission. Messages were developed by partners based on the set of behaviors identified and prioritized in this process.Facing an outbreak of a disease new to the Americas, public health institutions and organizations found themselves conducting research while simultaneously launching interventions and programs. At the time programs were rolling out, there were limited data to guide SBC programming and messaging for the most effective preventive actions for individuals and communities. These circumstances often led to a lack of cohesion in promoted behaviors and SBC messages. To more effectively coordinate the Zika response among implementing partners and increase the rate of behavior adoption among target populations, the Breakthrough ACTION + Research Projects, in collaboration with USAID, developed an evidence-based process to identify priority behaviors with the highest potential for preventing Zika acquisition and transmission. Stakeholders across disciplines and involved in various levels of programming were engaged throughout to ensure buy-in, harmonize priority behaviors and their SBC messages, and ensure a more effective Zika response. Existing research could be leveraged because the transmission dynamics for Zika were similar to other arboviruses and sexually transmitted infections; preventive behaviors targeting the vector  that also transmits arboviruses such as dengue and sexual transmission. A team of experts in SBC programming and vector control was enlisted to categorize and refine the behaviors. All of the Zika prevention messages were first grouped together by behavior to create a condensed version of about 15 behaviors. Through an iterative review process including experts and discussions with partners, the list was distilled to 7 key behaviors (Aedes aegypti-borne diseases (such as Zika) or reducing Aedes aegypti mosquito populations (after a quick literature scan and input from experts) or due to other criteria.A list of more than 30 Zika preventive behaviors (or their variants) that USAID implementing partners across USAID-supported countries were promoting was compiled by informally reviewing numerous project materials and documents. All of these preventive behaviors were related to the transmission dynamics of Zika virus—transmission by a vector .Aedes mosquito mainly bites in the daytime).There was limited evidence of the behavior's efficacy .The behavior was not supported by USAID  because of the lack of effectiveness of the behavior  or because it was not feasible within the scope of the program .In determining the most promising behaviors to review further, behaviors were excluded from the list if:At this stage, 5 behaviors were selected for a full evidence review that fell into the broad categories of (1) personal protection and (2) household and community vector control. A third category, prevention-enabling behaviors, was created for 2 additional behaviors that did not undergo a full evidence review here but were still recommended. Prevention-enabling behaviors create the potential for exposure to Zika prevention counseling by trained and trusted health care providers and include antenatal care and family planning counseling. Investigation of enabling behaviors was not pursued in the next phase of the process, as extensive existing literature supported the efficacy of antenatal care and family planning counseling on uptake of sexual and reproductive health services , and their promotion was already ongoing through reproductive health programs in the region. Although it is not necessarily clear how the Zika-specific context may have affected uptake of these behaviors, there was no research available at the time to review.Aedes-borne diseases published between 2012 and 2018. Any seminal papers published before 2012 and Zika-related gray literature and unpublished data  were also reviewed. Because of the recency of the Zika outbreak, a limited number of relevant papers had reached publication, so relevant articles on any Aedes-borne diseases  were also compiled. Literature on malaria was excluded because it is transmitted by Anopheles mosquitoes, not Aedes, calling for different interventions. A PRISMA diagram ; how the outcome was measured ; whether programs targeted specific sub-populations ; and whether interventions targeted multiple behaviors . These additional factors were considered to guide interpretation of the study findings based on context and better understand the generalizability of results . If the article had insufficient detail, we also contacted research authors to clarify the specific steps required in the behaviors assessed in their studies.In addition to efficacy and program effectiveness criteria, a third criterion was developed to assess whether the behavior was easy to do and amenable to change to consider the contextual realities of how behaviors were being promoted and adopted. As noted in the bottom panel of To summarize, the prioritization process took place over 7 months between October 2017 and April 2018. More than 30 variants of behaviors and messages were identified and narrowed down to 7 through extensive discussion with the technical working group and input from technical experts to reach an initial consensus. To narrow down to these 7 behaviors, we used a combination of expert opinion, initial scan of available evidence, and factors such as whether USAID was supporting the behavior by procuring the materials necessary. Two of the final 7 behaviors were considered enabling behaviors and recommended but not included in the evidence review, given the substantial existing evidence on these behaviors. Five behaviors were explored in a full literature review to ascertain their relative effectiveness. During the literature review phase, partners provided additional gray literature where applicable. The full findings were presented at a subsequent SBC Technical Working Group meeting to ensure a consensus on the findings. The initial purpose of presenting the findings was to further narrow down the prioritized behaviors, but after partners expressed significant pushback, the full list of 7 behaviors were agreed upon. Any feedback from implementing partners regarding how behaviors were carried out, contextual challenges, or unpublished effectiveness findings was integrated into the final recommendations, and the results were disseminated to all USAID implementing partners.More than 30 behaviors and messages were narrowed down to 7 after reaching consensus within the Zika SBC Technical Working Group.The findings and conclusions for Zika SBC programming drawn from this process are presented below for each of the 5 preventive behaviors reviewed, as well as the 2 enabling behaviors.Applying mosquito repellent: Application of mosquito repellent is highly efficacious in preventing mosquito bites, and thus the potential of vector transmission of Zika to an individual. This behavior is within the control of pregnant women and male partners of pregnant women. It is recommended that users be thoroughly counseled on proper product application. Women intending to become pregnant should also consider using repellent.Applying effective mosquito repellant properly and consistently is highly efficacious in preventing mosquito bites and potential transmission of Zika.-,Applying mosquito repellent  and using each product as directed is a highly effective method in preventing mosquito bites and reducing risk of Zika transmission through mosquito bites. DEET is considered the gold standard repellent, showing greater than 95% efficacy in preventing mosquito bites for 5–11 hours.10 USAID and CDC approved 3 additional repellents  based on evidence suggesting they have an efficacy comparable to DEET.11 These 4 repellents are the only ones for which evidence of effectiveness has been recorded. The use of these repellents was also considered safe for use during pregnancy.Using condoms to prevent sexual transmission: Condom use to prevent sexual transmission of Zika is highly efficacious, but sexual transmission may be a small portion of overall transmission. This behavior should be prioritized for pregnant women and their partners because pregnant women are at risk for negative pregnancy outcomes.Using condoms to prevent sexual transmission of Zika is highly efficacious and important during pregnancy to prevent congenital Zika syndrome.--,-There is evidence that Zika is transmitted sexually.Regularly removing unintentional standing water both inside and outside the house: This is a potentially efficacious behavior to reduce mosquito populations and reduce the potential for individual- and population-level risk of Zika transmission. Promotion of the behavior must be accompanied by specific, focused instructions that target the highest density breeding sites and be conducted weekly in homes and communal areas to be effective. Efficacy is highest in areas with strong community engagement, including active mosquito searches in homes and communities and awareness of the mosquito life cycle.Ongoing community clean-up campaigns to remove unintentional standing water can reduce the mosquito population and thus reduce potential Zika transmission.Aedes mosquito population. One study found a greater than 70% reduction in the adult mosquito population following a very strict intervention, involving community campaigns and visits from trained volunteers, to remove stagnant water.Aedes mosquito population, and thus Zika transmission, it requires an ongoing, collective effort,--Aedes mosquitoes.According to the evidence, performing this behavior is highly efficacious in reducing the adult Covering water storage containers at all times with a tight-fitting cover that does not warp or touch the water: Covering long-term water storage containers has moderate potential efficacy in reducing breeding sites if a tight-fitting, long-lasting lid is available. Covering short-term water storage containers has less potential efficacy, as frequent lid use can result in wear and tear and render the lids ineffective or counterproductive.Covering long-term household water storage containers with tight-fitting, long-lasting lids can prevent mosquito breeding sites.Aedes mosquitoes.Aedes breeding sites.The focus of this behavior is on long-term storage items such as barrels or other large household water-storage containers used less than once per week. A small number of studies suggest that the correct use of lids is associated with a significant reduction in pupal infestation if the containers are used infrequently.Eliminating mosquito eggs from water-storage container walls weekly: Thorough cleaning of water-storage containers can remove mosquito eggs, significantly reducing the population and, thus, Zika transmission, but easy access to effective materials cannot be assumed.Aedes aegypti mosquitoes lay their eggs in water storage containers, such as washbasins and metal drums, located inside or outside the house, increasing the risk of transmission of diseases such as Zika to households. As a result, cleaning containers is often recommended, but historically a lack of specific instruction has led to mixed results. For example, the World Health Organization recommends scrubbing containers with a brush, but does not mention whether a cleaning solution (such as bleach or detergent) should be applied, and cites studies that do not isolate the effect of cleaning from other behaviors (such as using lids).-The Untadita method, tested in a randomized controlled trial,The second method, developed in the Negociación de Prácticas Mejoradas trial,If the first 2 cleaning methods are not possible to carry out due to lack of bleach, the third technique of cleaning the walls of the container with detergent alone  should be implemented. This technique requires fully emptying the container.Lastly, scrubbing the container walls with a brush (only) is recommended if neither detergent nor bleach is available.Seeking antenatal care. Seeking antenatal care is known to contribute to healthy pregnancies. In this setting, seeking antenatal care enables counseling on Zika prevention by trained health care providers, allowing for early diagnosis and treatment, as well as access to information about effective protective measures to reduce the risk of transmission of Zika from mother to child.Using contraception voluntarily. Seeking family planning (for those not intending to get pregnant) is also a critical behavior, linked to prevention of sexually transmitted infections and prevention of sexual transmission of the Zika virus. Both of these behaviors—seeking family planning and antenatal care—are routinely promoted for adoption of healthy behaviors among pregnant women and women of reproductive age.The results of the literature review and the consultative process discussed in this article helped to identify behaviors with the greatest promise for preventing the acquisition and transmission of Zika. A critical part of this process was the consensus-building process with partners, ensuring input from those working on the ground. The SBC Technical Working Group engaged partners throughout the process. Partners requested focusing on behaviors that families and communities could do themselves (“locus of control” at the household). Points of contention often centered around behaviors that were being promoted already and were perceived by partners to be effective but had mixed evidence. For example, covering water storage containers was found to effective in the literature only for long-term water storage containers, but ultimately short-term containers were included in the guidance based on conversations with partners. This was mainly because partners perceived this behavior (covering water storage containers with a lid) to be effective based on this behavior being implemented already and being received positively. However, for other behaviors, for example use of bed nets, we clarified that although these are effective for malaria, they are not effective for Zika, and this was ultimately agreed upon and the guidance accepted. Through the process, partners were reminded that choices had to be made to prioritize key behaviors; although all of them were potentially effective, we were looking for relative effectiveness to prioritize the most effective ones and focus on them. Where there was pushback, we asked for field data to inform the decision to modify the final guidance.Aedes aegypti vector control and mosquito bite prevention behaviors are included in this resource, partners working on dengue and chikungunya can use it. The documents will be continually updated as new developments emerge, reflecting input from implementing partners, to ensure that the materials address realities on the ground. For example, based on input from the field, guidance on correct disposal of repellents was recently added.These results were summarized in the Zika Prevention Behavior Matrix, a document widely disseminated through the Zika Communication Network (ZCN), a platform for sharing Zika-related resources and media products in the LAC region. A Technical Specifications Content Guide (a companion to the matrix) was also developed to detail the evidence-based technical requirements and steps to follow for each of the 7 behaviors  and curriculum are available on the ZCN. Country-specific adaptations to the job aid, such as including language for dengue and chikungunya prevention, are also available on the ZCN.The conditions brought on by climate change, international travel, urbanization, deforestation and other global and regional trends may result in new emerging diseases, as well as the spread of existing diseases to previously unexposed populations.The behavior prioritization process was developed to identify focal behaviors for prevention to harmonize SBC programming efforts for greater impact.Aedes aegypti mosquito viruses were prevalent in the region, specifically, dengue, which had been circulating in the region for more than 50 years, and chikungunya had emerged in 2013. Thus, information regarding best practices to prevent transmission could be gleaned from previous mosquito control and behavior change programming from LAC and southeast Asia and could be adapted to Zika prevention efforts.The first 2 criteria of the process assessed the state of the evidence available for selecting key behaviors to promote. The availability and quality of evidence depends on how long the disease has been around and whether it is occurring in a new region or sub-population. For example, in the 2002 severe acute respiratory syndrome outbreak, there was no evidence as the world was contending against a newly encountered disease. During the West Africa Ebola outbreak, researchers were attempting to understand the behaviors to target to interrupt transmission, while programmers were developing prevention and treatment programs and messages in real-time; research and evidence generation came later. In the case of Zika, the disease was previously known in east Asia and the Pacific but was entirely new to LAC. However, other Aedes aegypti population, there were little to no published data available regarding sexual transmission of Zika or its impact on pregnancy outcomes. Sexual transmission and the link with congenital malformations meant we needed to consider specific behaviors and messaging directed to pregnant women and women of reproductive age. To address this gap, evidence was gleaned and adapted from the literature on sexually transmitted infections and from research findings as they emerged from the field (before publication).Two unique characteristics of Zika presented themselves when applying the evidence. First, unlike dengue or chikungunya, Zika is mostly asymptomatic or presents mild symptoms; LAC is the first setting where it has been widely associated with adverse pregnancy outcomes (and Guillain-Barré syndrome). Second, it was found that Zika can be transmitted sexually, but other arboviruses, such as dengue and chikungunya, cannot. Although we were able to adapt an extensive evidence base for vector control of the The third criteria of the process comprised a set of 3 components related to behavior amenability to change in the LAC setting. This step was an important contextual step because the effectiveness of a behavior is largely dependent on whether people are able and willing to perform it and was additionally complicated because Zika was new to the region. Because the behavior prioritization process was started after a year of Zika response, local partners had field experience to advise on which priority behaviors were feasible to implement and which materials were available and affordable. A consensus-building approach with USAID and its implementing partners was developed to categorize the relevant behaviors by criteria. Although the evidence review highlighted behaviors with potential to prevent diseases with the same modes of transmission or reduction of the same vector population as Zika, it was critical for each focal behavior to be assessed through the lens of this third criteria to ensure partner buy-in—that recommendations were realistic and reflected the context on the ground. The result of the process was a set of priority behaviors based on a combination of research evidence and context from partners with local knowledge.The result of the process was a set of priority behaviors based on a combination of research evidence and context from partners with local knowledge.Supplement 1], Technical Specifications Content Guide [Supplement 2], and a job aid). These products were useful for SBC programs to reconfigure and refine their messages as they continued to implement their SBC interventions. Many partners reported that the guidance provided a basis to focus their limited remaining resources and to attain the needed specificity for each behavior to be effectively implemented. The iterative, collaborative process of defining behaviors across all stakeholders was critical to ensuring a more harmonized and feasible response. This process strives to incorporate evidence where it is available and was refined as the work developed, allowing it to be responsive to new evidence and contextualized based on input and expertise from those on the ground. This process can identify and select behaviors with the most potential to reduce transmission, is designed to be adapted to local contexts, and is flexible and based on consensus building with local and international stakeholders. Our experience developing this process provides a potential model for future public health emergencies, as it highlights a way forward in prioritizing behaviors in evidence in situations where direct evidence is limited or absent, time is constrained, and there are many key stakeholders.The behavior prioritization process was developed to streamline the Zika response but began mid-implementation, by which time many partners were already deploying various behavior change recommendations. Any suggested changes to SBC programming that stemmed from the findings of this necessitated midcourse corrections. All 5 behaviors were prioritized by the USAID Zika response across 20 countries, and included interventions such as regional mass media campaigns, household and school-based SBC activities, household visits by vector control technicians, community fairs, and health care provider counseling. Several key products were developed and made available to implementing partners to facilitate the incorporation of the findings of the process, including a list of priority behaviors and detailed guidance on how to correctly perform each behavior for maximum effectiveness (through a Behavior Matrix [


Decision making in dynamic environments requires discounting old evidence that may no longer inform the current state of the world. Previous work found that humans discount old evidence in a dynamic environment, but do not discount at the optimal rate. Here we investigated whether rats can optimally discount evidence in a dynamic environment by adapting the timescale over which they accumulate evidence. Using discrete evidence pulses, we exactly compute the optimal inference process. We show that the optimal timescale for evidence discounting depends on both the stimulus statistics and noise in sensory processing. When both of these components are taken into account, rats accumulate and discount evidence with the optimal timescale. Finally, by changing the volatility of the environment, we demonstrate experimental control over the rats’ accumulation timescale. The mechanisms supporting integration are a subject of extensive study, and experimental control over these timescales may open new avenues of investigation. In a dynamic environment old evidence could be outdated. Here, the authors investigate the ability of rats to integrate and discount evidence provided by auditory clicks to infer a hidden, dynamic, state of the world and model the consequence of sensory noise to explain the source of errors. A well characterized decision-making paradigm is that of evidence accumulation or evidence integration referring to the process by which the subject gradually processes evidence for or against different choices until making a well defined choice. Evidence accumulation is thought to underlie many different types of decisions7, meaning equal weighting of all evidence across time. However, real world environments change over time. Crucially, in a dynamic environment older observations may no longer reflect the current state of the world, and an observer needs to modify their inference processes to discount older evidence. Previous studies have demonstrated that humans can modify the timescales of evidence integration, adopting leaky integration, which discounts old evidence9. These observations open many questions related to why and how subjects might alter their integration timescales, and whether the strategy adopted by humans is the optimal one. Recent studies developed a connection to normative drift-diffusion models (DDMs), and examined evidence accumulation in dynamic environments either in humans10, or in ideal observer models11. Glaze et al.9 found that while humans discount evidence, they did not appear to adopt the optimal discounting timescale. Here, we expand on a recently developed modeling framework11 to demonstrate that the optimal discounting strategy depends not only on the environment’s volatility, but also on the level of noise in sensory processing. Previous work11 incorporated stimulus statistics into their models, but did not incorporate sensory noise. We found that once both volatility and sensory noise are taken into account, rats adopt the optimal integration timescale. We furthermore show that rats can dynamically modulate their integration timescale according to changing environmental statistics. Our findings establish rats as an adequate animal model for studying evidence accumulation and discounting in a dynamic environment.Most behavioral studies to date have focused on evidence accumulation in stationary environments. In stationary environments, the normative behavioral strategy is perfect integration14. Within each trial, the dynamic environment is in one of two hidden states S1, and S2, each of which has an associated left and right click generation rate (S1: rates S2: rates S1 and S2 were symmetric (r1 and r2). Each trial starts with equal probability in one of the two states, and switches stochastically between them at a fixed hazard rate h. On each time step, the switch probability is given by hΔt, . At the end of the stimulus period, the auditory clicks end, and the center light turns off, indicating the rat must make a left or right choice by entering one of the side reward ports. The rat is rewarded with a water drop for correctly inferring the hidden state at the end of the stimulus period . The stimulus duration is variable on each trial (0.5−2 s), so the rat must be prepared to infer the current hidden state at all times. Figure We developed a decision-making task that requires accumulating noisy evidence in order to infer a state that is hidden, and dynamic. Rats were trained to infer during the course of a trial, which of two states the environment was in at that moment. In each trial of our task, we first illuminate a center light inside an automated operant chamber to indicate that the rat may start the trial by nose-poking into the center port. While nose poking, auditory clicks play from speakers positioned on the left and right sides of the rat. The auditory clicks are generated from independent Poisson processes. Importantly, the left and right side Poisson rate parameters are dependent on a hidden state that changes dynamically during the course of each trial. This is in contrast to previous studies where the Poisson click rates are constant for the duration of each trialh = 1 Hz, and click rates r1 ≈ 38 Hz, and r2 ≈ 2 Hz were kept constant. The click rates were chosen to be as difficult as possible while keeping rat accuracy above 70%. The chosen parameter values correspond to a high difficulty; as described below, the performance of the optimal agent for these parameters is ≈77% .Before the derivation, it is worth building some intuition. Because the hidden state is dynamic, auditory clicks heard at the start of the trial are unlikely to be informative of the current state. However, because state transitions are hidden, an observer doesn’t know how far back in time observations are still informative of the current state. Our derivation derives the optimal weighting of older evidence. We first consider observations in discrete timesteps of short duration Δ11, the probability of being in State 1 at time t, given all observed samples up to time t:Following ref. t, P(S1|εt1…), is proportional to the probability of observing the evidence sample at time t given State 1, P(εt|S1), times the independent probability that we were in State 1 given evidence from timesteps 1…t − 1, P(S1|εt−11…). This second term has two components which depend on the probability of remaining in the same state from the last time step, (1 − hΔt)P(S1|εt−11…), and the probability of changing states after the last time step, hΔtP(S2|εt−11…).We can interpret this equation as the probability of being in State 1 given all observed evidence up to time Rt) of the current state given all previous evidence samples εt1…:Combining the probability of each state, we can write the posterior probability ratio  and variance (diffusion), transforming Eq. . In our task a momentary sample εt is the result of two independent Poisson processes and can take on four possible values: a click on both sides, a click on the right, a click on the left, or no clicks. Evaluating the evidence term for these four conditions:This differential equation describes the evolution of the log-probability ratio of being in each of the two hidden states, See Fig. . The eviming Eq.  into theA click on both sidesNo clicksA click on the rightA click on the leftκ to be the increase in the log-probability ratio from the arrival of a single click on the right, given click rates r1, r2. The function κ tells us how reliably each click indicates the hidden state. This is easily seen when letting Δt → 0, so r1 and r2 are very similar  then we expect many distractor clicks , so an individual click tells us little about the underlying state. On the other hand, if the click rates are very different (so κ is large) then we expect very few distractor clicks, so an individual click very reliably informs the current state. In the limit of one of the click rates going to zero: κ → ∞, and a single click tells us the current state with absolute certainty. In our task, the two click rates r1 and r2 always sum to 40 Hz. Figure κ as a function of the click rates.We define the function κ and using δL/R,t to represent the left/right click times, we can summarize across all four conditions:Re-writing the log-evidence term in Eq.  in termsκ, let We can then rescale Eq.  by κ, leδtR, − δtL,, while accumulated evidence is discounted, h, and the reliability of each click, κ. This interpretation also allows for a simple assay of behavior: do rats adopt the optimal discounting timescale? We will present two quantitative methods for measuring the rats discounting timescales. However, before examining rat behavior, we need to examine the impact of sensory noise on optimal behavior.Equation  has a siκ tells us how reliably each click indicates the underlying state as a function of the click generation rates r1 and r2. The computation above of κ assumes that each click is detected and correctly localized as either a left or right click with perfect accuracy. Previous studies using pulse-based evidence demonstrate that rats have significant sensory noise17. Sensory noise was measured through parametric models that included a parameter noise due to each pulse of evidence. The exact biological origin of this noise remains unclear. Regardless of its origins, sensory noise is a significant component of rodent behavior. However, previous inference models in dynamic environments have not incorporated sensory noise.11The function n:We now show that sensory noise decreases the reliably of each click. While sensory noise can be modeled in many ways, primarily the mislocalization of clicks changes the click reliability. We analyze the cases of Gaussian noise on the click amplitudes and missing clicks, and provide a general argument for mislocalization in Supplementary Note A click on the rightA click on the leftκ. We can simplify the expression for κ by letting Δt → 0:The terms for no clicks, or clicks on both sides evaluate to 0. As in the case with no sensory noise, the log-evidence is either 0, or has value n decreases κ. If n = 0, we recover the original κ derived without noise. If n = 0.5, then each click is essentially heard on a random side, and therefore contains no information so κ = 0. If n = 1, then we simply flip the sign of all clicks.Sensory noise decreases how reliably each click informs the underlying state in the trial, increasing 17. We computed an estimate of the average sensory noise for the rats in ref. 12, finding an average value of n = 0.35. Figure κ against n and click rates r1, r2, and highlights the average rat sensory noise from ref. 12. Click mislocalization for each rat was estimated by fitting a parametric model introduced below and in ref. 12. See methods for estimation of click mislocalization from model parameters. Two lines of evidence suggest that our estimate of the level of sensory noise is reliable. First, ref. 12 predicted performance on single-click trials based on each rat’s sensory noise . Second, we found click mislocalization levels were constant across a wide range of click rates in ref. 12 ). We simulated the optimal inference agent on a large dataset of trials assuming either no sensory noise (black), or average rat sensory noise (pink). For both agents we computed the distribution of when changes of mind happen relative to changes in the hidden state of the trial. Figure The optimal inference equation attempts to predict the hidden state. As the hidden state dynamically transitions, we expect the inference process to track, albeit imperfectly, the dynamic transitions. From the perspective of a subject this dynamic tracking leads to changes of mind in the upcoming choice. Through the optimal inference process we can predict the timing of changes of mind by looking for times when the sign of the inference process changes . In addition to the click integration and linear discounting that was present in our normative theory, this model also parameterizes many possible sources of noise. Each click has multiplicative Gaussian sensory noise, C. The adaptation process is parameterized by the adaptation strength ϕ, and a adaptation time constant τϕ. If ϕ > 1 the model has facilitation of sequential clicks, and if ϕ < 1 the model has depression of sequential clicks. The accumulation variable a also undergoes constant additive Gaussian noise W with variance σa2. Finally, the initial distribution of a has some initial variance given by σi. See ref. 12 for details on the development and evaluation of this model. The only modification to the model from previous studies is the removal of the sticky bounds B, which are especially detrimental to subject performance given the dynamic nature of the task. This model is a powerful tool for the description of behavior on this task because of its flexibility at characterizing many different behavioral strategies14. We parameterized the model with linear discounting, rather than nonlinear discounting in the full theory for three reasons. First, the linear discounting model has been fit to rat behavior in static environments, allowing a direct comparison to previous results. Second, the linear model has an analytical solution that greatly facilitates analysis. Third, the linear model has comparable accuracy to the nonlinear model with less parameters, simplifying the fitting procedure and providing a more parsimonious description of rat behavior.At each moment in a trial, the model generates a distribution of possible accumulation values 18. Supplementary Table λ . The evidence discounting strength parameter λ shows a striking difference between the two rat populations  to a static environment (h = 0 Hz), and then back. The rats trained in each environment for many daily sessions (minimum 25 sessions). In each environment, we quantified the rats’ behavior using the model of Eqs. , which gave us the opportunity to examine how quickly it adapted to the new environment, by fitting the trial-by-trial model in consecutive blocks of 7500 trials. Only the evidence discounting parameter λ was fit to each block, the other parameters we fit separately to the first set of 0.5 Hz trials. Figure Previous studies have demonstrated that rats can optimally integrate evidence in a static environment of Eqs.  and 15)12. Here 24. These studies have showed that subjects can gradually accumulate evidence for decision-making, adopting long integration timescales. Trial-by-trial modeling12 revealed that errors originated from sensory noise, not from the evidence accumulation process. Using a high-throughput automated rat training, we trained rats to accumulate and discount pulse-based evidence in a dynamic environment. Extending results from the literature11, we formalized the optimal behavior on our task, which critically involves discounting evidence on a timescale proportional to the environmental volatility and the reliability of each click. Importantly, our pulse-based analysis allowed us to separate evidence reliability into experimenter imposed stimulus statistics and sensory noise. We find that once sensory noise is taken into account, the rats have timescales consistent with the optimal inference process. We used behavioral modeling to investigate rat to rat variability, and to predict a moment-by-moment estimate of the rats’ accumulated evidence. Finally, we demonstrated rats can adjust their integration timescales in response to changing environmental statistics. Our findings open new questions into complex rodent behavior and the underlying neural mechanisms of decision making.Previously accumulation of evidence has been studied in a stationary environment. These studies have given behavioral and neural insights into the ability of rats, monkeys, and humans to optimally accumulate evidence9 examined human decision making in a dynamic environment, and found that humans show nonlinear evidence discounting, but do not match the optimal inference process. Quantifying the subject’s estimates of environmental volatility, rather than the discounting rate, they found that subjects typically underestimated the volatility . This finding is consistent with the role of sensory noise decreasing the discounting rate. Incorporating models of human sensory noise into their analysis could potentially explain deviations from optimality. However, our rats performed more trials than the human subjects in ref. 9. Human subjects with more experience may more closely match optimal processes. Unlike ref. 9, we did not examine whether our subjects demonstrated nonlinear evidence discounting because the linear approximation in our task is very close to the nonlinear theory  as a potential substrate for upcoming choice memory33. Many circuit mechanisms have been proposed, from random unstructured networks35, feed-forward syn-fire chains31, and recurrent structured networks of many forms37. The task developed here allows for experimental control of the putative neural integrator’s timescale within the same subject. Measurement of neural activity in different dynamic environments, and thus different integration timescales, may shed light into which mechanisms are used in neural circuits for evidence integration. For instance, unstructured networks, or feed-forward networks may re-tune themselves via adjusting read-out weights. Networks that integrate via recurrent dynamics; however, would re-tune themselves via changes in those recurrent dynamics. Alternatively, measurement of neural activity in different dynamic environments may reveal fundamentally new mechanisms of evidence integration. For instance, ref. 13 proposed multiple integration networks with different timescales to account for behavioral changes in response to cortical inactivations. Our task may allow further investigation into the structure and dynamics of neural integrators.Second, normative behavior in a dynamic environment requires tuning the timescale of evidence integration to the environmental volatility. There is a large body of experimental and theoretical studies on neural integrator circuitsAnimal use procedures were approved by the Princeton University Institutional Animal Care and Use Committee and carried out in accordance with NIH standards. All subjects were adult male Long Evans rats  placed on a controlled water schedule to motivate them to work for a water reward.We trained 14 rats on the dynamic clicks task , this creates one smooth click rate for each trial. The filter had a standard deviation of 5 ms.The computation of the reverse correlation curves was very similar to methods previously reportedThen, the smooth click rate on each trial was normalized by the expected click rate for that time step, given the current state of the environment. This gives us the deviation (the excess click rate) from the expected click rate for each trial.Finally, we compute the choice triggered average of the excess click rate by averaging over trials based on the rat’s choice.aebt to each curve. The parameter a is a scale parameter. The parameter b is the discounting rate, while 1/b is the integration timescale. All exponential fits were computed using the MATLAB package fit, which used a least squares fit on a linear scale. 95% Confidence intervals on the exponential fits are shown in Supplementary Figure fit package.The excess rate curves were then normalized to integrate to one. This was done to remove distorting effects of a lapse rate, as well to make the curves more interpretable by putting the units into effective weight of each click on choice. To quantify the timescale of the reverse correlation curves, we fit an exponential of the form 12 have included sticky bounds which absorb probability mass when the accumulated evidence reaches a certain threshold. We found this sticky bounds to be detrimental to high performance on our task, so we removed them. The removal of the sticky bounds facilitates an analytical solution of the model. The model assumes an initial distribution of accumulation values P is Gaussian distributed with mean μ and variance σ2 given by:R is the number of right clicks on this trial up to time t, and R(i) is the time of the ith right click. C(R(i)) tells us the effective adaptation for that clicks. For a detailed discussion of a similar model, see ref. 38.Previous studies using this behavioral accumulation of evidence modelB, we can compute the left and right choice probabilities by:Given a distribution of accumulation values θ were fit to each rat individually by maximizing the likelihood function:These choice probabilities are then distorted by the lapse rate, which parameterizes how often a rat makes a random choice. The model parameters σi and accumulation noise parameters σa in some rats, but strongly supported keeping the parameters in other rats. Due to the presence of large discounting rates, these parameters are difficult to recover in synthetic datasets. Given the mixed BIC analysis, we included these parameters but constrained them with a half-gaussian prior on the initial noise σi and accumulation noise parameters σa. The priors were set to match the respective best fit values from ref. 12. Removal of these priors did not alter our conclusions about discounting strength, λ. The numerical optimization was performed in MATLAB, using the function fmincon. To estimate the uncertainty on the parameter estimates, we used the inverse Hessian matrix as a parameter covariance matrix.18 To compute the hessian of the model, we used automatic differentiation to exactly compute the local curvature39. See Supplementary Table 12 extensively analyzed how well this model recovers generative parameters, finding the model contains one maximum likelihood point in parameter space (See Section 2.3.3-6 of the supplement to ref. 12).BIC analysis supported a reduced model without the initial noise ϕ, and τϕ), we computed the average level of sensory adaptation on each click 〈C〉. Then, we computed what fraction of the probability mass would cross 0 to be registered as a click on the other side (See Supplementary Note Given the model parameters (The code and software that support the findings of this study are available from the authors upon request.Supplementary InformationPeer Review File


N-acetylgalactosamine-4-sulfatase (arylsulfatase B). The heterogeneity and progressive nature of MPS VI necessitates a multidisciplinary team approach and there is a need for robust guidance to achieve optimal management. This programme was convened to develop evidence-based, expert-agreed recommendations for the general principles of management, routine monitoring requirements and the use of medical and surgical interventions in patients with MPS VI.Mucopolysaccharidosis (MPS) VI or Maroteaux-Lamy syndrome (253200) is an autosomal recessive lysosomal storage disorder caused by deficiency in 26 international healthcare professionals from various disciplines, all with expertise in managing MPS VI, and three patient advocates formed the Steering Committee group (SC) and contributed to the development of this guidance. Members from six Patient Advocacy Groups (PAGs) acted as advisors and attended interviews to ensure representation of the patient perspective. A modified-Delphi methodology was used to demonstrate consensus among a wider group of healthcare professionals with expertise and experience managing patients with MPS VI and the manuscript has been evaluated against the validated Appraisal of Guidelines for Research and Evaluation (AGREE II) instrument by three independent reviewers.A total of 93 guidance statements were developed covering five domains: (1) general management principles; (2) recommended routine monitoring and assessments; (3) enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT); (4) interventions to support respiratory and sleep disorders; (5) anaesthetics and surgical interventions. Consensus was reached on all statements after two rounds of voting. The greatest challenges faced by patients as relayed by consultation with PAGs were deficits in endurance, dexterity, hearing, vision and respiratory function. The overall guideline AGREE II assessment score obtained for the development of the guidance was 5.3/7 .This manuscript provides evidence- and consensus-based recommendations for the management of patients with MPS VI and is for use by healthcare professionals that manage the holistic care of patients with the intention to improve clinical- and patient-reported outcomes and enhance patient quality of life. It is recognised that the guidance provided represents a point in time and further research is required to address current knowledge and evidence gaps.The online version of this article (10.1186/s13023-019-1080-y) contains supplementary material, which is available to authorized users. Mucopolysaccharidoses (MPS) are part of a clinically heterogeneous group of diseases known as lysosomal storage disorders (LSDs), of which there are over 60 different types. Symptoms of MPS occur because of deficiencies in enzymes that break down glycosaminoglycans (GAGs) –3. EleveN-acetylgalactosamine-4-sulfatase . Birth prevalence has been reported to range from 1 in 43,261 live births in Turkish immigrants living in Germany ), which may, in part, be attributed to growth in young patients  or penetrating keratoplasty [PK]) should be made on a case-by-case basis. There is some evidence from the general population to suggest that rejection is more likely to occur following PK than DALK [han DALK –108; as Considerations for intervention (e.g. if surgery) Monitored anaesthesia care with appropriate sedation and support with nasal CPAP/NIPPV may be used when performing eye surgery in patients with MPS VI. Signs of rejection require prompt ophthalmic assessment to prevent graft failure. Following corneal transplantation, long-term topical treatment is needed, as is regular  ophthalmic assessment to determine the health of the corneal graft, assess for recurrence of corneal deposits and astigmatism control. Follow-up is also required to monitor for optic neuropathy due to raised intracranial pressure. This can be indicated by a reduction in visual acuity, new onset of visual field defect, abnormal pupil reactions, new onset of optic nerve swelling or (more commonly) optic atrophy and visual evoked potential abnormality.Rationale and evidence base Carpal tunnel syndrome (CTS) is a common condition in patients with MPS VI, which if left untreated, can lead to loss of nerve function. Trigger finger can also present in association with CTS in patients with MPS VI. Surgical decompression of CTS, especially when performed early, has reduced signs and symptoms of compressive myelopathy and improves the chance of preserving hand function [function , 110. Evfunction . The prePatient selection for intervention CTD should be performed in patients who display restriction of hand function, including increased flexion contracture of the PIP and DIP joints, indicative of an increase in ‘scarred’ and tethered subsynovial connective tissue around the flexor tendons in the carpal canal. Clinical symptoms of hand pain/numbness include night pain, biting and/or slapping of fingers and pain or numbness in the thumb, index or middle finger. It is important to note that if a patient reports numbness in all fingers it is unlikely to be CTS, and pain in the little finger is suggestive of compression higher up than the carpal canal. Neurophysiological tests should be performed to rule out cervical compression before initiating surgery.Considerations for surgery CTD should be performed by a hand surgeon with a basic understanding of MPS and of the clinical presentation associated with this group of disorders. In most cases, CTD may be performed with regional block plus sedation, with or without the use of LMA.Considerations for post-surgery monitoring Regular post-surgical physical therapy may facilitate maintenance of increased hand movement. All patients who undergo CTD surgery should be assessed (pre- and post-surgery) for QoL, improvement in ROM and self-reported change in function.Rationale and evidence base Cardiac valve disease, which occurs as a result of valve thickening from deposition of storage material, occurs commonly in patients with MPS VI [h MPS VI  and is ah MPS VI –116. Such MPS VI , but sucPatient selection for intervention (refer to the recommendations for routine assessments and monitoring) The performance and interpretation of findings of echocardiography should be completed by individuals familiar with the expected pathological findings in patients with MPS VI. Valve replacement decisions should be based on current European/American (AHA) guidelines [idelines , 119 in Consideration for surgery Cardiac surgery in patients with MPS VI should be performed in a centre of excellence with a team experienced in both managing patients with MPS and performing high-risk valve replacement surgery. When possible, an anaesthetic specialist with experience in managing patients with MPS should assist the cardiac anaesthetic team during pre-operative assessment in formulating an MPS-related anaesthesia care plan. Similarly, the anaesthetic care plan for cardiac catheterisation should be formulated jointly by the cardiologist and anaesthetic care team specialist. Airway care, including the need for tracheostomy, should be assessed on a case-by-case basis.Rationale and evidence base ENT manifestations are common in patients with MPS VI and often involve hearing disorders, otitis media and upper airway obstruction [truction . Permanetruction , 120. Retruction , 121. Thtruction . Developtruction , 122. Evtruction . AdvanceConsiderations for surgery Vaccinations against respiratory pathogens causing influenza and pneumococcus infections are recommended to prevent pneumonia. Insertion of ventilation tubes should be performed according to the guidelines for the general paediatric population [pulation , 124. Papulation .Recommendations for the management of MPS IVA: systematic evidence- and consensus-based guidance).Where evidence is scarce, systematic approaches are required to ensure the evidence-base is as extensive as possible. This programme involved a validated systematic approach to the development of guidance statements and the resulting publication therefore addresses this unmet need by creating a robust, holistic set of recommendations for healthcare professionals managing patients with MPS VI. A detailed discussion on the development of the guidance methodology, strengths and limitations of the programme, future directions, facilitators/barriers to support the application of the guidance including cost considerations, and conclusions is provided in the discussion of the companion article of this publication Additional file 2:Oxford CEBM grading for MPS IVA: Tables detailing the evidence levels given to each reference supporting the MPS IVA guidance statements and the Evidence Grades applied to each guidance statement. Evidence levels were assessed using the Oxford Centre for Evidence-based Medicine and were based on the quality of evidence of each reference. For each guidance statement, an overall Evidence Grade was applied, based on the evidence levels of the supporting references. (DOCX 41 kb)Additional file 3:Oxford CEBM grading for MPS VI. Tables detailing the evidence levels given to each reference supporting the MPS VI guidance statements and the Evidence Grades applied to each guidance statement. Evidence levels were assessed using the Oxford Centre for Evidence-based Medicine and were based on the quality of evidence of each reference. For each guidance statement, an overall Evidence Grade was applied, based on the evidence levels of the supporting references. (DOCX 41 kb)Additional file 4:Modified-Delphi voting Round 1. Full results of the first round of the modified-Delphi voting, which was used to demonstrate consensus of the guidance statements. (DOCX 151 kb)Additional file 5:Modified-Delphi voting Round 2. Full results of the second round of the modified-Delphi voting, which was used to demonstrate consensus of the guidance statements. (DOCX 41 kb)


P = 0.04; medial fractures: 0.82 vs. 0.68±0.03 in session 1 only, P = 0.04; caudal fractures: 0.41 vs. 0.29±0.03, P = 0.03), and inexperienced assessors classified medial fractures more accurately in session 2 . However, effect sizes were small for deviations and even experienced assessors lacked accuracy when assessing caudal fractures. Unexpectedly, deviations tended to be assessed more accurately in session 1 than in session 2, regardless of assessor status , suggesting that prolonged assessment contributes to errors. Prior experience decreased specificity and precision of fracture assessment (more unfractured keels were classified as fractured) even though overall accuracy was greater. Intra-rater consistency was fair to good (0.55 to 0.67) for deviations and medial fractures, but poor to fair (0.36 to 0.44) for caudal fractures, and unaffected by prior experience (P = 0.49 to 0.89). In conclusion, experience improves accuracy to a limited extent but does not guarantee high accuracy for all types of damage. Future research should determine if other training methods  improve accuracy.Accurate assessment is essential when evaluating keel bone damage. Palpation is commonly used to assess keel bone damage in living hens. However, there is little information on the accuracy of assessment of deviations and fractures on different parts of the keel, and on the consistency within, and agreement between, assessors. Crucially, although the importance of experience is commonly emphasized, knowledge on its effect is scarce. Ten assessors with or without prior experience palpated the same 50 75-wk-old hens for deviations, medial fractures, and caudal fractures (scored as present/absent). Accuracy, sensitivity, specificity, precision, and negative predictive value were determined by comparing palpation scores to post-dissection assessment, and then compared between experienced and inexperienced assessors. To determine the effect of the experience gained during the experiment, hens were subsequently re-assessed. Consistency within, and agreement between, assessors were also determined. Assessors with prior experience were more accurate (proportion of accurately assessed deviations: experienced 0.83 vs. inexperienced 0.79±0.01,  Keel bone damage is a major welfare problem facing the laying hen industry due to its high prevalence  of keel bone damage assessment by palpation. We hypothesized that experienced assessors would be more accurate than inexperienced ones when assessing deviations, medial fractures, and caudal fractures. In addition, we hypothesized that at least the inexperienced assessors would become more accurate as the experiment progressed and they became more familiar with the technique.This experiment involved 10 assessors, 4 of which had previous experience with performing all 3 types of keel bone assessment. Two of these assessors had been trained by an expert assessor as part of a European research project . The third assessor had been trained by the first 2, and in turn trained the fourth. Experienced assessors 1 to 3 had each scored approximately 60 flocks in the 2 yr before the experiment, whereas the fourth assessor had scored approximately 20 flocks. On these occasions, 50 to 100 hens were assessed per flock and the assessors regularly compared their scoring to each other to improve agreement. One of these assessors compared palpation scores to post-dissection scores to improve palpation accuracy. This palpation-dissection comparison was only performed on a single occasion. A fifth assessor had been trained by a different international expert to assess medial fractures only, and had subsequently gained experience several years before the experiment by scoring 20 flocks (100 hens/flock) and by making regular comparisons between palpation and post-dissection scores (15 hens/flock). This assessor was considered an experienced assessor for medial fractures only. However, this fifth assessor had performed palpations on a very limited number of hens in the 7 yr prior to the experiment. Therefore, his status as “experienced” was somewhat questionable and the analyses of medial fractures were repeated without this assessor's results. The remaining 5 assessors had no previous experience with keel bone assessment, but all had experience handling poultry. Prior to the experiment all assessors were given a 30-min audio–visual training in which the assessments were explained but no samples were handled.Intact carcasses of 50 hens (Lohmann Brown Classic) obtained from a commercial aviary system with outdoor access when 75 wk old were frozen until palpation (2 mo later). All birds were thawed and then palpated to assess keel bone deviations, and healed fractures on the medial and caudal tip of the keel bone. Palpations were performed as described by Heerkens et al. : the cauP > 0.10) interactions and main effects were removed from the models. When interactions occurred, pairwise comparisons were made using step-up Bonferroni correction. Pairs differing in 2 factors were not compared .All statistics were performed in R 3.3.3 . Also, assessment tended to be less accurate in the second session than in the first (P = 0.060). Sensitivity, specificity, and precision were affected by an experience*session interaction . Inexperienced assessors were less sensitive in the second than in the first session . Within this session they also tended to be less sensitive than experienced assessors. The interactive effect on specificity  did not result in significant pairwise differences. The precision of the experienced assessors tended to be lower in the second than in the first session . Negative predictive value tended to be lower in inexperienced assessors and was significantly lower in the second session .The true prevalence of keel bone deviations was 60%, close to the percentage classified as deviated by our experienced assessors . On average, our inexperienced assessors were also close to the true prevalence, although their range was wide . Performance of the binary classification was good (>0.68) according to all statistics in both sessions and for both inexperienced and experienced assessors Figure . HoweverInter-rater agreement of deviation scoring during the first session was good for experienced assessors but poor for inexperienced assessors  after the exclusion of assessor 5. All inexperienced assessors on the other hand underestimated this prevalence and their range was relatively wide .As our sample mainly included fractured keels, accuracy, sensitivity, and precision mainly (or wholly) depended on the correct classification of truly fractured keels. The good to very good (0.65 to 0.93) scores for these 3 statistics show that our assessors had relatively little difficulty classifying the fractured keels. They had more problems exclusively classifying the small percentage of truly unfractured keels as such, as reflected by the lower scores for specificity (<0.54) for experienced assessors and the poor negative predictive value (<0.36) for all groups Figure .P = 0.036 and 0.019, respectively). The improvement in both statistics between the first and second session reached significance for inexperienced assessors only . Specificity and precision were higher in inexperienced assessors than in experienced ones . Negative predictive value was higher in the second session .Accuracy and sensitivity were affected by an experience*session interaction  led to similar outcomes, although more pairwise differences occurred Table . NotablyInter-rater agreement of medial fracture scoring was fair between inexperienced assessors, but poor between experienced ones in both sessions Table . HoweverP = 0.031 and P = 0.005, respectively). However, experienced assessors also had a lower specificity and tended to have a lower precision . This misclassification of fractured keels tended to be less common in the second session, as shown by increased specificity and precision in session 2 (both P = 0.051). The negative predicted value (the proportion of keels classified as unfractured that were classified correctly) was not affected by experience or session (P > 0.10).The true prevalence of caudal tip fractures was 85.1%. Hens were often found to have caudal as well as medial fractures . All assessed hens had either a caudal fracture or a medial one. Our experienced and inexperienced assessors respectively classified only 37% (min–max: 34 to 40%) and 16% (min–max: 4 to 25%) as having a caudal fracture, thus underestimating prevalence markedly. In line with this accuracy, sensitivity and the negative predicted value were poor  as well as fractures . As all our hens had at least 1 type of keel bone fracture we did not evaluate the accuracy of the pooled keel bone fractures , as the impossibility of true negatives and false positives would distort the results.Palpation is an indispensable technique to assess keel bone damage in situations where other techniques like dissection or radiography are impossible or highly impractical. Although palpation accuracy was not perfect, we found that not only extensive but even minimal experience  with palpation techniques can lead to a relatively accurate assessment of deviations and medial fractures. The technique also showed good consistency within assessors, flock level prevalence estimates close to true prevalences, and fair inter-assessor agreement (at least after the first session). This supports the continued use of palpation for deviations and medial fractures in cases where other methods are not feasible. The wide range in individual inexperienced assessors’ flock level deviation prevalence estimates (and their poor inter-assessor agreement in the first session) suggest that lack of experience can lead to both underestimation and overestimation, depending on the individual assessor. In contrast to the favourable results for deviations and medial fractures, both experienced and inexperienced assessors were inaccurate when assessing caudal tip fractures. They markedly underestimated their prevalence, showed poor agreement between assessors and a consistency within assessors that was only just fair. This suggests that, unless different training methods can improve the accuracy of caudal tip palpation, this technique cannot be recommended for use.Both types of assessors scored deviations with high accuracy (0.78 to 0.86) in both sessions. But as expected, experience prior to the experiment improved this accuracy. More specifically, experienced assessors had a higher negative predictive value overall and a higher sensitivity in the second session, showing they were less likely than inexperienced assessors to overlook deviations. Although inexperienced assessors’ accuracy of deviation assessment was high and their intra-rater consistency fair, their inter-rater agreement was poor in the first session, showing that different inexperienced assessors made different mistakes. Experienced assessors on the other hand combined a high accuracy and consistency with a good inter-rater agreement in the first session. Somewhat surprisingly, their inter-rater agreement was lower in the second session. This may mean that they were reverting to their own style of palpating or their original training (which was likely somewhat different for each assessor as they had been trained by different trainers at different times). Alternatively, it may mean that the experienced assessors lost their concentration after prolonged scoring. Such an effect seems to have affected the inexperienced assessors as well. Their performance was expected to increase in the second session (as they were now somewhat more experienced). However, the opposite was found: during the second session accuracy tended to be lower. More specifically, inexperienced assessors were less sensitive during the second session , whereas experienced assessors were less precise . In addition, both types of assessor had a lower negative predictive value in the second session . For inexperienced assessors, it seems probable that their recollection of the training prior to the first session was diminished during the second session. However, this seems less likely for experienced assessors (as their assessment should not be as strongly influenced by the training which was only a reminder for them). Decreased concentration after scoring many hens may also have contributed to the lower accuracy of both types of assessor in the second session. Taken together, these findings suggest that although deviation assessment by palpation is a technique that can be applied reliably after a short training, it can be improved by increased experience. Furthermore, the lower accuracy in the second session suggests that both experienced and inexperienced assessors may benefit from (reminder) training just before starting an assessment session or from avoiding prolonged assessments.The accuracy of our experienced assessors' keel bone deviation assessment (0.80 to 0.86) was in between the values reported in previous studies using a single experienced assessor  inter-rater agreement between the experienced assessors was fair. Before his exclusion inter-rater agreement had been poor (showing that this assessor scored markedly different than the others) and combined with his relatively low accuracy, this could suggest that his palpation skills had decreased due to lack of recent practice. Alternatively, it may indicate that for some assessors even rigorous training and extensive experience may not be sufficient to assure reliable application of keel bone palpation. In either case, this shows that regular feedback on palpation accuracy is of importance.In line with our other hypothesis that inexperienced assessors would improve during the experiment, their accuracy of medial fracture assessment was higher in the second session. Petrik et al.  found a The accuracy of our experienced assessors medial fracture assessment (0.82 to 0.85) was slightly higher than the 0.74 previously reported by Heerkens et al. , but subWith an accuracy of 0.29 to 0.43 caudal tip fractures were clearly more difficult to assess correctly than medial fractures, in line with previous results by Casey-Trott et al. , althougThe low sensitivity and negative predictive value show that many caudal fractures were not detected, which would lead to an underestimation of the prevalence if the palpation method is applied without further post-dissection assessment . In line with this, the flock level prevalence was markedly underestimated by experienced as well as inexperienced assessors . However, the accuracy of caudal tip fracture assessment by our experienced assessors (0.39 to 0.43) was much lower than in previous studies . More rigorous training methods  may be preferable to the now commonly used method of consensus training . The benefit of such methods should be evaluated in future studies. When assessing medial fractures, assessors lacking any prior experience may benefit somewhat from applying palpation techniques to a test set before doing real assessments, as this can improve their accuracy. However, an opposite effect was found for deviations (possibly due to loss of concentration). Assessment of caudal tip fractures was inaccurate even when performed by experienced assessors and, unless this can be improved by other training methods, it cannot be recommended for use.


Dirofilaria immitis and Acanthocheilonema reconditum are common parasites in dogs but have also been reported parasitizing humans. The differential diagnosis and epidemiological evaluation of these zoonoses are important to the development of efficient public health policies and control strategies. The purpose of this study was to carry out an epidemiological survey by using molecular methods for the specific identification of filarid parasites of domestic dogs in the Marajó mesoregion, State of Pará (PA), Brazil. A total of 418 canine blood samples from Marajó mesoregion (Northern Brazil) were collected, submitted to DNA extraction, polymerase chain reaction (PCR) with “pan filarial” primer, subsequent sequencing and sequence analysis using BLASTn software comparison with previously deposited sequences in GenBank. After that, a phylogenetic analysis by Maximum Parsimony was performed to aid the specific diagnosis. The obtained sequences showed the occurrence of 9 (2.15%) dogs infected with D. immitis and 30 (7.18%) by A. reconditum, with a confidence interval of 95%, there were no cases of co-infection. We observed that male dogs were more likely to D. immits and A. reconditum infection. However, age was not significant to both infections. This study reports for the first time the occurrence of A. reconditum in the northern region of Brazil and confirmed the presence of D. immitis in the Marajó mesoregion. Acanthocheilonema reconditum, Cercoptifilaria bainae, Dipetalonema grassi, Dirofilaria immitis and Dirofilaria repens . The blood was collected via cephalic vein puncture, using disposable sterile needles and stored in tubes with Ethylenediamine tetraacetic acid (EDTA) in a freezer at -20°C, for molecular analysis. The blood samples were collected from dogs with respective owner’s agreement, who were informed about the purpose of this research. Blood samples were collected following the order of houses within randomly chosen neighborhoods for sampling. In houses containing more than one dog all animals were sampled except for puppies less than 3 months of age.® Green . Canine blood samples were considered positive for filariasis when one or more bands were observed.All blood samples were submitted to DNA extraction using phenol-chloroform method . The ext®GEM-T  which in turn was introduced into Escherichia coli TOP 10 electrocompetent cells  and sequenced with ABI 3130 model automatic DNA analyzer , in conjunction with a BigDye®Terminator v3.1 Cycle Sequencing Kit , according to the manufacturer’s specifications.The PCR products were cloned into vector pBioEdit program [Ascaris lumbricoides (AB571301.1) and Trichuris trichiura (KC877992.1) sequences being used as an outgroups. Published sequences from Dipetalonema reconditum, now stated as Acanthocheilonema reconditum (AF217801.2), Dipetalonema dracunculoides (DQ018785.1), Dirofilaria repens (AY693808.1) and Dirofilaria immitis (EU182330.1) were also used for comparison with the sequences obtained in this work and for the construction of the phylogenetic tree.For a specific diagnosis, the obtained sequences were edited and aligned in the  program . Compare program  additona program  in MEGA  program  to aid i program . AscarisAll obtained sequences in this work were submitted to GenBank (accession numbers are shown in p<0.05).After specific diagnosis, we conducted an epidemiological analysis to investigate parameters that might influence the distribution of these infections. The prevalence of infected dogs was estimated based on specific diagnosis performed through phylogenetic inference. During sampling, information regarding age and sex of all dogs were obtained for association analysis with canine filariasis cases throughout Pearson chi-square test (X2), using the BioEstat 5.0  softwareThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the “Conselho Nacional de Saúde, Comissão Nacional de Ética em Pesquisa, Fundação Hemopa, Ministério da Saúde” (Permit Number: 0003.0.324.000–10).Among the 418 canine blood samples analyzed, 39 (9.33%) were considered positive for canine filariasis based in the presence of visible bands after PCR and electrophoresis. However, of the positive samples, we distinguish 9 samples with bands of approximately 500 bp and another 30 samples with bands of approximately 400 bp. None of the samples presented mixed bands; Rishniw et al.  also obsD. immitis sequences (between 91% to 100%), and the other 30 (7.58%) positive samples presented high identity values to A. reconditum (between 80% to 100%). Interestingly, the difference between the band patterns observed in this work was similar to that found by Rishniw et al.[D. immitis and 400 bp A. reconditum.Thus, all amplicons, were subjected to nucleotide sequencing and BLASTn analysis  and 9 , with 73% of Bootstrap.Clade I: formed by a politomy between the 30 sequences of this work and D. immitis (EU182330.1), with 83% of Bootstrap.Clade II: formed by a politomy between the 9 sequences of this work and D. immitis. However these authors did not identify A. reconditum in their analysis.Furtado et al.  performeD. immitis infection, but no specific method for species identification was performed. Thus, it is also possible that cases of misidentification by morphological analysis or cross-reaction in immunoenzymatic essays occurred in those studies. However, an epidemiological study in two municipalities of Marajó Archipelago , using classical and molecular methods for diagnosis, confirmed D. immitis parasitizing dogs in these municipalities and highlighted the endemic character of this zoonosis in this area [This is not the first description of canine filariasis in northern Brazil. Microfilaremic dogs have been reported in several cities, example, in Belém metropolitan region, State of Pará, based on the presence of microfilariae in blood smears ; Salvatehis area .D. immitis in Anajás, São Sebastião da Boa Vista and Portel, with a lower prevalence in Soure and Chaves, additionally, A. reconditum was detected in all municipalities studied  and Sebastião da Boa Vista (6.67%) [Previous studies in Marajó mesoregion, using molecular techniques, PCR and sequencing, have identified only  (6.67%) .D. immitis and A. reconditum infection in Marajó mesoregion, with males having a higher probability of acquiring the infection. On the other hand, the overall age was not significant in both infections. Specifically, in Soure municipality, only age (3 or more years old) was significant for infection by D. immitis. In contrast, in Chaves, just males showed to be more susceptible to the infection. The same was observed in Portel, where males were more susceptible to A. reconditum infection.The dog gender showed statistically significant association for This findings agree with studies performed by Furtado et al.  in the sHowever the differences observed here and the factors associated with heartworm transmission in dogs need to be studied in the Amazon Region, since a complex system of interaction between domestic dogs and wild animals can provide differentiated scenarios in the dispersion of infection, as previously described .A. reconditum . And this infection seems to be related to gender, but not to age, when analyzing the studied municipalities. None of the work reported the presence of this species of filarid in the Amazon, for this reason we cannot compare our results.The data presented in this paper describe for the first time in the Amazon Region the presence of dogs infected with A. reconditum only described the occurrence, without presenting consistent epidemiological data on this infection. Unfortunately, it was not possible to evaluate if this infection is recent or has been neglected in previous studies in the Amazon, since previous work have only used morphological and immunoenzymatic techniques which may lead to an erroneous species identification. Furthermore, the distribution patterns of D. immitis also need to be minutely studied in the Amazon, because, the correct diagnosis and dynamic of infections in the dog population will be important to the development of efficient public health policies, diagnosis and control strategies of zoonotic cases.Previous studies conducted in Brazil about D. immitis and A. reconditum are morphologically different and occupy distinct niches in the definitive host [A. reconditum in the Amazon Region opens up new possibilities for research on morphological and molecular differentiation of this filarid species.Adult stages of ive host . Howeverive host –34. Althive host , 35–36, Besides the diversity of canine filarid species in the extensive geographic area of the Amazon, other factors may have influence on the dynamic of those infections in the Amazon, and should be considered, as for example: (1) the high prevalence observed in previous studies; (2) the close association between humans and domestic animals cohabiting both urban and wild environments; and (3) the neglect of these infections by the public health authorities.A. reconditum was described for the first time in this study, other species not yet recorded, or new species, may be detected in future studies in this area.In the same manner that sequences of S1 Appendix(DOCX)Click here for additional data file.