Drosophila. Our characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network. We deconstruct movements into a syllabary of co-active muscles and identify specific syllables that are sensitive to neuromodulatory manipulations. We find that muscle activity shows considerable variability, with sequential increases in stereotypy dependent upon neuromodulation. Our work provides a platform for studying whole-animal behavior, quantifying its variability across multiple spatiotemporal scales, and analyzing its neuromodulatory regulation at cellular resolution.Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity. Brain and muscle activity naturally correlate via the output of motor neurons, but apart from simple movements it has been difficult to define behavior in terms of muscle contractions. By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single-cell resolution, we here describe a multiphasic behavioral sequence in  How do we find out how the brain works? One way is to use imaging techniques to visualise an animal’s brain in action as it performs simple behaviours: as the animal moves, parts of its brain light up under the microscope. For laboratory animals like fruit flies, which have relatively small brains, this lets us observe their brain activity right down to the level of individual brain cells.The brain directs movements via collective activity of the body’s muscles. Our ability to track the activity of individual muscles is, however, more limited than our ability to observe single brain cells: even modern imaging technology still cannot monitor the activity of all the muscle cells in an animal’s body as it moves about. Yet this is precisely the information that scientists need to fully understand how the brain generates behaviour.Fruit flies perform specific behaviours at certain stages of their life cycle. When the fly pupa begins to metamorphose into an adult insect, it performs a fixed sequence of movements involving a set number of muscles, which is called the pupal ecdysis sequence. This initial movement sequence and the rest of metamorphosis both occur within the confines of the pupal case, which is a small, hardened shell surrounding the whole animal. Elliott et al. set out to determine if the fruit fly pupa’s ecdysis sequence could be used as a kind of model, to describe a simple behaviour at the level of individual muscles.Imaging experiments used fly pupae that were genetically engineered to produce an activity-dependent fluorescent protein in their muscle cells. Pupal cases were treated with a chemical to make them transparent, allowing easy observation of their visually ‘labelled’ muscles. This yielded a near-complete record of muscle activity during metamorphosis.Initially, individual muscles became active in small groups. The groups then synchronised with each other over the different regions of the pupa’s body to form distinct movements, much as syllables join to form words. This synchronisation was key to progression through metamorphosis and was co-ordinated at each step by specialised nerve cells that produce or respond to specific hormones.These results reveal how the brain might direct muscle activity to produce movement patterns. In the future, Elliott et al. hope to compare data on muscle activity with comprehensive records of brain cell activity, to shed new light on how the brain, muscles, and other factors work together to control behaviour. A major goal of neuroscience is explaining how nervous systems generate and organize behavior. This requires describing behavior in terms that can be correlated with neural activity. The dynamics of brain activity can be observed in whole brains at single-cell resolution , but beh++ indicators and has been demonstrated at single-cell resolution in hydra 01289 (aka CG9432), that expresses robustly from early larval stages through adulthood, which we call hulk (hlk). We generated a Trojan exon insertion in the hlk gene that co-expresses LexA:QF and combined it with the Ca++ biosensor LexAop-GCaMP6s to report muscle activity. With this hlk-LexA>LexAop-GCaMP6s line , we imaged muscle Ca++ activity through the clarified puparium for approximately 90 min to capture pupal ecdysis behavior, in vivo  movements of P1 , Video 2, and P3 . Traces e phases , and osce phases . The Life phases . Posterie phases , as preve phases . These d++ imaging revealed a phase of random muscle activity prior to the onset of pupal ecdysis  in motor neurons, where it localizes to the neuromuscular junction . The sequences are thus not entirely random. Variability was evident between animals  and for We used the SequenceMatcher algorithm to evaluate the stereotypy of syllable activation during movements in each of the three phases . Surpris++ activity from the dorsal and ventral views, which permits the reconstruction of the movement from the posterior to anterior end of the animal . These syllables are shown in e animal . In gene++ activity represented by the PMEs provide a general description of the pupal ecdysis movements, not all body wall movement is a consequence of local muscle contractions. This is because the pliable cuticle of the pupa provides little rigidity. Like other animals reliant on a hydroskeleton  and peak fluorescence intensity (ΔF/F) for each muscle contraction in segments A3–A5 for each ecdysis phase. Increases in fluorescence only moderately correlated with muscle shortening , moving from A5 to A3 (Comparing ΔL/L with the corresponding average (ΔF/F) reveals hemisegmental differences  includinA5 to A3 . This trA5 to A3  as has bA5 to A3 . Each SwA5 to A3 . While aA5 to A3 .++ activity of M12 is idiosyncratic in P1 and does not contribute to ΔL/L. The coordinated contractions of M12 with other muscles during the Swing, together with the recruitment of additional syllables and higher Ca++ activities, explain why RollCons result only in deflections of the dorsal body wall while Swings cause hemisegmental compaction to bend the entire animal , and two overlapping populations of neurons expressing CCAP and Bursicon. The former manipulation disrupts P1 initiation by blocking abdominal lifting, while the latter blocks initiation of P2  results in normal Ca++ activity during P0 and P1, but P2 and P3 activity is absent  generalized increase in muscle activity during P2; (2) coordination of syllables along the A-P axis that facilitates full body swings; and (3) coordination of activity across the D-V axis, as indicated by the desynchronization of PME2 activity.Suppressing CCAP-secreting neurons  has effects similar to NCCAP suppression: P1 activity is normal, but P2 and P3 are not correctly executed  and competing physiological needs, are absent for pupal ecdysis. In addition, proprioceptive cues, while they may tune ecdysis behavior, are not essential for generating it in that a fictive sequence is generated by an excised pupal brain treated with ETH . FinallyDrosophila embryos prior to hatching  swing-like movement by blocking its progression into the anterior compartment. Additionally, the distribution of CCAP-R appears to reflect mechanisms for selectively regulating distinct compartments across the D-V axis. CCAP-R is expressed selectively in motor neurons that innervate dorsal and ventral muscles. These motor neurons have dendrites that occupy similar positions on the myotopic map and share similar synaptic inputs . This diThe putative source of CCAP for the muscles of PME2 are the type III terminals on muscle M12 , which iDrosophila muscles are targets of a variety of neuropeptides including myoinhibitory peptides, which have also been implicated in pupal ecdysis behavior .Further information and requests for resources and reagents should be directed to and will be fulfilled by Benjamin White  and housed at 25°C and 65% humidity in a 12 hr light/dark cycle. Both males and females were used in this study and all experiments analyzed animals at stages L3 (third instar), P2 01289 gene. To make hlk-T2A-LexA and hlk-T2A-Gal4 lines, either PBS-KS-attb1-2-PT-SA-SD-1-T2A-LexA:QFAD-Hsp70 or PBS-KS-attb1-2-PT-SA-SD-1-T2A-Gal4-Hsp70 was injected into y w; l(2)01289MI05738/Cy; + embryos (Bloomington stock# 42105) and adults were screened for loss of the y+ marker. All injections were made by Rainbow Transgenic Flies, Inc . The CCAP-Gal4 line was a kind gift from J. Ewer . All other fly lines, listed in the Key Resources tTable, were obtained from the Bloomington Drosophila Stock Center at Indiana University.Vinegar flies of the species tar), P2  ~6 hr aftar), P2 , ETHRB-Gtar), P2 . hlk wasThird instar larvae and stage P4 pupae with an air bubble and visible gut movement indicating ecdysis proximity  were isohlk-LexA::QFAD and LexAOP-GCaMP6s, respectively. Control animals bearing the UAS-Kir2.1 transgene, but no Gal4 driver, perform the ecdysis sequence normally, and animals in which the 410-Gal4, CCAP-Gal4, ETHRB-Gal4, and Burs-Gal4 drivers express mCD8-GFP are healthy and viable to adulthood.All neuronal suppression experiments were conducted using two copies of UAS-Kir2.1 in a parental line generated by combining insertions on chromosomes II and III with P4 stage pupae were isolated as for immunohistochemistry, rinsed with 50% bleach for 3 min, rinsed in PBS, and immersed in a custom-chamber with 2,2-thiodiethanol , which rendered the puparium transparent and immobilized the animal with the dorsal, ventral, or lateral side facing the objective . Muscles++-free controls were conducted with SIM without Ca++ (Sigma-Aldrich). CCAP  stock solutions were prepared by dilution to 1 mM in water.P2 stage pupae  were colFor experiments requiring GCaMP6s and jRGECO1a fluorescence, animals were prepared as intact animals above  and imaging was conducted on a Nikon Ti epifluorescence microscope with a 10×/0.5 NA air objective, Cairn twin-cam, two PCO edge 4.2 sCMOS cameras, and filters for GFP and RFP. Images were acquired at 2 Hz for 60–90 min.++ traces were extracted from ROIs defined as the entire animal excluding the puparium, or as single muscles/neurons where indicated, and normalized to F0, defined here as the average fluorescence intensity over the first 50 frames in the image series. Raster plots were generated from activity peaks identified from Ca++ traces using a peak finding algorithm in Python with manually determined thresholds for GCaMP6s and jRGECOa1.Unless otherwise noted, all live experimental image series were background subtracted in Fiji ImageJ2 . Where vhlk>GCaMP6s experimental dataset (N = 16) were annotated frame-by-frame by expert raters to identify the muscles newly activated in each frame. These data were used to determine the frequency and order of activation for each muscle. Movements were annotated manually for these four animals plus one more by visual inspection of muscle activity pattern and deflections of the body wall with respect to the puparium from raw image series (before background subtraction) that were contrast-enhanced to allow visualization of autofluorescence from the body wall. Bouts were identified manually from a subset of 10 hlk>GCaMP6s animals as the start of a sequence of muscle activations flanked on either side by ≥ 2 frames of no new activations, which were defined as inter-bout intervals. The criteria for defining PMEs were determined empirically based on frame-by-frame analysis of 10 complete image series of the pupal ecdysis sequence. Groups of muscles that were repeatedly observed  to be co-active in a regular pattern were subsequently analyzed for the duration of co-activity. A group in which the activity of all muscles overlapped for three or more frames was defined as a PME. Post-hoc analysis revealed that three frames on average represent approximately 15% of the total period of co-active duration for PMEs.A subset of four animals from the total https://anaconda.com, versions 2–2.4.0). Individual statistical methods and parameters are reported in the figure legends.Sample sizes were predetermined using power calculations with 85% power from ecdysis phase 2 behavior duration data presented in hlk>GCaMP6s image. For every frame, the MIP was a 1D vector of length H for an HxW image. Then we computed the location of the mode of the MIP vector on the y axis. An intensity mode indicates the location of the most dominant muscle activation. Therefore, the locations of the modes give an estimate of the direction of motion during the muscle activity period. Note that we are interested in finding the location of maximum motion, which is efficiently captured by the mode of the intensities, not by the mean. Once the intensity modes were identified for every frame using MIPs, we computed the number of modes above and below a threshold, indicating P->A and A->P motion, respectively. The threshold is automatically computed as the median of all modes. We then calculated the ratio of the number of modes below the median line to the number of modes above the median line and used this ratio to determine if the direction of activation is posteriorly or anteriorly directed.To find direction of activation (A->P or P->A) , we firshlk>GCaMP6s image frame. The network model is a modified version of Inception-v3  to estimate the motion at one frame. Therefore, the training data for each frame consisted of a 403 × 129 × 25 matrix. The CNN model was then trained by predicting the motion of the center (13th) frame.We used a CNN to automatically estimate the type of motion in each ption-v3 . To fit Since there were only five animals available for training, we used all possible overlapping frames to augment the training data. Categorical cross-entropy with the Adam  optimizehttps://docs.python.org/3/library/difflib.html).The annotated muscle datasets were sorted by onset frame number and then by muscle. Then, cells were concatenated into strings by bout, movement, or syllable, and assessed pairwise via the Python SequenceMatcher algorithm to score for similarity in string order. Other behavioral features were similarly analyzed using the onset times of syllables, movements, or compartments to order the sequences. Documentation for this algorithm is provided by the difflib library  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:Drosophila. Pupal ecdysis has benefits in that it occurs in the confines of the puparium and muscle activity of the entire animal can be followed through the entire behavioral sequence. The authors have developed tools to monitor both the responses of individual muscle fibers and the activity in the endplates of the motoneurons that innervate them during the course of the behavioral sequence. They have identified a discrete set of motor "syllables" that underlie the various movements and then begin to dissect how removal of different components of the peptide cascade results in the recruitment of these syllables and, hence, the behaviors.The insect ecdysis sequence is a long-standing model for understanding the role of neuromodulators in organizing complex goal-directed blocks of behavior; here it is elegantly explored in Decision letter after peer review:Drosophila" for consideration by eLife. Your article has been reviewed by 4 peer reviewers, including Chris Q Doe as the Reviewing Editor and Reviewer #1 and the evaluation has been overseen by Ronald Calabrese as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: James W Truman (Reviewer #2); Paul Taghert (Reviewer #3); Aref Zarin (Reviewer #4).Thank you for submitting your article "Pupal behavior emerges from unstructured muscle activity in response to neuromodulation in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential Revisions (for the authors):1) In figures 4, S4, and 5 you show GCaMP activity patterns, but it does not appear to be ratiometric with a muscle marker. How can you be sure the changes in GCaMP you are seeing are not due to muscle movement? I understand that the pupa is not translocating, but every muscle contraction will generate some movement in surrounding muscles. It is unrealistic to redo the experiments for ratiometric imaging, but you should explain how you minimize artifactual muscle activity (or loss of activity) due to local body wall movements.eLife. Pupal ecdysis in Drosophila is quite specialized and it would help to place it in context of other insect ecdyses. The complexity and temporal ordering of the "ecdysis sequence" have been nowhere better described than in the paper by Joe Carlson on cricket ecdysis  J. Comp. Physiol. 115:299): it illustrates the three major phases  and how each phase involves the recruitment of diverse motor elements in a characteristic order but with scope for variability. These three phases occur in all ecdysis sequences that have been studied and correspond to the P1, P2 and P3 phases of pupal "ecdysis" in higher flies.2) The background for the paper could be improved for a general journal such as 3) I found it very difficult to relate their findings back to the behaviors that they are trying to explain. The paper does a good job of describing the "trees" but it does not then pull back to place the trees in the context of the "forest". I found it very exciting that they could deconstruct the complex movements shown through the three major phases of the pupal ecdysis sequence into a small number of elemental components . However, it was not obvious as to how these syllables were then combined to generate the 8 basic behaviors they illustrate in Figure 2A. Table S2 is critical because it summarizes the syllables that are involved in building these 8 basic movements. . What I find lacking, though, is clear description the phase relationships of the various syllables during each of these basic behaviors. An attempt to reassemble the behaviors is presented in Figure 7 for two of the behaviors. We are shown the order of appearance of the PMEs, but not their phase (and overlap?) relative to each other during a typical bout. As described below for the figures, I do not think that their color coding works very well, even if the images were large enough to resolve the detail. The authors might consider using a more traditional representation, such as seen in papers defining phases of muscle contraction during a locomotor cycles. This would be a phase diagram using blocks to show the phase and duration of each muscle contraction [in this case the authors would have syllables {individual muscles or PMEs}] during a cycle [variance could also be indicated at the ends of each block]. Although there may be unique events at the two ends of the body [which cannot be resolved in the figures], the authors still focus on a segmental unit in their representations. They could anchor their descriptions on a typical mid-body segment  and show the timing of syllable recruitment during a behavioral bout.Reviewer #3 (Recommendations for the authors):The technical values of this work are very high and the argument that stereotyped sequences of muscle activation arise from smaller stochastically-produced elements (termed syllables) is novel and most interesting. Likewise, the manuscript effectively presents interesting observations on release activity of neuromodulatory inputs and relates these to long-standing ideas about checkpoint controls in ecdysis.Reviewer #4 (Recommendations for the authors):1) In the "Automated movement analysis" section in Methods, the last sentence in the first paragraph is missing a "was" ("The CNN model was then trained by …").2) The keyword "neural circuits" might not be a good match to the purpose of this paper. The study examined thoroughly on muscle activity and the role of neuromodulation; however, it did not investigate multiple layers of neural circuits.Drosophila, especially them having only minor role in larval ecdysis but critical role in pupal ecdysis.3) The authors could, in the introduction, briefly describe the role of ETHR and CCAP in 4) In Figure S4A, plotting both ΔF/F and ΔL/L over time might result in a better visual effect. Essential Revisions (for the authors):1) In figures 4, S4, and 5 you show GCaMP activity patterns, but it does not appear to be ratiometric with a muscle marker. How can you be sure the changes in GCaMP you are seeing are not due to muscle movement? I understand that the pupa is not translocating, but every muscle contraction will generate some movement in surrounding muscles. It is unrealistic to redo the experiments for ratiometric imaging, but you should explain how you minimize artifactual muscle activity (or loss of activity) due to local body wall movements.++ signals with muscle movements. Although technical constraints prevented us from routinely collecting simultaneous images in two channels, we took several steps to insure that the Ca++ signals we measured were correctly associated with the muscles in which they were generated and that changes in signal were not artifacts of muscle displacement due to generalized movements of the body wall. First, because the pupa has half the muscles of the larva, it was possible to measure the GCaMP signals in ROIs that included only the muscle of interest, and not neighboring muscles. Such ROIs were manually identified and the signal quantified for all of the single muscle data reported in the figures referred to. We are thus confident that there is no cross-contamination of signal from adjacent muscles during movement. Second, artifacts due to shifts in image plane during movement were avoided by using low magnification (4x) and widefield illumination. This insured that light was collected not from a single image plane, but from a large volume that included the side of the body wall facing the objective and all of its muscles. Ca++ signals from muscles in the body wall on the opposite side of the animal were excluded from the volume due to scattering and occlusion by internal tissues, which we confirmed by imaging z stacks. We have updated the Methods to clarify our methodology (ll. 980-989 [1056-1065]).We fully appreciate the reviewers’ concern and acknowledge the merits of ratiometric imaging in correlating CaDrosophila is quite specialized and it would help to place it in context of other insect ecdyses.2) The background for the paper could be improved for a general journal such as eLife. Pupal ecdysis in We thank the reviewer for this suggestion and have expanded the background on ecdysis research for a wider audience. We have expanded the Introduction (ll. 51-89 [51-93]) to describe the broader research on insect ecdysis, including the paradigmatic and detailed descriptions provided by Carlson for crickets and Hughes for locusts. Incorporating the suggestion of Reviewer 3 below, we also take the opportunity to place the work on ecdysis in the context of behavioral endocrinology. We note the long history of work on the hormonal control of behavior in both invertebrate and vertebrate preparations of which ecdysis research is a part.3) I found it very difficult to relate their findings back to the behaviors that they are trying to explain. The paper does a good job of describing the "trees" but it does not then pull back to place the trees in the context of the "forest". I found it very exciting that they could deconstruct the complex movements shown through the three major phases of the pupal ecdysis sequence into a small number of elemental components . However, it was not obvious as to how these syllables were then combined to generate the 8 basic behaviors they illustrate in Figure 2A. Table S2 is critical because it summarizes the syllables that are involved in building these 8 basic movements. . What I find lacking, though, is clear description the phase relationships of the various syllables during each of these basic behaviors. An attempt to reassemble the behaviors is presented in Figure 7 for two of the behaviors. We are shown the order of appearance of the PMEs, but not their phase (and overlap?) relative to each other during a typical bout. As described below for the figures, I do not think that their color coding works very well, even if the images were large enough to resolve the detail. The authors might consider using a more traditional representation, such as seen in papers defining phases of muscle contraction during a locomotor cycles. This would be a phase diagram using blocks to show the phase and duration of each muscle contraction [in this case the authors would have syllables {individual muscles or PMEs}] during a cycle [variance could also be indicated at the ends of each block]. Although there may be unique events at the two ends of the body [which cannot be resolved in the figures], the authors still focus on a segmental unit in their representations. They could anchor their descriptions on a typical mid-body segment  and show the timing of syllable recruitment during a behavioral bout.We appreciate the Reviewer’s detailed comments on this important point and are grateful for the suggestion that we represent the phases of syllable activity during movement generation. Based on this and other reviewers’ comments, we recognize that our attempt to bridge the gap between syllables and movements in the summary figure  of our original manuscript was unsatisfying. In the revised manuscript, we present results from the kind of phase analysis suggested by the reviewer and think that these results both strengthen the link between the syllables (“trees”) and the movements (“forest”) and illustrate this link in a relatively straight-forward way.As the reviewer alludes to in his comments, analyzing syllable phases is subject to several constraints. In the absence of an automated method for syllable detection, the required measurements of onset and offset times for each syllable need to be made manually—a daunting task for all syllables in the entire pupal ecdysis sequence! In addition, many pupal movements, particularly those of P3, involve distinct activity in different segments—or even in different halves of the same segment. There is thus no single, “representative” hemisegment on which to conduct the analysis. This is quite different from the peristaltic contractions of larval locomotion, which can be conveniently captured by analyzing muscle contractions in a single hemisegment. To accommodate these constraints, we performed a restricted analysis that focused on the relatively simple movements of P1 and P2. These movements involve muscle activity that is similar across hemisegments (as for the RollCon and Swing) or similar within the segments of the posterior compartment (as for the Lift). By confining the analysis to two hemisegments, one (A6) in the posterior compartment and one (A4) in the anterior compartment, we could thus resolve syllable dynamics for both movements of P1 and could monitor the progression of the P2 Swing as it traverses both the posterior and anterior compartments. To do so, we measured the phasic activity of all syllables visible from the lateral view for A6 and A4 in a set of representative bouts of P1 and P2. These analyses are now presented as phase diagrams in Figure 7D and E of the revised manuscript. As suggested by the Reviewer, we used blocks to show the phase and duration of each syllable during a bout and indicated the standard deviations at the ends of each block. In the accompanying text (ll. 246-274 [273-301]), we describe how the phasic activity of the syllables gives rise to the movements executed. We also now provide a supplemental figure  that illustrates the syllable dynamics of a Swing movement for muscles of all hemisegments of the pupa. In addition, we have taken the reviewer’s suggestion to elevate the former Table S2, describing the syllable composition of all movements, to Table 1 in the main text. We have also tried to improve the color-coding in figures throughout the manuscript to resolve potential ambiguities. In an effort to rationalize the use of color, we chose from the palette of colors recommended for the color-blind.Reviewer #4 (Recommendations for the authors):(1) In the "Automated movement analysis" section in Methods, the last sentence in the first paragraph is missing a "was" ("The CNN model was then trained by …").Thanks very much for catching this detail. We have inserted the missing word (l. 1078 [1154]).(2) The keyword "neural circuits" might not be a good match to the purpose of this paper. The study examined thoroughly on muscle activity and the role of neuromodulation; however, it did not investigate multiple layers of neural circuits.We appreciate the reviewer’s point, but certainly hope that our paper will be of interest to others who are mapping neural circuits. Motor neurons represent the final common path for all circuit activity and our goal was to define activity in this path as an entry point for understanding activity at higher levels in the pupal ecdysis circuit. We hope that others will see the merits of using muscle activity as a proxy for motor neuron activity to gain traction on how circuit-level activity generates behavior.Drosophila, especially them having only minor role in larval ecdysis but critical role in pupal ecdysis.(3) The authors could, in the introduction, briefly describe the role of ETHR and CCAP in In the expanded description of ecdysis requested by several reviewers, we now outline the roles in ecdysis played by neurons targeted by ETH and CCAP in the Introduction (ll. 79-86 [80-90]).(4) In Figure S4A, plotting both ΔF/F and ΔL/L over time might result in a better visual effect.To show how these values change over time, we have now separated them out by phase .


The provision of post-discharge malaria chemoprevention (PMC) in children recently admitted with severe anemia reduces the risk of death and re-admissions in malaria endemic countries. The main objective of this trial was to identify the most effective method of delivering dihydroartemesinin-piperaquine to children recovering from severe anemia.This was a 5-arm, cluster-randomized trial among under-5 children hospitalized with severe anemia at Zomba Central Hospital in Southern Malawi. Children were randomized to receive three day treatment doses of dihydroartemesinin-piperaquine monthly either; 1) in the community without a short text reminder; 2) in the community with a short message reminder; 3) in the community with a community health worker reminder; 4) at the facility without a short text reminder; or 5) at the facility with a short message reminder. The primary outcome measure was adherence to all treatment doses of dihydroartemesinin-piperaquine and this was assessed by pill-counts done by field workers during home visits. Poisson regression was utilized for analysis.Between March 2016 and October 2018, 1460 clusters were randomized. A total of 667 children were screened and 375 from 329 clusters were eligible and enrolled from the hospital. Adherence was higher in all three community-based compared to the two facility-based delivery . This was observed in both the SMS group  and in the non-SMS group . Although adherence was higher among SMS recipients (98/148 66·2%] vs. non-SMS 82/144 (56·9%), there was no statistical evidence that SMS reminders resulted in greater adherence . When compared to the facility-based non-SMS arm (control arm), community-based delivery utilizing CHWs resulted in higher adherence .Community-based delivery of dihydroartemesinin-piperaquine for post-discharge malaria chemoprevention in children recovering from severe anemia resulted in higher adherence compared to facility-based methods.NCT02721420; ClinicalTrials.gov. Severe anemia in under-five children is defined as low hemoglobin level of less than 7g/dl . It affePost-discharge malaria chemoprevention (PMC) is the targeted use of antimalarials in children with severe anemia to create a malaria prophylactic window period post-transfusion and during recovery . SimilarUtilizing community health workers (CHW) to deliver interventions for childhood diseases such as pneumonia and malaria has been reported to be effective in reducing childhood mortality and improved access . ComparePMC is an intervention that could offer substantial public health gains in managing under-five children who are at risk of dying. Although PMC is a relatively simple intervention, implementation will require appropriate delivery strategies so that it reaches the population that need it most. However, a major concern is how it can be delivered in an effective and sustainable way . HoweverThe aim of this trial was to identify the most effective delivery mechanism for PMC by comparing community and facility-based delivery systems with and without reminders using short-text message service (SMS) and using CHWs also known as health surveillance assistants (HSAs) in Malawi.The study was conducted at Zomba central hospital (ZCH), a referral government hospital with high pediatric admissions rates due to severe malarial anemia located in southern Malawi with perennial malaria transmission. The hospital provides health services to a population of over one million and it is the only government health facility that provides blood transfusions and health services in Zomba district free of charge. Zomba Central Hospital was selected because previous related studies had been conducted there and is was particularly suitable for the delivery trial because of the existing linkages with community based health care providers and rural clinics within their catchment areas. This was a five-arm open label cluster-randomized trial. Children aged less than five years who were admitted to ZCH pediatric ward were included if they had severe anemia (defined as a hemoglobin (Hb) concentration of <5g/dL) , and havThe unit of randomization was the village cluster and it was computer-generated by the trial statistician prior to the trial. All of the 1486 villages which were defined to be in the catchment area of ZCH were randomized to each delivery method, as it was not known which village prospective participants would reside prior to the study. Therefore, all children from the same village cluster would be assigned to the same delivery arm. The villages were randomized to one of the following five arms: 1) community-based without an SMS reminder (Com + No SMS); 2) community-based with an SMS reminder (Com+SMS); 3) community-based with an HSA reminder instead of SMS reminder (Com+HSA); 4) facility-based delivery without SMS reminder (Fac-SMS); or 5) facility-based delivery with SMS reminder (Fac+SMS). Detailed descriptions of the methods have been provided in In the SMS arms, the text of the SMS was customized for each child with the following message “Remember to give (child’s name) medication starting from tomorrow for three days.” It was translated into the local language of Chichewa, the most widely spoken language in Malawi. The reminder was pre-tested on a random sample of caregivers coming with their children to the pediatric out-patient department prior to commencement of the trial. Considering that this was an implementation trial, we wanted to reflect the “real life” situation and therefore mobile phones were not provided to caregivers. However, for all study participants; we collected mobile phone numbers of the nearest contact person. Consenting caregivers were instructed to inform the owners of the mobile phone numbers that their child was participating in the trial Caregivers and study staff was aware of the allocation. However, the study statistician who performed the analyses was blinded to treatment allocation.®, Sigma-Tau, Italy) dispersible tablets prescribed in standard doses for three days every month. The monthly treatment courses were prescribed at two (PMC-1), six (PMC-2) and ten weeks (PMC-3) after discharge enrolment regardless of the presence of symptoms. The maximum number of doses that could be administered was nine. Standardized verbal instructions about administration were given to the primary caregiver and specific dates of administration were documented in the child’s health book and the drug blister pack. After enrolment, all the participants were requested to come back to the study clinic at 15 weeks for the end of study assessment.The study medication was co-formulated 20 mg dihydro-artemisinin and 160 mg piperaquine  electronic case report forms (eCRF). Stata 15 was used for analysis.The study was designed to determine whether delivery of PMC through community-based methods resulted in higher adherence than when delivered through facility-based methods and the sample size was based on this. The main effects of “place of delivery” and “use of mobile phone reminders” were of primary interest. It was estimated that each village cluster would contribute 2 to 4 children with severe anemia. We assumed an intra-cluster correlation coefficient (ICC) of 0.1 and allowing for 20% loss-to-follow-up or efficiency loss due to varying cluster sizes, a sample size of 125 clusters (villages) of an average of 3 children per village , had 80% power to detect a 25% absolute increase in coverage from an estimated 50% in the standard out-patient groups to 75% in the arms supported by mobile phone reminders (α = 0.05). The ICC of 0.1 is slightly more conservative than the ICC in a previous trial of delivery approaches for IPTc in the Gambia (0.08). A statistical analysis plan (SAP) was written and agreed upon by all investigators before the data were analyzed. However, additional analyses were included in the SAP during analysis to explore effects of different cut offs of adherence.We used appropriate descriptive statistics to summarize data, with mean or median (IQR) for continuous data, and frequencies and percentages for categorical data. For the primary outcome, our analysis was by intention to treat at cluster and participant levels. We used Poisson regression for the total number of doses administered. The unadjusted and adjusted incidence rate ratios (IRR) measuring the percentage of children receiving PMC according to schedule in each arm were obtained and compared between arms and the 95% CI for the IRR have been reported. The estimates of the IRR were adjusted for baseline characteristics and accounted for clustering with 95% CIs are provided for all measures of effect.We also employed a factorial design analysis for further analysis to investigate the interaction between the effects of community compared to facility delivery and the pooled effect of SMS; and interaction terms were included in models to assess the strength of interaction. We also did several subgroup analyses for the primary and secondary outcomes adjusted for clustering, socioeconomic and demographic variables stratified by each intervention arm with the Fac-SMS group as the reference group.Our primary outcome of the study adherence to 7 to 9 doses of the study drugs. The secondary outcomes of the trial were: 1) the proportion of those with medium adherence defined as administration of 4 to 6 doses of the study drug; 2) the proportion of those with low adherence defined as the administration of less than 3 doses of the study drugs; 3) all-cause mortality and 4) all cause sick visits.For the secondary outcomes, the levels of adherence were compared between the five groups. Full details of the study design and statistical methods are provided in detail in the protocol , which iNCT02721420).The study was approved by the research ethics committees of the College of Medicine in Malawi . Written informed consent was obtained from legal guardians of the study participants prior to enrolment. The trial was registered at ClinicalTrials.gov . A total of 351 children (93.6%) were followed successfully until the end of the study; caregivers of four children (1.1%) withdrew consent prior to the first home visit while four children died and twenty did not attend the end of study visit at 15 weeks. Mobile phone ownership in the household or with a neighbor was reported in 39.7% of the caregivers, with the Fac+SMS arm reporting highest (45.2%). The mean distance to the hospital was similar among all the arms (19.7 ±SD 9.5 km). Out of the 150 participants who were expected to receive SMS reminders, only 25 [18.0% (10 in COM+SMS and 15 in FAC+SMS)] reported to have received at least one SMS.The primary outcome was to compare the PMC delivery across the five intervention arms. The endpoint is based on the total number of doses that were actually used. Adherence to all PMC courses was reported in 63.1% of all the children. A higher proportion of children in the community-based arms reported full adherence to compared to facility-based arms . Children receiving PMC in the community in the SMS arm reported the highest proportion of full adherence (79.7%) compared to those in the community but receiving no SMS (63.2%), Com+HSA (68.4%), Fac-SMS (51.3%) and Fac+SMS (52. %) in the univariate analysis. The proportion that was administered at least six doses representing medium adherence was 22.1% among all the children and Fac-SMS arm had the highest proportion of medium adherence (27.6%), and Com+SMS arm (12.2%) lowest for medium adherence. On the other hand, only 2.9% of the children reported very low adherence which was representing less than 30% of the total doses  Files.In the mixed effects model, village did not have a significant random effect, the likelihood ratio test was 2.9e-05, p = 0.498. In fact the Intra-Cluster Correlation coefficient (ICC) was very small 7.56e-06, indicating that there were no correlations for individuals coming from the same village relative to those of different villages. Thus, the available data is as good as the independent data and give the same results as independent data analysis.When modelled as count data to assess whether the incidence of number of doses used per patient differ across the groups, a mixed effects Poisson regression was fit to the data. When PMC was delivered through the community-based methods, adherence was greater than facility-based methods  and this was observed in both the SMS recipients  and in the non-SMS recipients  Figs .Overall, a total of 83.6%, 84.1% and 78.0% of the children received complete courses of PMC1, PMC2 and PMC3 respectively. Generally, most of the children who received the first dose of each PMC course also received the second and the third dose. A small proportion (1.3%) received the first and second dose but not the third dose of PMC 1, only one participant (0·3%) took only the first dose of PMC 2 and another one (0.3%) took only the first and second doses of PMC 2. All participants who took the first course of PMC 3 completed all the three doses.Community-based delivery arms had the highest proportions of participants receiving PMC2; (86.4 to 91.9%) and PMC3 (78.4 to 89.4%) compared to facility-based arms; who reported up to 78.7% receiving PMC2 and less than 70% for PMC 3. There were no significant differences in the proportion of participants across the arms in administration taking the PMC1 (p = 0·56) and PMC2 (p = 0·08) but administration of PMC 3 was statistically different across all the groups.As shown in Figs There was no difference in adherence between facility-based delivery with SMS and without SMS   and therThere were total of four deaths ; two each in the Com-SMS and Fac+SMS. Two deaths were caused by severe anemia; one due to sepsis and one death caused by drowning. The total number of reported sick visits was 90 of which 46.7% were due to uncomplicated malaria. The lowest number of adverse events was observed in the Com+HSA arm. The number of deaths was too low to make meaningful association to adherence.nd and 3rd course of PMC. These findings are consistent with findings reported from other African countries. Studies in Kenya, Uganda and Ghana reported that malaria treatment distributed by CHWs resulted in reduced malaria incidence and increased access to treatment [In this study, we found that children who had their monthly DHP in the community provided by caregivers irrespective of reminders resulted in 24% higher adherence compared to facility-based delivery where caretakers were asked to return to the clinic to collect their 2reatment ,25,26. Sreatment . In the reatment .Although utilizing CHWs has been supported by WHO and has proved to be effective for high adherence and reduced incidence of disease, the effectiveness has shown to decline for subsequent doses. In Mali, seasonal malaria chemoprevention (SMC) delivered through routine programmes utilizing existing CHWs reduced malaria prevalence; 84% of the targeted children received the first course but only 54% received four complete SMC courses . AnotherWhereby there are limited studies that have examined adherence to medication when utilizing caregivers to administer longer-term malaria interventions at home, in our study, we found that community-delivery utilizing caregivers resulted similar adherence without requiring HSA reminders. One study in Sierra Leone reported that caregivers are capable of administering anti-malarial medication to the children with basic instructions and supervision of the first dose by the CHW . CaregivUse of mobile phone reminders has been hailed as an important tool to achieve adherence, optimal treatment response, case management and outcomes in resource poor settings such as SSA ,36. HoweOur findings are important because PMC is a relatively new intervention in the Malawian and other developing countries health care system where established delivery systems currently do not exist. Because the timing of PMC medication administration is sensitive, it would be a challenge for a caregiver to rely on receiving timely reminders on a personal phone or rely on others to receive the reminder and pass on the message. A study in Tanzania reported that unsupervised malaria treatment given at home by caregivers was effective without the utilization of any reminders . ContrasAlthough we found that SMS reminders had no effect on adherence, it must be noted that the proportion of caregivers that reported received the SMS reminder was very low. This was a major limitation in our study as we were not able to conclusively state that they are not effective in improving adherence regards to PMC in our setting.This is the first study to assess adherence to PMC, which is an innovative intervention for managing sick children during the post-discharge period. We found that using community-based strategies, caregivers were able to administer PMC medication to their children without additional reminders. As they routinely administer medication to their children at home, this may be a cost-effective strategy for delivery of PMC. Although utilizing HSAs has proven to be effective for improving adherence for other malaria interventions, adherence to subsequent doses has tended to decline and may remain challenging for PMC delivery utilizing HSAs reminders, which is interval sensitive.S1 Checklist(DOCX)Click here for additional data file.S1 File(XLS)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(XLS)Click here for additional data file.


The present study aims to evaluate dosimetric and clinical risk factors for the development of maxillary osteoradionecrosis (ORN) in head and neck adenoid cystic carcinoma (ACC) patients treated with carbon ion radiotherapy (CIRT).D), with an increment of 10 Gy (RBE), and additional clinical factors were correlated to the incidence of ORN with univariate analysis (Chi-square test). The logistic regression model was subsequently applied for multivariate analysis. Treatment plans calculated with a local effect model (LEM)-based optimization were recalculated with the modified microdosimetric kinetic model (MKM), and compared with literature data from the Japanese experience.Clinical data and treatment plans of ACC patients, consecutively treated from January 2013 to September 2016 within the phase II clinical trial CNAO S9/2012/C, were retrospectively reviewed. ORN and other treatment-related toxicity were graded according to the Common Terminology Criteria for Adverse Events (CTACE), version 4.0. The maxillary bone was contoured on the planning CT, and only patients receiving more than 10% of the prescription dose at their maxilla were considered for the analysis (67 patients). The volumes of maxilla receiving doses from 10 Gy (RBE) to 60 Gy (RBE) . Maxillary ORN was observed in 11 patients (16.4%). Grade 1 ORN was observed in 2 patients (18.1%), G2 in 4 (36.3%), G3 in 4 (36.3%) and G4 in 1 (9.3%). From univariate analysis, the site of the tumor, the presence of teeth within the PTV and acute mucositis correlated with the development of maxillary ORN. VThe volume of maxilla irradiated with high dose values was relevant for ORN development in our cohort of ACC patients. These results are in line with previously published data obtained with a different radiobiological model. Our findings might be helpful to prevent the risk of ORN in patients receiving CIRT. Osteoradionecrosis (ORN) is described as a chronic wound that fails to heal with bone exposure. It is a treatment related complication primarily reported in the head and neck population treated with radiotherapy (RT) frequently associated with a substantial morbidity causing pain and infection. The pathophysiology of its occurrence is linked to the hypoxia, hypocellularity, and hypo vascularity that follow a RT course . There aThe incidence of ORN of the mandible when irradiating parotid tumors and also at the skull base when irradiating tumors localized at the nasopharynx, has decreased in recent years with the transition from 2 and 3D-RT to the modern intensity modulated-RT (IMRT), since the more localized deposition of the higher doses to the tumor target in respect to the surrounding normal tissues , 5. In tWhile numerous clinical and physical factors have been reported to be associated with ORN development after photon RT, the risk factors for maxillary ORN after CIRT are still under investigation. Sasahara et al.  reportedThe aim of the present study was to identify dosimetric and non-dosimetric risk factors correlated to ORN development for head and neck ACC patients treated at CNAO  with CIRT delivered with curative intent. At CNAO the local effect model (LEM—version I) is employed for RBE calculation as in other European CIRT centers , with trThe present study is a retrospective, observational study, a subanalysis of the prospective phase II clinical trial ongoing from January 2013 to September 2016 at CNAO (CNAO S9/2012/C) to assess safety and feasibility of CIRT with radical intent for ACC patients with partially resected tumors (R1/R2 margins), or unresectable tumors, or unfit to surgery . CNAO’s A total of 128 patients with head and neck ACC consecutively treated with CIRT along with their diagnostic imaging and treatment plan data were screened for selection in the present study. Only the patients with at least more than 10% of the total prescription dose of 68.8 Gy (RBE) to their maxilla were selected for the analysis as detailed in Sasahara et al. . On the A set of 2 mm thickness CT scan was acquired for treatment planning. Patients were immobilized with a personalized thermoplastic head–neck–shoulder mask equipped with bite-blocks and fixation points to the couch as previously reported . After tD doses , for both LEM and MKM calculated plans. For each VD parameter of the LEM-based plans, the median value was calculated over the entire patient cohort, and patients dichotomized accordingly, to test the correlation with the incidence of ORN. Furthermore, the patient cohort was divided into two groups according to the appearance of ORN, and the average DVHs were calculated for both groups and radiobiological models (LEM and MKM). In the work by Sasahara et al., the absolute maxillary volume receiving 50 Gy (RBE) (V50MKM) was found significant for the onset of ORN. We therefore extracted the V50MKM for each patient, from the MKM-DVH, and calculated the corresponding LEM-based dose received by the same volume of maxilla in each delivered plan, to compare our treated population with literature dosimetric data  increments (V10–V60) extracted from the LEM-based calculated plans, and statistically significant clinical factors. To increase the power of our logistic regression model, we then limited the variables in the analysis to the VD in 10 Gy (RBE) increments and the ORN incidence. If the estimated odds ratio was >1, the variable was assumed to be a risk factor because it increased the baseline risk. SPSS ver.19 software  was used for statistics.The Chi-square test was performed to investigate both clinical and dosimetric variables as predictors of ORN risk. For statistically significant variables only, the strength of association was estimated by Cramer’s V. The Kaplan–Meier method was adopted to evaluate the cumulative incidence of late post-radiation bone effects with respect to the only statistically significant variables in the univariate analysis. The difference that was observed between the cumulative incidence curves was compared with the Log-rank test. P-values <0.05 were considered statistically significant. The logistic regression model was subsequently applied to assess how the baseline risk of developing ORN varied according to the presence/absence of several given variables, namely, the VThe median follow-up time was of 50 months (range 8–82 months). ORN was observed in 11 patients (16.4%) with a median time to ORN development of 24 months (range 8–54 months). Grade 1 ORN was observed in 2 patients (18.1%), G2 in 4 (36.3%), G3 in 4 (36.3%), and G4 ORN in 1 patient (9.3%). In this latter case the patient had a microbial infection at the sphenoid bone not properly managed, and the necrosis expanded up to the right carotid channel approximately 10 months after CIRT. To avoid fatal bleeding due to carotid blow out, the patient had to perform carotid artery coiling. At the last follow up, the patient was alive without evidence of disease, and his necrosis stable. In 6 patients ORN started at the bone close to the teeth alveoli, and in one case followed the loss of one dental element. Majority of the cases had stable ORN at the last follow up (81.8%). In 2 cases with G3 ORN a surgical sequestrotomy was performed, while in one case the defect was repaired with a temporal muscle flap. Furthermore, 3 patients in the series took advantage of hyperbaric chamber treatment to stabilize the progression of their G2 necrosis after intermittent cycles of antibiotics for recurrent infections of the necrotic area.On univariate analysis, the tumor site, and acute mucositis ≥G1 developed during CIRT were found to be statistically associated with maxillary ORN  together with the presence of teeth in the irradiated PTV. The incidence of ORN always occurred in patients where the maxillary volume received dose higher than or equal to the median cut-off. D confirmed that the survival function from ORN decreases as the irradiated maxillary volume increases, both for low and high dose levels (data not shown). Among those patients who had no upper teeth within the PTV, none reported ORN. Among those who had maxillary teeth within the PTV, 36.7% suffered from maxillary ORN after CIRT .In D, the tumor site, the presence of teeth within the PTV and acute mucositis, the logistic regression model identified only V60LEM as a significant variable in differential risk compared with baseline risk . The risk of developing ORN increased by 43.1% if a maxillary volume ≥0.6 ml was covered with at least 60 Gy (RBE) dose. When limiting the variables in the regression analysis to the VD in 10 Gy (RBE) increments to increase the power of prediction, the V60 LEM was still identified as independent factor significantly associated to the ORN development in our series.Considering all the VLEM.When the cohort of the patients was divided into two groups according to the appearance of ORN, the average maxillary dose received by patients with ORN was higher at all volume levels, in both RBE languages  ± 2 SD.Finally, as for comparison with previous literature dosimetric data acquired with the MKM radiobiological model , the LEMBone necrosis is an important side effect, not only when irradiating with conventional photon RT, but also after CIRT. Here we analyzed for the first time the risk factors for ORN development in patients treated with LEM-based CIRT. Although we had a small sample of patients and of ORN events, we have still shown in our series the relevance of dosimetric parameters in predicting ORN risk after CIRT, with both the biological models mostly used in CIRT facilities worldwide for treatment planning, the LEM and MKM model. Tumor treatment, especially when curative, must not disregard ORN risk to preserve patient quality of life. However, sparing dose to the maxilla, should never be prioritized at the cost of target coverage.MKM was found to be a risk factor at multivariate analysis, along with the presence of teeth in the PTV, in a series with different radioresistant tumors including ACC treated with CIRT at a prescription dose of 57.6 Gy (RBE)  was not explicitly tested, but it is in good agreement with the dose value resulting significant at our multivariate analysis (V60LEM), considering all the uncertainties involved. As a limit, several sources of dose deviation need to be considered when comparing clinical outcomes from patient series treated with different techniques, such as the beam delivery, the different beam number per fraction and beam arrangements, physical and RBE-weighted dose calculation uncertainties, the small number of patients, and different clinical characteristics  . As poinGy (RBE) , when ine series , into theristics . FurtherMKM of the maxilla or mandible was 12.5 ml  for patients with Grade 0–1, and 12.5 ml  for patients with Grade 2–3 ORN, respectively, with a difference not statistically significant  prescription dose in 16 fractions equivalent to 89.6 Gy (RBE) at a fractionation of 2 Gy (RBE) per fraction, experienced 11.8% G3 ORN requiring surgery . Furthernificant .MKM–V45MKM at the mandible, and in case of V10MKM–V30MKM to the teeth (D (V10 LEM–V60 LEM) tested at univariate analysis were statistically correlated with the occurrence of ORN. Maxillary ORN cases occurred in the subgroup of patients with VD larger than- or equal- to the median volume for that DVH as it has been reported also in Sasahara et al. although with a different biological model  in 16 fractions, maxillary ORN was the most frequent late toxicity together with visual impairment, with G3 ORN reported in 18 patients (4%) and grade 2 in 36 (8%) .In our series, the presence of superior dental elements within the PTV correlated with the onset and development of maxillary ORN at univariate analysis. In Bhattacharyya et al. the number of teeth irradiated with more than 50 Gy (RBE) was a significant independent risk factor for the development of oronasal fistula, which is also a late complication of CIRT. In order to reduce the risk, the authors suggested minimizing the number of teeth at a maximum of 2 within the volume irradiated with more than 50 Gy (RBE) . These dIn our series, we investigated also pre-CIRT surgery as risk factor for ORN. According to Marunick et al., pre-RT surgical risk involves tissue trauma, reduced blood supply, cell death, collagen lysis, and greater metabolic challenge to repair. Surprisingly, here postoperative CIRT was not correlated with increased ORN risk differently from Marunick et al., where only the 4 patients included in the study who had jointly received both definitive tumor resection and neutron RT developed ORN at the residual maxillary orbital complex .LEM–V60LEM irradiated volumes as most important factor correlated with ORN. The limitations of our study include the small patient number and the short follow up. For the future, clinical trials with larger patients’ number and longer follow up are warranted. Based on our experience enriched by literature data, we might further summarize the recommendations for the optimal strategies to reduce ORN risk onset when irradiating tumors at the skull base, especially in facilities using the LEM-based CIRT: 1) to perform dental examination and prophylactic extraction procedures before CIRT if necessary, and close dentist follow up for all the 2-year period after CIRT more at risk of developing ORN, especially in case of patients with the maxilla covered for large volume by dose higher than 55 Gy (RBE); 2) to maximize the dose conformity to the maxilla by reducing volumes irradiated with dose higher than 55 Gy (RBE) and the dose bath below 50 Gy (RBE); 3) to avoid teeth in the PTV especially if the tumor does not involve the maxilla, and in case it is inevitable, to include not more than 2 teeth in the high dose PTV; 4) to prefer conservative measures and avoid surgical traumatic procedures after ORN onset. In case of unamenable necrosectomy after CIRT, our recommendation might be to remove the bone around the necrotic tissue by including at least the V55 LEM isodose and higher, in order to facilitate tissue post-surgical healing.In summary, among all the toxicities of patients treated with CIRT in the head and neck area, ORN is one of the most serious and debilitating chronic post-treatment sequelae. Here, we have pointed out the V55The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethical committee San Matteo Hospital, Pavia . The patients/participants provided their written informed consent to participate in this study.Conceptualization: BV, SR, and EO. Methodology and statistics: BV, SR, SM, EO, MM, and SR. Writing: MM and BV. Review and Editing: BV, SR, MM, MB, SRO, RI, AMC, SI, LP, MC, SM, and EO. Supervision: BV and EO. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The prevalence of gestational diabetes mellitus (GDM) is increasing, and only a few mobile health  applications are specifically designed to manage GDM. In this mixed-methods study, a follow-up study of a randomized controlled trial (RCT) analyzed a largely automated mHealth application-based lifestyle coaching program to (a) measure the application’s usage behavior and (b) explore users’ perceptions of its usefulness in GDM management. Quantitative data were collected from the 170 application users who had participated in the intervention arm of the RCT. Semi-structured interviews (n = 14) captured users’ experiences when using the application. Data were collected from June 2019 to January 2020. Quantitative data were analyzed descriptively, and interviews were analyzed thematically. Only 57/170 users (34%) logged at least one meal, and only 35 meals on average were logged for eight weeks because of the incorrectly worded food items and limited food database. On the contrary, an average of 1.85 (SD = 1.60) weight values were logged per week since the weight tracking component was easy to use. Many users (6/14 (43%)) mentioned that the automatic coach messages created an immediate sense of self-awareness in food choices and motivated behavior. The findings suggest that for GDM management, a largely automated mHealth application has the potential to promote self-awareness of healthy lifestyle choices, reducing the need for intensive human resources. Additionally, several gaps in the application’s design were identified which need to be addressed in future works. Gestational diabetes mellitus (GDM) is a well-established risk factor for future type 2 diabetes mellitus . GDM affMobile health  applications have been shown to be an effective alternative to face-to-face consultations for delivery of health-related information because the information can be delivered promptly. Additionally, mHealth applications have been shown to improve patients’ compliance with treatment and their self-management awareness ,5,6. ManGlobally, only a few mHealth applications are designed to support self-management of GDM ,15,16,17On the other hand, studies on the effectiveness of largely automated mHealth applications in managing GDM are limited, i.e., an mHealth application consisting of an interactive coaching system with minimal manual intervention from healthcare professionals. One such study is the SMART-GDM study . This stThe mHealth application, i.e., Habits-GDM, analyzed in this study is a largely automated mHealth application-based lifestyle coaching program minimizing the need for intensive human resources. Habits-GDM has several components built in to increase women’s self-awareness when managing GDM. The application has three key components: interactive educational lessons, tracking tools , and coaching . Table 1Health Belief Model constructs was used to develop the components of the Habits-GDM application ,23. The This study was a follow-up study of the SMART-GDM randomized controlled trial conducted at the National University Hospital, Singapore, which sought to examine the effectiveness of the Habits-GDM application designed for women with GDM . The SMART-GDM study recruited a total of 340 women with GDM (170 women in the intervention arm and 170 women in the control arm). The SMART-GDM aimed to determine whether the Habits-GDM application could prevent excessive gestational weight gain and improve glycemic control and maternal and neonatal outcomes in GDM  dietician had all the products on her shelf […] nurses even bought a bowl and showed us the amount that we could take […] application cannot replace them […]” (P14).The qualitative interview participants (7/14) named ‘Healthy eating’ as the most useful lesson because it helped them to remember the healthy foods (7/7) and guided them to make healthy food choices (7/7) when managing GDM. ‘Eating smart’ and ‘Eating out’ were the next most useful lessons. The quantitative data showed that the ‘Glucose monitoring’ lesson was most frequently accessed . It could likely be due to the main focus on blood glucose monitoring in GDM. Consistent with the quantitative data, the interview participants (9/14) named ‘Why exercise’ as the least useful lesson. They mentioned feeling tired due to pregnancy and hence did not exercise. Five out of the fourteen interview participants did not mention any lesson as the least useful when asked. Users were prompted to log at least three meals in the application for any two days of a week . However, among the 170 application users, only 0.88 (SD = 0.99) number of meals were logged per week on average, with only one user (0.6%) logging six or more meals. Among the 170 application users, 113 users  did not log any meals using the application. Only 57 users  logged at least one meal using the application and, on an average, 35 meals were logged by them for 8–10 weeks. The number of meals logged represents those meals logged using the application and do not include entries on the paper diary, which is part of usual care. As confirmed by the qualitative interviews, most participants (12/14) had negative experiences using the application when recording diet and discontinued its usage after a few days. It seemed that the application’s diet tracking component did not help them in managing GDM. Participants experienced difficulties with the search feature (3/12), measurement unit (8/12), and limited ethnic food database (9/12). For instance, the search feature did not bring up the commonly consumed items, and the everyday Chinese food items such as chicken rice were missing in the database:“[…] the food options are not very localized. The common local food, like chicken rice, cannot be found.” (P08)Another challenge was that the food item’s name was not worded in the commonly known way (1/12), and the imperial measurement (cup) in the application was not familiar to the participants. Instead, participants suggested to either allow them to upload pictures of their diet or to replace the measurement unit with “tablespoon” or “bowl”, which is the terminology commonly used by the dieticians who conduct the usual care education sessions. These participants (12/14) resorted to the usual procedure of a paper diary due to its ease of recording and convenience of viewing diet and blood glucose values side-by-side. Healthcare professionals tended to favor the paper diary because they had to navigate to different screens on the mHealth application to view the corresponding diet consumed for a particular blood glucose reading rather than being displayed on a single screen, as a paper diary does. Due to this, participants (12/14) perceived that their healthcare professionals were more interested in looking at the paper diary, which further discouraged them from using the application for data entry.Despite the above-mentioned challenges, a few participants (2/14) used the application more frequently due to their perceived responsibility to the research team. They coped with the database’s limitations either by breaking down the ingredients or by choosing the most similar food type available in the application’s database. Additionally, they also mentioned that although they were not able to capture the precise food eaten, tracking diet using the application gave them a sense of control (2/2) and confidence in their food choices (2/2). By cross-checking the food item when their blood glucose readings were high, they were able to avoid the food item in future meals.Users were asked to monitor their weight once a week. The average number of weight values logged per week among the application users was 1.85 (SD = 1.60), with 116/170 users (68%) logging weight at least once a week.Similarly, most interview participants (9/14) reported tracking weight using the application. Ease of use due to the automatic transfer of weight values from the digital weighing scale to the mobile phone (9/9) and GDM-related perinatal complications were the reasons reported for tracking weight. The graphical representation of the weight values recorded enabled straightforward interpretation and helped participants to track their progress over time (7/9).By looking at the graphical representation of weight values, participants (7/9) felt they were on the right track in managing GDM because by looking at the weight values they could estimate if they could continue to eat in the same pattern or not. This type of self-monitoring increased their awareness. Five out of the fourteen participants interviewed did not use the application to monitor weight. Of these five participants, two mentioned monitoring their weight during the consultation as a reason for not using the application to record their weight. Three out of these five said that they did not perceive monitoring weight as a health priority during pregnancy. The graphical representation increased their self-awareness when managing GDM because with the graphical format, they could see how well they were managing their weight values over time and they could also review the data which was entered retrospectively. One participant viewed otherwise, perceiving the graphical representation of weight to induce weight-related anxiety, causing women to restrict dietary intake unnecessarily.Of all the coach messages, 162/189 (86%) were replies typed by the research coordinator in response to the logistics issues  raised by the users. A minority  of these messages typed by the research coordinator were replies to the dietary clarifications raised by the users. The remaining messages were system update messages. Consistent with these findings, many qualitative interview participants (10/14) reported sending messages to the coach via the chat interface only when they faced logistic issues. Participants (4/14) reported not using the coach component to ask queries about diet and glucose monitoring. The option of emailing healthcare professionals (2/4) and healthcare professionals in charge of their care not having direct access to the dashboard messages were the reasons (2/4). The automatic messages in response to high blood glucose readings were non-judgmental and encouraged some participants (6/14) to manage their GDM. Participants attributed the automatic messages to creating an immediate sense of self-awareness (5/6) in their food choices and motivated them for behavior change. However, they (5/6) thought its usefulness was only temporary because the messages delivered were always the same. They suggested having “personalized” and specific messages. For example, they preferred the messages to highlight “four carbohydrate portions were taken instead of three”. On the contrary, a few others (2/14) who had to use insulin to manage their GDM did not like the idea of taking suggestions from an application. For example, when they failed to achieve their target, having the application continue to give negative feedback demotivated them:“The messages are always replayed and standard. Always they will say you have to lower your carbs. After a while, you get used to the messages.” (P07)The usage frequency of the application’s components varied greatly among the users. Tracking diet was the least commonly used component. The low usage was attributed to incorrectly worded food items and limited food database. However, a few interview participants mentioned perceiving a sense of control and confidence in their food choices when logging food using the application. On the contrary, the weight tracking component was the most used due to its ease of use. The automatic coach messages created an immediate sense of self-awareness in food choices and motivated behavior change according to some interview participants.To concretize the key takeaways of this paper, the key results are mapped to the constructs of the Health Belief Model, and we provide suggestions for improvements to enhance application usage  below. BEducational lessons in the mHealth application have a key advantage as they provide vast quantities of health-related information which patients can frequently access at their convenience . HoweverDiet data entry was a key topic that affected the usability of the diet tracking component. Our findings showed that participants preferred to record the precise food eaten . HowevermHealth coaching by a healthcare professional has been shown to improve blood glucose targets in type 2 diabetes mellitus patients, as evidenced in a recent study by Koot et al. . HoweverOur participants were, on average, 1.5 years postpartum when participating in this study. They may or may not remember their experiences with the application. However, firstly, to reduce recall error, we walked through the Habits-GDM application in detail with all the participants before conducting the interview. Additionally, participants were also allowed sufficient time for adequate recall of their experiences. Secondly, our study shares views of individuals who were interested in participating in the study. Therefore, it is possible that the results may be biased toward those individuals who chose to participate. Thirdly, only one author coded the majority of the transcripts. Nevertheless, we used specific strategies to ensure rigor and trustworthiness of the study, as described in the Methods Section of this paper. Lastly, due to the nature of qualitative research methods, caution should be taken when extending the findings beyond the sample.To our knowledge, this is the first time a usage study of a largely automated mHealth application for GDM has been conducted in the Asia-Pacific region. This study is valuable from a health systems perspective because firstly, at a micro-level, using a mHealth application to manage GDM has been shown to improve patients’ satisfaction with their care and reduce unnecessary workplace absenteeism and travel costs . MoreoveThe practical implication of this study is to understand if an mHealth application for GDM management can be incorporated in a clinical setting. mHealth interventions are complex because they involve multiple components with different behavioral constructs ,19. HencOur findings suggested that the automated coaching in the mHealth application is effective in promoting self-awareness on healthy lifestyle choices among women with GDM, while reducing the need for human resources. However, the implementation of a mHealth application for GDM management is far from ideal. The inadequacies in some components of the application’s design resulted in poor user experience and less than ideal user engagement. Dietary management is an important element in GDM management. From our study, we learned that users found it tedious to track their diet using the existing design of the application. The context relevance of the food database is also a critical component to ensure continuous usage. Future work on a similar application should anticipate these potential challenges and attempt to address them during the development phase.


Traditionally, the standard of care for medication refractory essential tremor has been to utilize omnidirectional deep brain stimulation of the ventral intermediate nucleus. The advent of directional stimulation allows for spatial restriction of the stimulation on selected targets without involving the neighboring structures, thereby limiting off-target side effects and improving clinical utility.We performed a retrospective review of patients between February 2017 and September 2019 who had received ventral intermediate nucleus deep brain stimulation that allowed for directional programming . Initial and final major programming sessions post-operatively (approximately 30- and 90-days post-surgery) were examined to determine frequency and reason for use of directional programming.A total of 33 total patients were identified. A little over half were males , with an average age of 68 years old (SD 9.3) at the time of surgery, and a disease duration of almost 30 years  with a wide range from 2–62 years. After initial programming, over 50% (17 of 33) of patients were using directional configurations. This increased to 85% (28 of 33) at the 90-day programming. Reasons for conversion to directional configuration included avoidance of side effects , paresthesia (5/33), dysarthria (1/33) and gait ataxia (1/33)) or improved tremor control (12/33).Our single-center experience suggests that in the large majority of cases, directional leads were utilized and offered advantages in tremor control or side effect avoidance. Essential Tremor (ET) is one of the most common neurologic movement disorders, with a prevalence of almost 1% in the general population and between 4–6% in persons aged 65 years or older . There iFigure 1). Stimulation of one or more segment(s) of the electrodes can change the shape of the stimulation field to maximize benefits and prevent off-target side effects due to current spread  [Surgical interventions are appealing, especially in patients with medically intractable essential tremor. Prior to the 1990s, the mainstay of surgical treatment for ET had been lesioning of the ventral internal medial (VIM) nucleus of the thalamus. Starting in the 1990’s, led by Benabid et al, deep brain stimulation (DBS) of the thalamic VIM nucleus started to become a much more common surgical treatment for tremor control . Lesiona ataxia) . Our cen ataxia) . We feelThe study was approved by Oregon Health and Science University Institutional Review Board (STUDY 00017680. Deep Brain Stimulation Data Repository). A retrospective electronic chart review was performed using the Oregon Health and Science University Deep Brain Stimulation Clinic Database between February 2017 and September 2019.At our center, surgical implantation is performed using asleep DBS techniques. For each participant in our review, a preoperative MRI  was obtained and merged for image guided DBS planning on the Medtronic Stealth Station . These were imported into a Stealth Station 7 . Surgical planning and intraoperative navigation were performed using Frame Link software . For the ventralis intermedius, the second contact on the electrode was targeted at 25% of the distance from posterior commissure (PC) to anterior commissure (AC), 13.5–15 mm lateral to the midline, and at the AC-PC plane. A post-implantation, intraoperative head CT was obtained as part of our standard image-guided DBS implantation protocol . As AbboFigure 2). Key information from the database and on chart review was then extracted including age at time of DBS implantation, sex, number of years since tremor onset, DBS configuration settings at initial programming approximately 30 days from date of surgery – including use of directional versus omnidirectional and the reason for choosing directional current, and follow-up settings at a visit approximately 90 days from date of surgery and again the reason for choosing directional current. Specific tremor rating or quality of life scales were not available as these are not generally done by most programmers and this was a review of clinical practice, not a specific research protocol. The programming strategies varied with different programmers and different patients, as did the degree of details documented in the chart.Our general protocol is to schedule patients for an initial programming visit approximately 30 days after implantation, and then again at 60 and 90 days post-operatively. All patients who had undergone VIM DBS between February 2017 and September 2019 (N = 42) were reviewed. We excluded patients who (1) had not completed all three parts of their initial programming at OHSU , (2) who had a condition other than ET that was likely to affect the clinical picture (one person excluded for ET/PD syndrome) and (3) who did not have the St. Jude/Abbott device (one person had a Medtronic system and one a Boston Scientific system) (see Table 1). Of these patients, the sex breakdown was 14 females and 19 males. The mean age at time of surgery was 68 years old (SD 9.3) with disease duration from time of symptom onset being 27 years (SD 18.9).Following review of the database, a total of 33 patients were identified as having met all inclusion and exclusion criteria for having had surgical implantation of Bilateral VIM DBS for Essential Tremor with Abbott Platform (see Table 2). The remaining patients (3/17) were configured to use directional settings as they were unable to achieve optimal tremor control as determined by the provider on solely initial omnidirectional settings. We reviewed these three charts in depth to get a sense of process. One chart had very little detail on programming specifics and was complicated by being done only a week after the surgery and patient having some micro-lesion effects. In the other two patients the programmer appears to conduct a mono-polar review of all leads and then a review of all the individual segments of the optimal lead on mono-polar review and noted that tremor control was improved with the directional stimulation.At time of the 30-day initial programming visit, directional configurations with segmented leads were used unilaterally in 6/33 patients and bilaterally in 11/33 patients, conferring a usage rate of 16.8% and 33.3% respectively. The remaining 16/33 patients were noted to be using traditional omnidirectional settings with ring configuration. All patients in this subgroup using omnidirectional settings were noted to be using the two middle contact levels out of the four possible levels – the two middle contacts can be converted to directional if needed in the future, whereas the top and bottom contact levels are non-segmented and do not have ability to use directional configuration. None of the omnidirectional patients had bipolar configuration at 30 days. Only one patient had a bipolar configuration at this time, and this was in a patient also utilizing a directional configuration. Reasons for use of directional configuration during initial 30-day programming included avoiding stimulation-related adverse effect such as paresthesia (5/17) or muscle contraction (9/17) (see Table 1). There were no significant differences between groups in any of these measures. Finally, we examined the most recent follow-up visit for the patients in our cohort. This averaged 24 months (SD 14) from the time of surgery. We found that one patient was transitioned away from a directional configuration and two patients were transitioned from omni-directional to directional settings (see Table 1).At 90-day follow up, all patients that were using directional settings (17/33) remained with directional configurations with slight increase in amplitude. Of the 16 patients that were using omnidirectional settings previously at initial 30-day programming visit, 10 were transitioned to directional settings for the purpose of achieving improved tremor control and one to reduce dysarthria. The changeover rate was 68.8%. We reviewed the charts of those where directional settings seemed to provide better tremor control to see what other parameters were adjusted. There was no consistent pattern seen and other changes tried included increase in PW, frequency, and current, as well as bipolar configuration. The remaining 5 patients continued to demonstrate optimal tremor control in omnidirectional settings. Only two patients were transitioned to bi-polar settings at the 90-day programming sessions, one in a directional patient and another in an omni-directional patient. We used a t test and Fisher’s exact test to compare age, sex, and disease duration between those with omni and directional DBS at 30 and 90 days . While bi-polar settings can adjust the stimulation field to make it narrower and less likely to cause off target side effects, directional stimulation offers more of a lateral shift of stimulation .The introduction of segmented leads to the field of deep brain stimulation programming brings the use of directional programming to the forefront. This newer technology has been shown to produce larger VTA in the desired hemi-plane and this is the reason that lower stimulation at a segmented electrode can produce similar effects on tremor control and may lower Total Electrical Energy Delivered (TEED) by the Internal Pulse Generator (IPG) . CurrentAbbott  was the first DBS platform to develop a directional system; however, later on in 2017, Boston Scientific’s directional Vercise DBS system was FDA-approved based on the results of the INTREPID study . Both ofIn this single center retrospective study, we demonstrate that the use of directional programming with segmented leads has a strong role to play in the future of managing medication refractory essential tremor. In a group of 33 patients, directional configuration was used in 51.5% of patients at the initial 30-day programming which rose to 84.8% of patients. Fairly similar rates were found in a recent publications from the Cleveland Clinic – 79% of patients utilized directional settings, but they did not indicate why the change was made [We found it interesting and somewhat surprising that so many patients seemed to have directional leads utilized for optimal tremor control. We suspect some of this was related to this being a clinical chart review rather than a research protocol where clinicians might be forced to push stimulation to where side effects are seen and then make choices more based on a therapeutic window. We also wonder if at times side effects where not documented as degree of details varied between individual providers and patients. In informal questioning of our faculty, it was not clear that TEED generally plays a major role in their programming process. Finally, it is interesting how infrequently bi-polar settings were utilized and it appears programmers tended to trial a directional lead prior to trialing a bi-polar configuration.Some limitations of this study include the retrospective nature of the study, the lack of objective tremor ratings scales such as The Essential Tremor Rating Scale – TETRAS  – to havOverall, in our experience, this retrospective review demonstrates that the use of directional stimulation with segmented leads appears to confer additional benefits to both patients and DBS programmers over traditional omnidirectional configurations for improved tremor control and avoidance of unwanted off-target adverse effects.