In Malawi, children under the age of five living in different geographical areas may experience different malaria risk factors. We compare the risk factors of malaria experienced by children under the age of five from Zomba district, who reside in lakeshore and highland areas.We conducted a case control study of 765 caregivers, cases being children under-five who were diagnosed with malaria, and obtained matched controls from local health facilities and communities. We used a multivariate logistic regression to identify individual and household risk factors.In lakeshore areas, risk factors were households located one kilometer or less away from stagnant water ; or if the household had obtained a mosquito bed net more than one year ago . In highland areas, risk factors were households which used a borehole/unprotected well , communal standpipe , and un-protected dug well in their yards  as their drinking water sources. In highland areas, caregivers not attending health talks on malaria prevention messages was a risk factor .Children under the age of five living in highland areas experience different malaria risk factors compared to children living in lakeshore areas. Settling away from stagnant/open water source in lakeshore and encouraging caregivers to attend health talks on malaria prevention in highlands can help reduce malaria transmission. Nevertheless, using a mosquito bed net that is more than one year old is a common risk factor in both locations. Using new mosquito bed nets can significantly reduce the risk of contracting malaria in children under the age of five. Malaria is a major cause of morbidity and mortality in Malawi and many other countries world-wide . In MalaThe government of Malawi distributes free Insecticide Treated Bed Nets (ITNs) to people living in rural and malaria prone areas to achieve and maintain universal coverage. Universal coverage of ITNs is defined as ownership of at least one net per two people living in a household , 6. Accoet al . Th. Th21]. Zomba health systems have 34 health facilities, including private, public and Christian Health Association of Malawi (CHAM). In this study we concentrated on public and CHAM health facilities. Public health facilities offer free care while CHAM facilities charge a contributory fee for health care services but they are relatively affordable and attract a good number of people of all kinds. Aside from being expensive, private health facilities are located in the town of Zomba, which is in the buffer region and thus excluded from the study. Seven health facilities were eligible in the lakeshore area, while six facilities were eligible in the highland area. Of the eligible facilities, CHAM and public facilities were grouped together for random selection in their respective stratums. According to each stratum (lakeshore and highland), simple random sampling was done and two facilities were picked from each stratum. Four health facilities included three public health facilities and one CHAM facility, of which Mkasala and Chingale health centers were located in the highlands, and Bimbi and Likangala health centers were located in the lakeshore area.In our study, cases were children under-five who tested malaria positive using malaria rapid diagnostic test (mRDT) during a health facility visit. Each case was matched to a control for under-five child. Controls comprised two groups; namely health facility and community based controls. Health facility controls were children who tested negative for malaria, but were diagnosed with other infections, at the same health facility. Community based controls were children who were randomly selected by visiting the village of a malaria case. Community based controls were children under-five who tested malaria negative with mRDT and had axillary temperature of less than 37.5°C.1 was at 90%.To determine if malaria risk differed between lakeshore and highland areas for children younger than five years old in Zomba district, Malawi. According to Dzinjalamala  malaria TM which was also used in the health facilities in to diagnose malaria. If the child tested malaria negative and their temperature was less than 37.5°C, then the caregiver was recruited as a community control. Those children, who had suspected clinical malaria, but tested malaria negative, including children who had been tested or treated for malaria in the past two weeks were excluded as controls. Children who had suspected malaria and tested positive were referred to the nearest health facility for proper management.We used a ratio of 1 case to 2 controls, and standard error of 0.05 to estimate a suitable sample size. For every case enrolled in the study, we recruited two controls from the same catchment area. This ensured that each health facility recruited equal numbers of cases and controls. After computing this in Epi-Info, we had a total sample size of 765 children under-five years according to Fleiss w/CC; 255 cases, 255 hospital based controls, and 255 community based controls. At each health facility, the study team worked in the outpatient department and identified caregivers of under-five children. After children and caregivers received care, they were invited to participate in the study. If the caregiver agreed to participate, a research assistant conducted a screening to determine if the participant was eligible or not. Participants were excluded if malaria was diagnosed using symptoms alone without a lab confirmation test, those who were severely sick and were referred for tertiary care, and those who were already interviewed for the same study. From the villages where the malaria case lived, we also enrolled community based controls by first contacting the village head, who identified the households with under-five children in the village. From the list, the research assistant randomly sampled the households that had an under-five child. If the village had no under-five children, the next closest village was considered until a matching control was found. The caregiver of the selected under-five child was invited to participate in the study. Since other children act as a reservoir for malaria, upon acceptance of the caregiver, a trained Lab Technician/nurse conducted a confirmatory mRDT, using similar SD Bioline malaria Ag P.f/pan (HRP-II) We recruited four research assistants, two who worked in the health facility and two who worked in the community. The two community based research assistants were certified lab technician/nurse, who could conduct mRDT tests. All the research assistants completed a two day training course on the data collection tool.Before data collection started, we piloted the data collection tool for validation. We selected one health facility  and conducted 15 interviews, five with caregivers of under-five children with malaria in the outpatient department; five with caregivers of under-five children not diagnosed with malaria , and five with caregivers in the community (community based controls). After the pilot, we modified all the questions that were difficult to ask and those which respondents struggled to understand.Data were collected using structured questionnaires to interview consenting caregivers of children under-five who were either cases, hospital or community based controls. Caregivers self-reported on ownership of a bed net (yes/no), attended health talks on malaria prevention (yes/no), how far they stayed from the nearest river stream or stagnant water (<1km/ >1km). Caregivers self-reported on the main material of the dwelling house , type of the floor for the dwelling ; sanitation of the household compound (clear weeds as soon as they grow/clear weeds once a month/clear weeds once a year/ never clear the weeds); literacy level of the household heads , main water source for the compound (piped water/unprotected well/protected well/river or lake or stream); knowledge of antimalarial prescription, assessed if the respondents were able to pick the correct dose and frequency. We assessed knowledge of antimalarial prescription by asking the caregiver to give the standard prescription dose of antimalarial (LA) used to treat uncomplicated malaria in Malawi. If caregivers gave the correct dose, they were coded as having knowledge of antimalarial prescription and vice versa. We assessed the preventive measures used .Data were collected over five weeks, starting from January 2016 to February 2016.The study was conducted in strict accordance with the principles of the Declaration of Helsinki. The protocol was approved by University of Pretoria Ethics Committee, South Africa and National Commission for Science and Technology (NCST) review board, Malawi, under references 485/2015 and NCST/RTT/2/6 respectively. The Zomba district health office in Malawi permitted the collection of data. Additionally, we obtained written informed consent from the caregivers who were the legal guardians of eligible under-five children (cases and controls) before conducting exit interviews.In the analysis, our response variable was a binary (y = 1 if case or 0 if control). We used Chi-square tests of association to examine the following explanatory variables to determine risk factors: economic status of the household including type of the dwelling houses; sanitation of the household compound; literacy level of the household heads, and main water source for the compound; the knowledge of how to administer prescribed antimalarial drugs for non-complicated malaria and preventive measures; malaria prevention health education talks; possession, and usage of bed nets; gender of the child and the caregiver.To compare the risk factors between children living in lakeshore and highland areas, we stratified data by geographic area and analyzed the risk factors separately for each area. We used summary statistics and frequency tables to quantify and describe the data. We used univariate and multivariate logistic regression analyses to model the effect of risk factors on malaria in the under-five children. For the multivariate regression models, we pre-selected potential explanatory variables if the univariate model indicated significance using a relaxed p-value of ≤ 0.25. We checked all the explanatory variables for co-linearity, and when a pair of co-linear variables were identified, then one of the two was dropped from the model. We tested all potential interactions for significance, and excluded all non-significant interactions from the model. We included all candidate explanatory variables in a saturated model, and removed non-significant variables in a stepwise fashion until we reached the final model.Malaria risk factors in the lakeshore areas were quantified using logistic regression to calculate univariate and multivariate odds ratios . AccordiAccording to the multivariate analysis, the following risk factors were statistically significant in the lakeshore area . ChildreMalaria risk factors in the highland areas were identified in the similar manner as done for the lakeshore area, according to the univariate analysis  the follAccording to the multivariate logistic regression analysis the following risk factors were statistically significant in the highland areas . ChildreIn our previous study  we reporet al [In lakeshore areas, knowledge of administering prescribed antimalarial drugs for non-complicated malaria suggest that children of caregivers who has the knowledge have a greater malaria risk. These findings are contradictory to Mathanga et al  who inveet al . In prinet al , 23. HowOur study showed that children under-five, who lived in lakeshore areas and lived close (<1km) to open water sources, such as rivers, swamps or unprotected wells had a higher malaria risk than those who lived far (>1km) from an open water source. Lakeshore areas are prone to flooding, which leads to more stagnant water bodies after the flooding period which eventually become ideal mosquito breeding habitats. Lakeshore areas are also usually flat, leading to the accumulation of stagnant water bodies. Many people living in lakeshore areas may fish for a livelihood, causing them to stay closer to water bodies, hence exposing themselves to malaria vectors.We also found that the means of obtaining a mosquito bed net influenced the risk of contracting malaria in highland areas. Most Malawians use free distributed mosquito bed nets, and in our study, we found that children under five living in a household that owned a free bed net distributed by Non-Governmental Organizations (NGO) had an increased malaria risk compared to those who had bought their own mosquito bed net. Freely distributed mosquito bed nets  may be oThis study also revealed that drinking water sources are a risk factor for contracting malaria in highland areas. Those caregivers who use unprotected dug wells in the yard, and those who use communal standpipes have a higher risk of contracting malaria. These water sources may harbor malaria vectors. The communal standpipe may lack control of water which spills over when used, water puddles may become stagnant and provide a conducive environment for mosquito breeding. Caregivers who use unprotected dug wells as a drinking water source probably live closer to these dug wells which increases their risk of being bitten by mosquitoes and contracting malaria.In highland areas, access to malaria prevention messages at health facilities was an important malaria risk factor for children under-five . The higThe pattern of malaria risk factors that emerged in this study, clustered according to geographic location, may be driven by other factors beyond those we examined. There may be delayed uptake of malaria prevention due to limited access to health care or distance to health facility, or contributory fee which is required in the CHAM health facilities.Malaria cases were diagnosed by mRDT. However, it is reported that, mRDTs sometimes fail to detect low density parasitaemia (<200 parasites/μl) . Our stuWe observed different variables associated with malaria diagnosis between highland and lakeshore regions, although there is a common risk factor shared across areas. These findings suggest that some interventions such as the use of new mosquito bed nets may help to reduce malaria risk among under-five children, and may be suitable to both geographic locations. People in lakeshore areas may potentially benefit by moving their households more than one kilometer away from water sources, whilst people living in highland areas should be encouraged to manage stagnant and open water sources close to their households and be encouraged to attend health talks on malaria prevention.S1 Questionnaire(DOCX)Click here for additional data file.S1 Dataset(DTA)Click here for additional data file.


In the original article, there was an error. A sequence was mistakenly labeled as originating from the Central African Republic (GenBank accession number HQ131960) but was actually a duplicate of the sequence Burkina Faso Pibaore 2014, obtained during this study.Abstract:A correction has been made to the “Peste des Petits Ruminants (PPR) is a viral disease affecting predominantly small ruminants. Due to its transboundary nature, regional coordination of control strategies will be key to the success of the on-going PPR eradication campaign. Here, we aimed at exploring the extent of transboundary movement of PPR in West Africa using phylogenetic analyses based on partial viral gene sequences. We collected samples and obtained partial nucleoprotein gene sequence from PPR-infected small ruminants across countries within West Africa. This new sequence data was combined with publically available data from the region to perform phylogenetic analyses. A total of fifty-five sequences were obtained in a region still poorly sampled. Phylogenetic analyses showed that the majority of virus sequences obtained in this study were placed within genetic clusters regrouping samples from multiple West African countries. Some of these clusters contained samples from countries sharing borders. In other cases, clusters grouped samples from very distant countries. Our results suggest extensive and recurrent transboundary movements of PPR within West Africa, supporting the need for a regional coordinated strategy for PPR surveillance and control in the region. Simple phylogenetic analyses based on readily available data can provide information on PPR transboundary dynamics and, therefore, could contribute to improve control strategies. On-going and future projects dedicated to PPR should include extensive genetic characterization and phylogenetic analyses of circulating viral strains in their effort to support the campaign for global eradication of the disease.”Introduction, paragraph five:A correction has been made to the “Due to transhumance and poorly controlled movement of animals across the region, we expect to find evidence of close phylogenetic relationship between PPRV strains in West Africa. We collected samples and obtained partial N gene sequences from PPRV-infected small ruminants across countries within West Africa. This new sequence data was combined with publically available data from the region to perform phylogenetic analyses and test this hypothesis.”Results, paragraph two:A correction has been made to the “Phylogenetic analyses showed that all the sequences obtained belonged to the lineage II (LII) of PPRV . The samDiscussion, paragraph three:A correction has been made to the “Phylogenetic analyses based on sequence data from this lineage can be used to study transboundary PPRV dynamics in the West African region, characterized by complex transboundary movements of animals through transhumance and trade. Indeed, our results showed that we could identify different genetic clusters within lineage II containing samples from more than one West African country, although good statistical support was obtained for only two of them. Some of these clusters consisted of samples from countries sharing borders (for example cluster 5 with samples from Ghana, Burkina Faso, Benin and Nigeria; Lastly, corrections have been made to The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Suboptimal HIV testing rates through available testing approaches such as HIV counselling and testing have directed research efforts toward recognizing the potential of HIV self-testing as an additional testing method. However, HIV self-testing is not readily available within HIV testing facilities and data on how HIV self-testing and HIV counselling and testing will co-exist within HIV testing facilities is limited. Therefore, this study sought to fill this knowledge gap.Forty consenting adults were exposed to HIV counselling and testing and HIV self-testing using a cross-over study design between February 2016 and February 2017 resulting in 80  interviews. Participants were randomly exposed to HIV counselling and testing first, followed by self-testing, or HIV self-testing first, followed by counselling and testing. In-depth interviews were conducted at baseline, and after each testing exposure, using a semi-structured interview guide. Interviews were transcribed and translated prior to doing the framework analysis.Support through counselling played a central role in the HIV testing process for some participants who desired support or were not confident to perform unsupervised HIV self-testing. The complementary relationship between HIV self-testing and HIV counselling and testing requires a combination of benefits such as availability of counselling, confidence, convenience and confidentiality (4 Cs) derived from HIV self-testing and HIV counselling and testing. Implementation of the 4 Cs will depend on the availability of unsupervised HIV self-testing and/or supervised self-testing with support from HIV counselling and testing.As treatment and prevention efforts expand, the reasons for and frequency of testing is changing and there is a need to develop differentiated models for providing HIV testing services to meet client’s needs. HIV self-testing is an important addition to enhance HIV testing efforts and should be offered in combination with HCT. The World Health Organization (WHO) recommends the use of HIV self-testing (HIVST) as an additional approach to HIV testing services  to enhanDespite the beneficial nature of HIVST in overcoming current drawbacks of HCT, the absence of counselling and accuracy when testing have been highlighted as potential challenges with unsupervised HIVST . Over thAccording to recent national HIV testing guidelines, in South Africa, HIVST should be viewed as an additional approach to existing testing services and its use is not intended to displace confirmatory facility-based HIV tests . Public This study was conducted at an urban and rural Centre for the AIDS Programme of Research in South Africa (CAPRISA) site in KwaZulu-Natal, South Africa, which have some of the highest prevalence and incidence rates of HIV . These sThe following study design was adopted to explore complementarity between HCT and HIVST and how HIVST can be introduced within the HIV testing framework. Obtaining informed consent from all participants preceded the determination of eligibility in this randomised cross-over designed study. Randomisation envelopes were developed electronically using a 1:1 ratio for participants to receive either standard HCT or HIVST. Once a participant was deemed eligible the research assistant assigned a randomisation envelope in sequential order to the participant. Demographic data was collected for all participants and each participant underwent three in-depth interviews (IDI) on the same day by trained research assistants. The first interview was conducted before the first HIV test, regardless of the assigned study arm; the second after the first HIV test  of testing (research testers). Eligibility criteria for this study included being HIV uninfected (per participant’s self-report) of unknown HIV status, men and women, > 18 years of age and willing to provide informed consent.The researchers followed interview guides which were developed based on a scoping review of acceptability of HIVST . BaselinThe framework analysis method described by Rabiee  was usedA total of 40 participants were enrolled in the study, resulting in 80 in-depth interviews following the participants’ exposure to HCT and HIVST. Cohorts were stratified into HIV testing naïve and regular testers as there were no distinguishable differences in reported experiences between repeat testers and research testers. We were unable to recruit male research testers due to limited HIV prevention studies recruiting men within our recruitment areas Table . Also, aWe used a cross-over study design to eliminate the potential for bias between novel and familiar HIV testing practices among regular and research testers. Through longitudinal analysis we found that as individual testing approaches, optimal use of HCT and HIVST is limited by several drawbacks. However, individually, HCT and HIVST display beneficial characteristics that complement each other and may allow them to co-exist within the HIV testing framework. The main factors associated with HCT and HIVST that influence complementarity are counselling, convenience, confidence and confidentiality.Although regular testers were previously exposed to HIV counselling, a need for counselling was still evident during HIVST to prepare a person for testing in the event of a positive result Table  which coSome participants believed that testing by trained professionals through HCT was more accurate and reliable Table . DespiteThe majority of regular and naïve testers believed that HIVST would offer a convenient testing opportunity for those who either did not have time to present at HIV testing facilities to access HCT or could not afford transport costs or both Table . In our Participants were concerned with lack of privacy and disclosure of their results to staff within the health care facilities which may result in exposure to stigma and discrimination Table . ThroughThe interrelationship of counselling, confidentiality, convenience and confidence associated with HIVST and HCT has led to the design of a HIV testing strategy to support the complementarity of HIVST and HCT, as well as their ability to potentially co-exist within a primary health care setting Fig. .Fig. 2FaWe found that some HIV testing naïve participants felt confident to perform unsupervised HIVST, but reported that they would have been more comfortable if they had support during the testing process Table . TherefoWhile self-confidence and use of self-testing instructions to perform unsupervised HIVST was evident, the absence of counselling in the event of a positive HIV result was raised as a concern by some participants who lacked confidence to manage a positive HIV result Table . TherefoSome participants felt confident to perform unsupervised HIVST and found associated confidentiality with HIVST beneficial to testing, but desired counselling to complete the HIV testing process. In these instances, counselling could again be offered through HCT or supervised HIVST in an attempt to support testing with HIVST. To invoke confidence in participants exposed to HIVST for the first time, supervised HIVST could be introduced. Once participants feel comfortable to perform HIVST independently, they can have a follow-up, unsupervised HIV test.The complementary relationship between HCT and HIVST suggests a place for differentiated testing options for HIV testing to provide a variety of options for individuals that would accommodate their testing needs that include the 4 Cs – counselling, confidence, convenience and confidentiality – together with options of supervised and unsupervised HIVST. To our knowledge, our study provided a novel strategy (4 Cs) toward introducing HIVST within the HIV testing framework and built on the WHO recommendation of introducing HIVST as an additional HIV testing approach . The 4 CCounselling plays a crucial role in the HIV testing process by providing support during testing, preparing an individual for a HIV test and management of positive results. This concurs with evidence from other studies within and outside sub-Saharan Africa –21. AbseBenefits such as convenience and confidentiality, underpin the robustness of HIVST as a complementary HIV testing method to that of HCT, which may have a positive effect on HIV testing rates by overcoming the barriers associated with HCT. Hard-to-reach populations who avoid testing at health care facilities due to long waiting times, lack of privacy and possibility of encountering stigma and discrimination  may beneThe strength of introducing a novel technology such as HIVST into primary health care facilities may not be fully appreciated until we are able to integrate HIVST into the current HIV testing model. This study provided insight and evidence for policymakers into the complementarity of HCT and HIVST to guide the introduction of HIVST within the primary healthcare domain. The study has several limitations; firstly, given the select sample the study findings are not generalizable, and secondly, we were unable to explore real-time complementarity as HIVST is not routinely available with HCT. Notwithstanding these limitations, the study does provide useful insights on introducing HIVST into primary health care facilities, as HIVST is not available in the public domain in KwaZulu-Natal, South Africa.HIVST is suitable as an additional testing approach and should be piloted in combination with HCT given the associated benefits of counselling. The use of HIVST and HCT as a complementary testing approach will need to be tailored to a person’s need by factoring in individual, population and contextual factors. At an individual level, comfort with HIVST will determine whether a combination of supervised and unsupervised HIVST is required, as well as to what extent characteristic components such as professional support is required. Human resource infrastructure to support the demands of interplay between HIVST and HCT will need to be determined at a population level as this may pose a challenge within resource-strained settings. Further research to evaluate real-time use of the complementarity between HCT and HIVST within public health care facilities is needed.


Stuttering is a complex neurodevelopmental disorder that has to date eluded a clear explication of its pathophysiological bases. In this review, we utilize the Directions Into Velocities of Articulators (DIVA) neurocomputational modeling framework to mechanistically interpret relevant findings from the behavioral and neurological literatures on stuttering. Within this theoretical framework, we propose that the primary impairment underlying stuttering behavior is malfunction in the cortico-basal ganglia-thalamocortical  loop that is responsible for initiating speech motor programs. This theoretical perspective predicts three possible loci of impaired neural processing within the cortico-BG loop that could lead to stuttering behaviors: impairment within the basal ganglia proper; impairment of axonal projections between cerebral cortex, basal ganglia, and thalamus; and impairment in cortical processing. These theoretical perspectives are presented in detail, followed by a review of empirical data that make reference to these three possibilities. We also highlight any differences that are present in the literature based on examining adults versus children, which give important insights into potential core deficits associated with stuttering versus compensatory changes that occur in the brain as a result of having stuttered for many years in the case of adults who stutter. We conclude with outstanding questions in the field and promising areas for future studies that have the potential to further advance mechanistic understanding of neural deficits underlying persistent developmental stuttering. Developmental stuttering  is a childhood onset speech disorder that affects approximately 5–8% of children and 1% of adults . Core syneurogenic, arising through stroke, neurological disease, or as a result of treatments for neurological diseases , which affects approximately 1% of the population and occurs in nearly all cultures and languages  for producing speech sounds, and an initiation circuit, which is responsible for turning the appropriate motor programs on and off at the appropriate instants in time. Following seminal work from the primate motor control literature (cortico-BG loop). Within our theoretical framework, then, the primary impairment underlying stuttering behavior is malfunction in the cortico-BG loop responsible for initiating speech motor programs  and non-vocal learning  species . Vocal lfunction .via projections from cortex to the putamen . Internal circuitry within the basal ganglia, including portions of the globus pallidus (GP) and substantia nigra (SN), performs the job of selectively exciting the correct motor program in the current context while inhibiting the competing motor programs. For example, if a mature, fluent speaker is currently producing the word “pet” and is in the process of producing “p,” the basal ganglia are monitoring the sensorimotor representations of the speech articulators for evidence of the impending completion of “p” ; when this occurs, a “completion signal” is sent to cerebral cortex to extinguish the initiation map node for “p”, at which time the cortico-BG loop selects “e” over competing motor programs and sends this information to SMA to activate the initiation map node for “e.”In the DIVA model, the initiation circuit is responsible for sequentially initiating phonemic gestures within a  motor program by activating nodes for each phoneme in an initiation map in the supplementary motor area (SMA), a region of cerebral cortex thought to be involved in the initiation of motor programs, including those for speech . In termBefore a new sequence (syllable) is fully learned by the basal ganglia, the motor system will rely heavily on cortical mechanisms to sequence through phonemic motor programs. This situation is schematized in Failure to recognize the sensory, motor, and cognitive context for terminating the current phoneme would result in a prolongation stutter since activity of the SMA initiation map node for the current sound will not be terminated at the right time. Failure to recognize the context for initiating the next phoneme would result in a block stutter since the initiation map node for the next phoneme in SMA will not be activated at the right time. If the initiation signal “drops out” momentarily, production of the next phoneme might begin but prematurely terminate and then restart, as in a repetition stutter. timing signals, since they indicate the right time to terminate/initiate movements. This characterization provides an insight into the frequent observation that stuttering is often greatly reduced or eliminated in situations where external timing cues are available, such as choral reading and metronome-timed speech  and an indirect pathway that has the overall effect of inhibiting cerebral cortex (needed to suppress competing motor programs). Striatal dopamine has opposite effects on the two pathways: it excites the direct pathway and inhibits the indirect pathway. Thus, excessive dopamine can lead to a situation in which there is insufficient inhibition to suppress competing motor programs, making it difficult for the correct motor program to be chosen over incorrect alternatives. Such a situation could delay the choice of the desired motor program, leading to a block or prolongation stutter, or it may lead to an unstable initiation signal that starts to increase but suffers dropouts that result in repetition stutters.This last finding fits well with one popular hypothesis regarding the neurogenesis of PDS, the In support of the dopamine excess theory of stuttering, it has been noted that antipsychotic drugs such as haloperidol and risperidone that block dopamine D2 striatal receptors are effective in treating symptoms of stutteringdecreased dopamine levels in the striatum could lead to stuttering dysfluencies, which would account for the onset of stuttering in individuals with Parkinson’s disease noted above. In this scenario, reduced excitation of the desired motor program through the direct pathway leads to a decrease in the competitive advantage of this motor program, which in turn leads to a delayed, weakened, and/or unstable initiation signal. It should be noted that many people with PD exhibit speech disruptions other than stuttering, further highlighting that the relationship between Parkinson’s disease and stuttering is not a simple one. Studies aimed at distinguishing the neural characteristics of PD patients with stuttering-like behaviors from those with other types of speech motor disruptions may help clarify this relationship.Relatedly, the computer simulations of These considerations suggest that there may be at least two subtypes of PDS: one characterized by an under-active indirect pathway and another characterized by an under-active direct pathway. Behaviorally, the former might be characterized by a tendency toward excessive motor activity due to reduced inhibition of movement from the indirect pathway, whereas the latter might be characterized by a reduced level of motor activity due to reduced excitation of movement from the direct pathway. This is similar to the proposal put forth by Further evidence of possibly impaired basal ganglia functioning in PDS comes from a functional magnetic resonance imaging (fMRI) study by The second potential source of impairment in the basal ganglia motor loop of PDS identified in via the cortico-BG loop. This interpretation is also relevant to the Neuroimaging results from several studies provide some support for this contention. Using diffusion tensor imaging (DTI) data acquired from CWS and age-matched controls, Atypical processing in corticostriatal circuits has also been shown through functional connectivity analyses of resting state fMRI data. In one study , the relCompared to CWS, clear evidence of impaired cortico-subcortical connectivity in adults who stutter remains relatively scarce. However, The third possible source of impairment in the basal ganglia motor loop of PDS is the network of cerebral cortical regions involved in speech production PDS-3 in . NeurogeStructural neuroimaging studies of PDS further support this assertion. For example, Further support for the view that left hemisphere impairments in PDS result in increased right hemisphere involvement during speech comes from functional neuroimaging studies. Anomalous functioning in left hemisphere inferior frontal cortex in adults with PDS during single-word production was identified using magnetoencephalography by Consistent with the view that left hemisphere anomalies underlie PDS, In the first longitudinal DTI study in childhood stuttering, via corticostriatal projections , and deficits in the cerebral cortex. Neuroimaging data to date from both children and adults who stutter have provided evidence to support deficits in each of these areas relative to fluent speakers. The differences that are present in the literature based on examining adults versus children give important insights into potential core deficits associated with stuttering versus compensatory changes that occur in the brain as a result of having stuttered for many years in the case of adults who stutter. Below we discuss some promising areas of investigation that have the potential to further our understanding of the role of the basal ganglia in stuttering pathophysiology.Anatomical and functional anomalies involving the left hemisphere premotor cortex, IFG, SMA, and putamen have been found in both adults and children who stutter, suggesting that these impairments may represent the primary deficits underlying stuttering. By contrast, differences between adults and children who stutter have been identified, including auditory cortex deactivation relative to controls during speech and decreased compensation to auditory perturbations in adults who stutter but not CWS. Currently, this assertion has only been supported in a small number of studies involving CWS, but if this pattern holds up in additional studies, it suggests that decreased auditory feedback processing during speech in adults who stutter is a secondary effect that may develop over years of stuttering; perhaps, as a compensatory mechanism that decreases the probability that a stutter will be induced by the detection of minor inaccuracies in speech output through auditory feedback. More generally, studies that directly compare adults and CWS as well as longitudinal studies of individuals who stutter (including both persistent cases and those that resolve over time) are needed to tease apart primary deficits from the many secondary behaviors and neural anomalies that have been identified in adults who stutter. In turn, this knowledge will aid in the development of effective treatments aimed squarely at the root causes of the disorder.system-level problem rather than the result of impairment in a particular neural region or pathway.The preceding review makes clear that neural anomalies in stuttering have been identified in a number of different portions of the cortico-BG loop. Furthermore, individual connectivity studies often report disparate neural pathways that differ in individuals who stutter compared to controls. These considerations are indicative of the fact that stuttering is likely a One implication of this is that network-level analyses, such as those studied in graph theory, may provide a more reliable and effective means of identifying the neural bases of the disorder. For example, Apart from MRI-based studies, we expect that increasing attention will be given to the temporal dynamics of neural communication, including anomalies in these dynamics in stuttering speakers. According to the “communication through coherence” hypothesis of neural communication , neural Specific to speech, beta oscillations in the motor cortex during speech preparation reflect the communication of the speech plan to the motor effectors and to the sensory regions required for monitoring speech output . CoherenIn summary, extant research suggests that beta oscillations may provide a mechanism for coordinating auditory and motor components of the cortico-BG loop when producing speech sequences that are internally timed, and this mechanism may be impaired in stuttering speakers. Future studies investigating this aspect of stuttering should provide more fine-grained temporal information on how the basal ganglia and its connectivity with cortical areas differ in stuttering. This information can provide the basis for intervention development, which may involve better synchronizing and in turn inducing better communication across the basal ganglia, motor, and auditory regions to help achieve more fluent speech in people who stutter.Speech production abilities in humans are uniquely complex, making it difficult to study its mechanisms in an animal model. However, research involving animal models – in particular those that present with learned vocal abilities – presents critical opportunities to investigate the biological mechanisms relevant to stuttering in a tractable model. As mentioned in a previous section, song nuclei and the connectivity among vocal learning pathways found in songbirds have important parallels to brain areas and neural pathways supporting human speech production . SelectiApart from songbirds, mouse vocalizations have been studied as a possible animal model for human speech as well. Mouse vocalizations are innate rather than learned  and diffThe genes identified so far associated with persistent developmental stuttering include not only GNTAB mentioned above but also GNTPG, NAGPA, and AP4E1 , which hAlthough the studies reviewed herein provide broad support for basal ganglia-thalamocortical loop involvement in PDS, it is noteworthy that differences between fluent and stuttering individuals have been found for neural structures not in this loop, most notably the cerebellum e.g., , includiThe basal ganglia and their connections to cortical regions involved in speech form critical networks that support fluent speech production. Anatomy and function of this cortico-BG loop have been found to be atypical in an increasing number of studies of speakers who stutter, pointing to possible deficits within the basal ganglia proper; connections between cortex, basal ganglia, and thalamus; and in the cortical circuitry involved in speech production. Future studies that examine in greater detail neurological deficits in the morphology, interconnectivity, functionality, and developmental time course of the cortico-BG network have great potential to further our knowledge on possible neural vulnerabilities for chronic stuttering and for distinguishing core deficits from anomalies/compensatory deficits that develop after years of stuttering. These studies will in turn help pave the way to developing neuroscience-guided treatments for stuttering that may not only help alleviate stuttering in adults who stutter but also help prevent chronic stuttering during childhood with early intervention.S-EC and FG planned, drafted, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A Task Force was commissioned jointly by the European Academy of Allergy and Clinical Immunology (EAACI) and the Respiratory Effectiveness Group (REG) to develop a quality assessment tool for real-life observational research to identify high-quality real-life asthma studies that could be considered within future guideline development.The resulting REal Life EVidence AssessmeNt Tool (RELEVANT) was achieved through an extensive analysis of existing initiatives in this area. The first version was piloted among 9 raters across 6 articles; the revised, interim, version underwent extensive testing by 22 reviewers from the EAACI membership and REG collaborator group, leading to further revisions and tool finalisation. RELEVANT was validated through an analysis of real-life effectiveness studies identified via systematic review of Medline and Embase databases and relating to topics for which real-life studies may offer valuable evidence complementary to that from randomised controlled trials. The topics were selected through a vote among Task Force members and related to the influence of adherence, smoking, inhaler device and particle size on asthma treatment effectiveness.Although highlighting a general lack of high-quality real-life effectiveness observational research on these clinically important topics, the analysis provided insights into how identified observational studies might inform asthma guidelines developers and clinicians. Overall, RELEVANT appeared reliable and easy to use by expert reviewers.Using such quality appraisal tools is mandatory to assess whether specific observational real-life effectiveness studies can be used to inform guideline development and/or decision-making in clinical practice.The online version of this article (10.1186/s13601-019-0255-x) contains supplementary material, which is available to authorized users. Randomised controlled trials (RCTs) are required to establish the efficacy and explore the safety of asthma treatments, but are insufficient to provide holistic evidence on the benefit/risk ratio of interventions when offered, initiated or used in the heterogeneous asthma patient populations and care settings that exist outside the trial environment. Registration asthma and allergy RCTs are designed to maximize chances of observing efficacy . To do sIn observational research as well as in RCTs, ensuring high-quality methodology is crucial to avoid biases that would compromise the reliability and validity of results . FollowiA number of frameworks have been proposed to help characterise the extent to which a study design reflects pragmatic aspects or real-life patients and practice , 21. OneTo address this, REG and the European Academy of Allergy & Clinical Immunology (EAACI) convened a joint Task Force to set and test quality standards for observational comparative effectiveness research (CER) in asthma, and to assess the extent to which the evidence from such studies might complement the results of RCTs. The aim of the current manuscript is to provide an overview of how this Task Force developed a quality assessment tool and applied it to selected PICOT questions for which RCTs provided only limited evidence. The purpose of this paper is not to provide an extensive description of the tool development process, nor the detailed results of the literature review; these will be the topics of additional publications  development and assessment process are detailed in a dedicated article . In summA first set of 21 PICOT   questionPapers considered of sufficient quality were sent to at least two raters each with a reading grid  for 8/25 items when first piloted among Task Force members. Iterative refinement was then based on rater feedback and removal of ambiguity in time language to avoid potential for uncertainty or differing interpretation. This resulted in a marked improvement in concordance between raters, to ≥ 73% for all primary and ≥ 69% for all secondary items when averaged over the three rating groups. Further details on concordance are available in the accompanying methodology paper .The four selected PICOT questions (numbered following their rank during the selection process) dealt with the influence of adherence/persistence on asthma outcomes, the influence of smoking on asthma outcomes and treatment effectiveness, the impact of the inhaler device on asthma outcomes and the relation between particle size of maintenance therapy and asthma outcomes Table .Table 2SAltogether, 46 relevant articles were identified: n = 24 for PICOT question 1 , 42–59, A summary of the literature analysis is presented in Tables All papers retained for analysis following RELEVANT-based quality assessments reported an association between lower adherence to maintenance therapy and poorer control, as assessed by SABA use or markers of exacerbations . More specific findings relating to drug classes were reported: oral leukotriene receptor antagonist (LTRA) therapy appeared to be more effective than ICS in obtaining control in non-adherent patients; the opposite was true for adherent patients . Some asPapers dealing with device choice suggested that limiting the number of different inhaler device types prescribed to an individual patient was associated with better asthma outcomes, while switching devices outside the context of a dedicated medical visit was associated with a greater risk of a loss of control , 62. OneA prospective cohort study did not find differential treatment effects of an ICS-LABA fixed dose combination for patients with different smoking behaviours, but asthma patients with a current smoking status had poorer outcomes overall . AnotherSeveral analyses, mostly by the same group , 67–71, The REG-EAACI Task Force on quality standards in asthma comparative effectiveness research defined target quality standards and developed a quality assessment tool—RELEVANT—to appraise published observational effectiveness studies. The definition of standards and development of the tool aimed to provide a simple assessment grid for paper-based or web-based use, specifically designed for the evaluation of published effectiveness research. They were based on previous initiatives in this area –27 and iFour PICOT questions were selected through a poll among the Task Force and members of REG and EAACI, and related observational studies identified through a systematic literature search among bibliometric research databases. Retrieved records were then quality appraised using the RELEVANT tool. Several of the papers were assessed as being of sufficiently good quality to allow an increase in their level of evidence from low to moderate following a GRADE-like process. For all PICOT questions of interest, assessed observational studies yielded results with possible impact on clinical practice in areas where similar evidence from RCTs is lacking. Altogether, this suggests that RELEVANT could become a useful tool to appraise the evidence from observational studies as part of a guideline development process as well as offering wider utility for more general literature reading and appraisal, and to inform research development.A quality assessment instrument needs to offer ease of use and sufficient robustness to ensure confidence in its grading. This requires the items to be clear, easily understood and rated similarly by different raters. While the detailed tool development process is the topic of a dedicated paper , it can An interesting observation permitted by this process was the high number of PICOT questions n = 21) identified during the first round of selection by the Task Force and its REG and EAACI correspondents. This outlines the evidence gaps that are still present regarding asthma treatment, despite the considerable research interest for this common but still insufficiently controlled disease. This also contrasts with the high number of RCTs assessing the efficacy of various components of asthma care, including both medications and non-pharmacological approaches such as education or integrated care. This discrepancy illustrates that classical RCTs are not sufficient to answer all questions regarding real-life effectiveness of therapeutic strategies, and need to be complemented by dedicated studies designed to reflect better the range of patients and care settings that exist in routine clinical practice  identifi, 13, 14.The initial steps of the process confirmed that conducting a search specific to observational effectiveness research is challenging given the lack of standardised and specific terminology/nomenclature and requires the input of specialists of bibliometric research databases. Despite this, the number of published articles available for each of the PICOT questions was surprisingly low given the long period that was scrutinized (10 years), especially for PICOT questions 2  and 3 . This illustrates that observational effectiveness research in asthma is still infrequently performed or reported despite the clear need for real-life data to complement classical RCTs by answering different study questions including to, better assess the extent to which their results can be generalised, and identify the best target populations. Inhalation technique is known to be poor overall with at least 50% of the patients making manipulation and/or inhalation errors, which is associated with poor control of asthma and chronic obstructive pulmonary disease (COPD) –80. ThisThe number of articles published in the 10-year period of interest regarding the influence of adherence and particle size on the effectiveness of maintenance ICS therapy was higher than for the two previous topics mentioned above. However, it remained insufficient given the importance of these issues. More specifically, although poor adherence has been associated with both poor inhaler technique and poor disease outcomes , 85, theA common limitation of analysed papers was the lack of precise clinical data available to ensure that patients of compared groups were 100% comparable at baseline. To minimize the impact of confounding (bias), most studies used statistical strategies including matching (exact or propensity score-based) and adjustments for variables differing between groups during the baseline period. These strategies could not, however, account for variables that were entirely absent from the databases. For instance, in most studies lung function data  or questionnaire-measured level of asthma control were not available; so proxies for control were used although they were not necessarily strictly concordant with questionnaire results, e.g., due to time frame differences. In addition, some studies did not report sufficient detail on baseline population characteristics, making it difficult to assess their quality with a high level of confidence. These points reflect the lack of 100% concordance between raters, as outlined above. To deal with this difficulty, a third rater intervened in all cases of discordance between the two initial raters. Ultimately, 48%  came from a single research team  found that asthma outcomes were better in real-life patients receiving ICS or ICS-LABA combinations delivered by extra-fine rather than fine particle formulations , 67–72. This is the first initiative to develop and use a quality-assessment tool specifically designed to aid appraisal of observational asthma research. Although RELEVANT could be used in other fields, testing was performed using articles reporting results from effectiveness studies in asthma. The Task Force systematically reviewed previous quality assessment tools for observational research, selected items and revised the incorporated quality domains and items based on pilot and extended pilot testing. This provided an opportunity to improve item selection and formulation and to reduce and refine the items classification into primary and secondary items to aid inter-rater agreement.The final literature analysis served as a validation process confirming the applicability of the tool and its relevance for quality assessment with the aim of informing guidelines and clinicians. Some limitations need to be acknowledged. First, the process could be applied only to a limited number of PICOT questions considering the high burden of each set of analyses. To limit the impact of a biased selection of questions, a consensus on prioritisation was reached not only within the Task Force but also through a poll among REG and EAACI members. Similarly, a large number of REG and EAACI members contributed to the literature analysis, to limit reviewing biases. Systematic literature appraisal is time- and resource-consuming. Ultimately the use of standardised tools such as RELEVANT should facilitate this activity but the process still requires the involvement of experienced reviewers. The limited number of high-quality real-life comparative effectiveness research studies retrieved here underlines the need for a more extensive “real-life research culture”.Comprehensive quality assessment tools are now available, not only for efficacy trials  , but alsThe EAACI-REG Task Force developed and tested RELEVANT, a quality assessment tool for real-life comparative effectiveness observational research. The tool was specifically used to evaluate literature relating to PICOT questions pertinent to current evidence gaps around existing asthma interventions, but can also be used in other areas of medicine. It is hoped that the availability of this tool will assist the expansion of high-quality real-life effectiveness research in the field of respiratory medicine and allergology. Importantly, several results of the literature analysis conducted using the RELEVANT tool could lead to changes in clinical practice.Additional file 1: Table S1. Taskforce Activity Schedule Summary. Table S2. Literature review search terms: used to identify a list that would include asthma observational comparative effectiveness studies. Table S3. Reading grid used by reviewers to summarize selected articles. Table S4. List of REG and EAACI contributors. Table S5. Literature Review Assessment Overview: All papers.


Identifying robust markers for predicting the onset of psychosis has been a key challenge for early detection research. Persecutory delusions are core symptoms of psychosis, and social cognition is particularly impaired in first-episode psychosis patients and individuals at risk for developing psychosis. Here, we propose new avenues for translation provided by hierarchical Bayesian models of behaviour and neuroimaging data applied in the context of social learning to target persecutory delusions. As it comprises a mechanistic model embedded in neurophysiology, the findings of this approach may shed light onto inference and neurobiological causes of transition to psychosis. Persecutory delusions, defined as unfounded beliefs that others are deliberately intending to cause harm, are core symptoms of psychosis and a burden for patients . PersecuA recent approach to treatment of psychosis focuses on early detection and prevention. However, a fundamental problem for research on the early phases of psychosis is identifying robust markers for transition to psychosis from the clinical high-risk state (CHR) . The CHRPrevious studies have examined the predictive value provided by neuroimaging methods including structural –10 and fAlthough current methods link transition risk with particular differences in genetic polymorphisms or brain structures, they do not allow for quantifying the probability that a particular disease mechanism is present. This, however, is the basis for targeted treatment.One solution for identifying disease mechanisms is to pursue a computational modelling strategy and employ generative models that focus on core symptoms, such as persecutory delusions. Generative models describe mechanisms that could have generated the observed behaviour or neuroimaging data. Individual differences in behaviour—potentially related to disease mechanisms—can be uncovered by estimating individual model parameters based on participants’ behaviour . In addiOne class of generative models, which can be fit to noninvasive measurements  or functional magnetic resonance imaging (fMRI)), is models of effective connectivity such as dynamic causal modelling (DCM), describing causal (directed) influences between neurons or neuronal populations . DCMs exHere, we introduce a computational framework that focuses on a central symptom of psychosis, namely persecutory ideation. This framework integrates computational models of behaviour with neural circuit models, which describe the neuronal causes of aberrant learning and can be fit to EEG and fMRI data. It makes specific predictions about pathophysiology in psychosis, which may be used to predict transition to psychosis in CHR individuals and treatment response in FEP patients.Delusions in general are conceptualised as false beliefs based on incorrect inference about the external world, which persist in the face of disconfirmatory evidence. Two major computational theories exist, which assume specific mechanisms of delusional belief genesis and persistence.First, a popular notion is that patients with psychosis attribute inappropriately high aberrant salience to irrelevant events. This theory posits a key role of the dopamine system in mediating the misattribution of salience . It is A second and related theory of delusions focuses on the Bayesian brain hypothesis and the interplay between prior beliefs and “correction” signals or prediction errors (PEs) , 25. TheRecent theories of perceptual abnormalities in psychosis have built on hierarchical Bayesian frameworks, extending the concept of aberrant salience by highlighting the role of uncertainty  , 30–33. Fully developed delusions could be understood as implausible beliefs with overly high precision, which function to attenuate aberrant sensory evidence . Recent In the context of psychosis, the most-prominent delusional beliefs pertain to the social world and result from inference about the mental states of others, specifically that their intentions are of a persecutory nature , 43. A pComputational models of persecutory delusions must be based on existing cognitive models. Key cognitive predispositions for persecutory ideation are in line with the hypothesis of an initially uncertain predictive model of others’ intentions .N-methyl-d-aspartic acid (NMDA)-receptor dependent synaptic plasticity and neuromodulatory systems like dopamine and acetylcholine  about which option to choose. In order to perform well, they not only have to predict the accuracy of current advice, but also the adviser’s intention and how it might change over time  , thIn the context of learning about intentions, different hypotheses about how participants took decisions  were formalised in terms of a model space, which comprised different models of learning and belief-to-action mapping, including reinforcement learning models, which were formally compared . The modIn previous studies, the inferred adviser fidelity and volatility of intentions, - estimated with the HGF, - reflected participants’ overtly expressed beliefs about the adviser’s intentions at different times during the task. Furthermore, the learning parameters describing each individual’s belief updates predicted participants’ ratings of their own perspective-taking tendencies, suggesting that the model captures key aspects of social cognition , 65.According to this model, surprising advice outcomes have a greater impact on the agent’s internal representation (should have more influence on the belief update) when the sensory precision from the level below In the context of social learning, we demonstrated a dichotomy between low- and high-level precision-weighted PEs as they were related to dopaminergic and cholinergic systems . WhereasNot surprisingly, as social inference is particularly impaired in individuals at risk for psychosis , the regAs persecutory delusions predominate in major psychotic disorders and contribute to symptom severity, computational models that explain their formation and persistence may shed light onto the neural mechanisms that mark the different stages of psychosis.In the context of social learning, we predict that the high-risk state is defined by an imbalance between the precision of beliefs at low compared with high levels of the processing hierarchy, as suggested by recent studies of perceptual inference in relation to delusions , 73. ThuBased on neuroimaging results in the healthy population , 63 and In the later stages of psychosis, the presence of delusions might reflect a compensatory response to the aforementioned deficiencies of hierarchical inference. Thus, in individuals who exhibit persecutory delusions, we predict an increased representation of high-level belief precision about the other’s intentions Fig. . This noAn alternative hypothesis is that the pathophysiology underlying persecutory delusions is unrelated to precision, but instead to social PEs. Accordingly, individuals with persecutory delusions regard the adviser as purposely misleading, and therefore place greater weight on negative advice PEs. At the neural level, this would be expressed as biased predictions and enhanced PE signals for misleading advice.We propose two experimental designs to test our hypotheses: (1) Individuals with high-risk of developing psychosis and patients with persecutory delusions could be compared in a cross-sectional design. However, although generative modelling approaches may be useful for identifying inference and neurobiological processes leading to psychosis, validation studies are needed to determine their clinical utility. Regardless of how well a model may capture a putative pathophysiology, it needs to support differential diagnosis or prognosis, for example, by predicting transition to psychosis or treatment response with sufficient accuracy and in individual patients. (2) This can only be tested in prospective studies where CHR individuals and FEP patients who receive first-line treatment are assessed at multiple time points and model parameters are used to predict transition to psychosis or treatment response, respectively.From previous studies of aberrant learning in psychosis, it is unclear whether alterations in social inference are specifically required to explain persecutory delusions. In fact, alterations in higher-level inferential processes that are not necessarily specific to social contexts may affect processing of socially relevant information and produce delusions. To address this question, a control task that removes the aspect of intentionality may be needed. We have previously included such a control task  with bliMechanistically interpretable generative models like the ones outlined here allow for model comparison and testing of competing hypotheses as well as inference on disease mechanisms in individual patients at different stages of psychosis. Furthermore, the computational quantities derived from the model—such as the low- and high-level, precision-weighted PEs—could be associated with distinct neuromodulatory systems, dopaminergic and cholinergic , respectSupplementary InformationSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3


Sexual selection is proposed to be a powerful driver of phenotypic evolution in animal systems. At macroevolutionary scales, sexual selection can theoretically drive both the rate and direction of phenotypic evolution, but this hypothesis remains contentious. Here, we find that differences in the rate and direction of plumage colour evolution are predicted by a proxy for sexual selection intensity (plumage dichromatism) in a large radiation of suboscine passerine birds (Tyrannida). We show that rates of plumage evolution are correlated between the sexes, but that sexual selection has a strong positive effect on male, but not female, interspecific divergence rates. Furthermore, we demonstrate that rapid male plumage divergence is biased towards carotenoid-based (red/yellow) colours widely assumed to represent honest sexual signals. Our results highlight the central role of sexual selection in driving avian colour divergence, and reveal the existence of convergent evolutionary responses of animal signalling traits under sexual selection. What factors explain variation in the pace and trajectory of evolutionary divergence between lineages? Here, the authors show that a proxy measure for sexual selection intensity predicts both the rate and direction of plumage colour evolution in a diverse radiation of New World passerine birds. In particular, despite a long history of study5, explaining the tremendous diversity of animal sexual signalling traits has proved particularly challenging, and our understanding of the processes driving the evolution of species’ sexual signalling traits remains highly incomplete7. Sexual signalling traits mediate the complex relationship between an organism and its environment in important ways8. For instance, signalling traits such as colour, patterns, and sounds are under strong selection to facilitate effective communication between individuals in a given environment, but such benefits are balanced against costs such as those associated with signal production, maintenance or display, including predation11. Furthermore, animal signalling traits often play a key role in reproductive decision-making processes such as mate choice, and particularly choice for a conspecific mate12. Evolutionary divergence in signalling traits has consequently been recognised as a potentially powerful mechanism facilitating reproductive isolation before and/or after speciation among diverging populations14. Understanding the processes driving sexual signal divergence across lineages therefore has important implications for explaining multiple facets of biodiversity, including patterns of phenotypic and species diversity among groups and across geographic regions15.A central problem in evolutionary biology is to explain the processes shaping the evolutionary diversification of species’ phenotypic traits12, as preferences for novel signals can theoretically drive continuous and often rapid divergence in sexually selected traits between populations18. This view is supported by empirical studies showing that young, closely related species often differ primarily in terms of male courtship traits22, implying a role for divergent sexual selection in driving the rapid evolution of  sexual signals during or after speciation. At a macroevolutionary scale, increased intensity of sexual selection is therefore expected to drive high rates of evolution in signalling traits. A limited number of studies have attempted to test these ideas directly using a comparative approach, but so far results linking sexual selection to elevated rates of sexual signal divergence have been mixed24.Sexual selection has long been viewed as a potent diversifying force in animal systems25. Signals of species identity and attractiveness are expected to be arbitrary in form but differ in terms of elaborateness. This is because the information conveyed does not depend on the exact nature of the signal, only that they are distinctive or attractive25. Signals of quality should converge on one or a few signal types that are more effective (or have lower costs) than others28. For example, receiver sensory biases or higher production and maintenance costs may make carotenoid-based  pigments more efficient or honest visual signals of individual quality than other types of colour producing mechanism 30. Thus if the evolution of particular signal types is promoted or constrained by selection for signals of quality, and perceptual, developmental or energetic differences between signal production mechanisms, then the direction of trait evolution under sexual selection should be to some extent predictable. The relative roles of selection for arbitrary  versus adaptive  signals on the rate and direction of ornament evolution remains unresolved25.In addition to driving trait divergence and increases in evolutionary rate, sexual selection may also play a role in determining the direction of signal evolution within lineages. Ornamental traits  such as feather colouration, signal at least three very different types of information: species identity, attractiveness, and individual quality32, few have explicitly assessed the importance of sexual selection for explaining variation in both the rate and direction of phenotypic evolution at broad phylogenetic scales, and whether these patterns differ between the sexes. Second, previous studies have generally been hampered either by small sample sizes or by a lack of high quality data on species’ signalling traits, limiting the potential scope and generality of the resulting inferences. Third, such studies have also typically failed to contrast the importance of sexual selection against the role of other factors, making it difficult to assess the relative importance of sexual selection in relation to alternative mechanisms 34.Despite these longstanding theoretical predictions, progress in resolving the role of sexual selection in signal divergence has been slow for a number of reasons. First, although previous studies have attempted to test the impact of sexual selection on signal elaboration/divergence directly35. Plumage dichromatism represents a widely used proxy for sexual selection intensity in birds36, but is likely to be an imperfect measure38. We therefore investigated the relationship between dichromatism values and independent measures of sexual selection from prior work, using both proxies to test the predictions outlined above. Our results show that sexual selection (i) is associated with elevated rates of signal divergence between related species, (ii) impacts divergence between males more than females, and (iii) generates biases in the direction of signal evolution. We conclude that sexual selection plays a key role in explaining variation in both the rate and direction of plumage colour divergence in birds.Here, we use a dataset of male and female plumage colouration based on calibrated UV/Vis digital images of museum specimens and a method for summarising species-specific rates of trait evolution across a phylogenetic tree to examine the evolutionary dynamics of plumage colouration in the Tyrannida—a large, morphological diverse radiation of New World suboscine passerine birds comprising tyrant flycatchers, cotingas, manakins, and their allies. Birds offer an ideal study system in which to test the role of sexual selection in signal evolution because of the general availability of phylogenetic, phenotypic and geographic data. Furthermore, the Tyrannida in particular are especially suited to our analysis because colour patterns (and underlying colour producing mechanisms) are highly variable across species, and because species in the group occupy a wide range of ecological and environmental niches, allowing investigation of the importance of alternative hypotheses for signal evolutionManacus and Pipra) and Cotingidae , as well as more isolated cases of exceptionally rapid male divergence within the Tyrannidae . In contrast, rates of female plumage colour divergence were found to be less extreme than in males, but nonetheless still varied by three orders of magnitude across lineages  of male and female plumage colour evolution estimated using a method summarising rate variation across the phylogeny (see Methods) revealed that rates of plumage colour evolution are correlated between the sexes, but that males typically diverge faster than females  to test several competing hypotheses for the evolution of plumage colouration across species. Our models revealed that a proxy for sexual selection (plumage dichromatism) has a strong positive effect on rates of male but not female plumage evolution that is independent of other key variables  intraspecific colour variation  and accelerated  plumage colour divergence across Tyrannida species. To test whether this pattern was driven by the differential response(s) of particular signal types, we re-assessed this relationship after first assigning each patch in our dataset to one of three non-overlapping categories broadly distinguishing colours consistent with two distinct colouration mechanisms , rates of plumage colouration are (i) correlated between the sexes, but typically faster for males than females (disproportionately so within fast-evolving species), (ii) significantly associated with proxies for sexual selection intensity (including dichromatism), with contrasting effects between the sexes, and (iii) disproportionately elevated towards carotenoid-based  colouration traits, particularly within body regions frequently observed to function in inter-sexual or intra-sexual displays in birds . These findings support the idea that sexual selection is the key driver in determining both the rate and direction of plumage colour divergence in birds.23 focused on a small yet broad sample of avian sister species pairs, but contrasts with a related clade-based analysis using different proxies for sexual selection intensity24 that found both positive and negative associations between sexual selection and ornamentation. Our results indicate that this effect holds within, as well as between avian lineages, and supports the longstanding interpretation that sexual selection accelerates the evolutionary diversification of male plumage signals involved in courtship and species recognition4. Furthermore, we found that on average male plumage traits diverge significantly more rapidly than female traits and that rates of female plumage divergence are not associated with dichromatism—results that are consistent with those of previous studies examining sex-specific relationships between sexual selection and plumage divergence/elaboration32. While overall differences in rate between the sexes may in part reflect greater constraints on plumage colouration in females than males5, a pattern of increasingly rapid plumage divergence rate associated with dichromatism in males  is more consistent with the general prediction that is due to sex differences in reproductive investment, sexual selection should typically be stronger on males than females12.We found strong support for a positive association between sexual dichromatism and the rate at which male plumage colouration traits diverge. This result is consistent with a previous study40. This is consistent with patterns observed for other traits, such as allometric increases in male-biased sexual size dimorphism in larger species that are driven by a correlation between evolutionary change in females and directional sexual selection on males, e.g., ref. 41. However, genomic studies indicate that genetic correlations between males and females may not represent a major evolutionary constraint44. An alternative explanation therefore is that correlated rates of female plumage divergence are the result of mutual selection on signalling traits in both sexes46. Evidence suggests that elaborate signalling traits are maintained in females as adaptive responses to sexual and social competition47. Correlated divergence rates across the sexes may therefore be the result of females co-opting (elements of) male sexual signals for use in intra-sexual or inter-sexual or social interactions. Irrespective of the underlying mechanism, however, finding that rates of plumage evolution are generally elevated in males but also correlated between the sexes suggests that sexual selection plays a role in explaining the evolutionary divergence not only of male but also female plumage traits.We find evidence for a strong between-sex correlation in plumage divergence rate that becomes disproportionately more male-biased as rate increases. One explanation for this pattern is that genetic correlations cause non-adaptive changes in female traits in response to strong divergent sexual selection acting primarily on males49. Second, we detected a positive but weak effect of confamilial sympatry, which is consistent with the idea that interspecific interactions may promote rather than constrain phenotypic evolution50. However, this relationship was absent when using an alternative tree topology and subset of the data. None of the variables included in our analysis were able to explain the significant effect of dichromatism on rates of plumage evolution and, taken together, our multipredictor modelling results imply that sexual selection is the key driver of rates of plumage colour evolution across the Tyrannida.Our results also reveal additional predictors of rates of plumage evolution across species. First, rates of plumage evolution were associated with time since divergence for both males and females. At face value, a negative relationship between rate and time since divergence is consistent with the hypothesis that changes in plumage colouration are associated with speciation events. However, we interpret this relationship with caution as it may alternatively be explained by a methodological bias towards inferring faster rates on shorter branches if there are errors in tree topology and/or measurement error in phenotypic data51, dichromatism represents an imperfect proxy for total sexual selection intensity. This is because other mechanisms can influence patterns of sex-differences in plumage colouration, including natural selection for female crypsis during incubation52 or social selection on females to signal quality in the context of male mate choice or female–female competition47. Furthermore, if trade-offs exist between signalling modalities, then dichromatism may underestimate sexual selection intensity, particularly in lineages emphasising acoustic over visual signalling traits37. However, when we tested an independent  index of sexual selection intensity based on social mating system, sexual size dimorphism and paternal care data for a subset of our dataset, we found qualitatively identical results. Moreover, this alternative index was significantly positively correlated with our dichromatism scores , suggesting that in this system at least, dichromatism provides an adequate proxy for underlying gradients in sexual selection intensity. Nonetheless, ideally we would use a more direct measure of sexual selection intensity across lineages than any of these proxies provide, for example, indices based on variation in male mating success . However, such measures are unavailable for most species, and thus quantifiable indices such as dichromatism currently represent the most practical proxy for measuring sexual selection intensity for large numbers of species54. Yet, given these issues, we suggest that the effects with respect to dichromatism we report here are likely to be underestimates, implying that the true effect of sexual selection on rates of signal evolution may in fact be stronger than revealed here.These conclusions are contingent on the degree to which dichromatism represents an accurate measure of variation in sexual selection intensity across lineages. Although dichromatism has been shown to correlate with independent measures of sexual selection such as social mating system and relative testes mass59. Avian carotenoid-based signals represent classic examples of sexually selected, condition-dependent signals of individual quality29. Although there is continuing debate regarding the precise mechanism through which carotenoid traits become honest signals60, field and behavioural studies have demonstrated the role of such traits in determining the outcome of competitive social interactions61. Furthermore, the genetic and biochemical basis of avian carotenoid signal production is becoming increasing well understood63, and phylogenetic evidence indicates that carotenoid pathways are deeply conserved across birds and can be readily up-regulated or down-regulated in response to relatively short-term changes in selection64. This flexibility contrasts with developmental and morphological constraints that potentially inhibit rapid evolution towards other types of conspicuous colouration 65, potentially explaining why we found that rates of evolution towards blue/violet colouration in males were significantly lower than expected compared with null expectations  colours, as well as steeper relationships between dichromatism and evolution rates for carotenoid-based patches than for other signal types . The strength of these effects was strongest in front facing body regions , consistent with a primary role for these body regions in close-proximity sexual or social signalling66, as well as in other animal lineages including fish and reptiles68, our results provide insight into the evolutionary forces responsible for explaining patterns of diversity and convergence in visual signalling traits across the animal kingdom.Overall, our results highlight the dominant role of sexual selection in driving the evolutionary divergence of avian plumage colouration, and reveal the existence of convergent evolutionary trajectories of avian signalling traits under strong sexual selection. Furthermore, our results suggest that signal divergence under sexual selection may be to some extent predictable, with rapid instances of signal divergence biased towards carotenoid-based colouration traits known to function as honest condition dependent signals of individual quality. Further work is required to assess the generality of these patterns, but given the prevalence of convergently-evolved carotenoid signals within and between avian lineages69) phylogenies  of the taxa represented in the Jetz et al. . Together these patches provide a comprehensive set of measurements capturing variation in overall plumage colouration for each specimen that is comparable across species72. To do this, we used a custom IMAGEJ macro to draw a series of polygons onto our images outlining the location of individual plumage patches on each specimen  for 10 plumage patches for a total of 1877 specimens covering 372 species.To convert linearised and normalised RGB values into avian colourspace values, we first estimated the spectral sensitivity of our cameras using data on the camera’s response to a range of colour patches  of known surface reflectance to estimate camera sensor sensitivity functions see . We thenn = 550 patches; see r = 0.92–0.96, P < 0.001 in all cases). Thus, we conclude that our image-based approach is valid and is capable of providing estimates of patch colouration that are highly similar to those generated using spectrophotometric techniques across the full avian visible range.To demonstrate the validity of our image-based approach for measuring plumage colouration, we tested whether relative receptor stimulation values extracted from our processed digital images (see below) matched those generated by spectrophotometric measurements of patch reflectance. For a sample of specimens  capturing the breadth of Tyrannida colourspace (74. This tetrahedron—in which the achromatic (luminance) dimension is removed and each vertex represents one of the four cones characterising avian colour vision —is the sensory equivalent of a morphospace, where similar colours fall in close proximity in the colourspace and disparate colours are far apart74. Given that relative cone stimuli are often correlated, following standard approaches65 we conducted a principal component (PC) analysis to represent the colourspace in fewer orthogonal variables. We used the first two PCs, which together accounted for 93% of the variation in the original colourspace variables  to generate species-level averages in each variable for each patch for each sex. Following previous studies, we represented chromatic variation among patches using a standard avian colourspace model in which raw cone catch values are converted to relative cone stimuli values and projected in a tetrahedron76 implemented in the software BayesTraits V2.0.2 (http://www.evolution.rdg.ac.uk/). To provide species-level estimates of plumage evolution rate that also incorporates information on the longer-term rate of trait evolution leading to the phenotype of a given species, we developed an approach for apportioning rate-scaled branch lengths among each tip of a phylogenetic tree77. Specifically, our approach involves calculating a weighted average of tip-to-root branch rates for each extant lineage, where, analogously to the equal splits approach77, the rate associated with each root-ward edge is down-weighted by a factor of ½:We estimated rates of plumage colour evolution using the multivariate version of the variable rates model of trait evolutionmi is the weighted average of tip i, Ni is the number of edges between tip i and the root of the tree, and lj is the length of each edge j, with j = 1 being the terminal edge leading to the species and j = Ni being the edge nearest the root. Thus, when applied to phylogenies where branch lengths are in units of rate of trait evolution (rather than time), such as those derived from the outputs of the variable rates model, then these tip-specific weighted average values provide a species-level estimate of trait evolutionary rate that also captures the longer-term rate of trait evolution leading to the phenotype of a given species. We refer to this rate-scaled tip metric as TRES  and apply the above formula to mean rate phenograms derived from outputs of the variable rates models described above.andES values) across species, we used phylogenetic reduced major axis regression based on a maximum likelihood optimisation of lambda78. To assess the effect of predictor variables on plumage TRES variation across species, following31 we used Bayesian phylogenetic mixed models in MCMCglmm79 to fit male and female plumage rates as the response variable using six variables  and their interaction with sex as predictors. We used the same approach to test for interaction effects between dichromatism and colouration category, except that here male and female datasets were analysed separately. For details of methods and data sources, including details regarding classification of patches with respect to signal type, see 78. We then compared the average rate of divergence associated with differing evolutionary trajectories in colour space to null distributions of rates generated assuming no relationship between rates and direction of colour evolution. Any deviation in the mean observed rate of evolution compared with the null distribution of rates within each discrete divergence trajectory suggests that rates are disproportionately higher or lower on average than expected relative to null expectations. For full details of this approach, see To assess the relationship between male and female plumage rates (TRSupplementary Information


Lead levels or stunting did not predict failing the screener after controlling for covariates. Though this tool was feasibly administered, it did not demonstrate preliminary construct validity and is not yet recommended for screening in high-risk populations. Future research should include a larger sample size and cognitive interviews to ensure it is contextually relevant.There is a need for developmental screening that is easily administered in resource-poor settings. We hypothesized that known risk factors would predict failed developmental screening on an adapted screening tool in East African children living in poverty. The sample included 100 healthy Ugandan children aged 6–59 months. We adapted a parent-reported developmental screener based on the Child Development Review chart. The primary outcome was failure to meet age-appropriate milestones for any developmental domain. Venous blood was analyzed for lead, and caregivers completed a demographics questionnaire. We used multivariate logistic regression models to determine if elevated blood lead and stunting predicted failure on the screener, controlling for maternal education level, age in months past the lower bound of the child’s developmental age group, and absence of home electricity. In the sample, 14% ( Worldwide it is estimated that 200 million children under 5 years of age do not reach their developmental potential annually . Nearly Children who experience poor nutrition early in life are more likely to have growth stunting ,9. In tuThe association of environmental lead exposure and poor neurocognitive outcomes is also well established . Even chChild development screening is limited or non-existent in many low- and middle-income countries . In thesIt is uncertain whether developmental screening tools standardized in Western industrialized nations are valid across cultures. Western developmental milestone tools adapted for use in African settings tend to be more reliable for gross motor items, compared to social and language development . There aTo date, no studies have been conducted on adapted developmental screening instruments that are easily administered in clinical or community settings and are appropriate for high-risk populations aged 6–59 months in East Africa. To begin to answer these questions, we implemented an adapted developmental screening tool to determine if known neurodevelopmental risk factors, specifically lead exposure and undernutriton as assessed by nutritional stunting, are associated with delayed developmental milestones. We piloted the screening tool as part of a larger survey on environmental heavy metal exposure among children living in the Katanga urban settlement . We hypoAs previously described , we receWith the assistance of study staff, each child and his or her caregiver were guided to Mulago Hospital where informed consent was obtained, a venous blood sample was collected, and a medical history form was completed that included the adapted developmental screening tool.Blood lead was measured in whole blood samples at the Senator Frank R. Lautenberg Environmental Health Sciences Trace Metal Laboratory at the Mount Sinai School of Medicine by LC-tandem mass spectrometry.To assess the developmental status of the children in our sample, we created a brief chart for age-related developmental milestones that could be administered in a caregiver-interview format, which has previously been found to be the most appropriate method of questionnaire administration for populations with lower literacy levels . The purCaregivers of all participants provided written informed consent. The study protocol was approved by the Institutional Review Boards of the University of Minnesota , the Research Ethics Committee of the Makerere University School of Biomedical Sciences, and the Uganda National Council of Science and Technology .We first conducted descriptive and bivariate analyses. We then constructed multivariate logistic regression models to determine if known factors associated with child development—specifically, blood lead levels and growth stunting—predicted failure on the developmental screening tool. Venous blood lead was measured as a continuous variable. For reference, elevated blood lead was defined as >5 μg/dL. Growth stunting was defined as height-for-age Z score of less than 2 standard deviations of the reference mean  . FailureIn this sample, 53 out of 100 children were male and the average age was slightly more than two years . The majWe applied an adapted and easily administered screening tool designed to identify developmental milestones of children living in the Katanga urban settlement in Kampala, Uganda. This screening tool identified 14% of children in the study as having potential developmental delays. Comparatively, in the United States, approximately 15% of children between the ages of 3 years and 17 years have a developmental disability . ContrarWe adapted the tool from widely used developmental screeners in the United States, including the Child Development Inventory (CDI). Similar tools have previously been adapted for use in East Africa as a low cost option for developmental screening, however these are typically time-consuming and require significant cultural modifications related to wording . Our resA limitation of this study is the small convenience sample of caregiver volunteers and the subsequent limited generalizability to other populations in East Africa. Other significant limitations include: reliance on caregiver-reported milestones and no requirement for primary caregiver participation in the study; the fact that we did not collect a concurrent gold-standard measure of child development with which to determine criterion validity of the screener; the lack of cognitive interviews with study participants to ensure the screeners’ contextual and cultural relevance; and the lack of inter-rater reliability for the study coordinators who administered the screener. Though the two local study coordinators who administered the items on the screening tool by verbal interview were fluent in both Luganda and English, possible communication issues or varying levels of comprehension should be considered, particularly given the wide range in educational backgrounds of the caregivers. Future studies of developmental screening in this population should correlate findings of the developmental screening tool with additional measures of neurodevelopmental risk factors, such as maternal and child health concerns. It was not possible at the time of this study inception to complete a prospective design with the primary outcome measure of developmental skills assessment given that this study was completed as part of a secondary data analysis to a larger cross-sectional study on blood levels of heavy metals in children. Future research should be completed on this screening tool with a prospective study design, controlling for moderators, such as stunting and elevated lead levels, along with a comparison gold-standard developmental assessment to better determine its validity prior to widespread use in community settings.To meet the need for accurate, brief, culturally appropriate methods to universally screen children’s development in high-risk global populations, more culturally flexible, validated screening tools are needed. These efforts should be paired with a focus on promoting therapeutic interventions for East African children. Addressing undernutrition, maximizing cognitive stimulation, and establishing community-based therapy efforts are effective, low-cost strategies that are likely to enhance child development . These e