Branchinecta lynchi (BRLY), the Midvalley Fairy Shrimp Branchinecta mesovallensis (BRME), and the Conservancy Fairy Shrimp Branchinecta conservatio (BRCO), and the Vernal Pool Tadpole Shrimp Lepidurus packardi (LEPA). We tested these assays using eDNA sampling protocols alongside traditional dip-net surveys to assess their viability as an alternative method to monitor vernal pool branchiopods. Based on occupancy modeling, each of our assays achieved a 95% or higher detection rate when using optimized sampling protocols.California’s vernal pools are declining ecosystems that support valuable native plant and animal diversity. Vernal pool branchiopods are particularly at risk from vernal pool habitat loss and conservation efforts have targeted their long-term protection through the establishment of preserves and conservation banks. These conservation strategies require repeated, perpetual monitoring of preserved habitat, which is currently carried out through dip-net surveys and visual identification of specimens. Dip-netting may be destructive and frequently requires some sacrifice of protected species. Environmental DNA offers a new, modern method to monitor many protected freshwater organisms. We designed qPCR-based species-specific assays for four of California’s vernal pool branchiopods: The Vernal Pool Fairy Shrimp  Vernal pool wetlands support high levels of biodiversity and provide a wide range of ecosystem services , but areA variety of conservation approaches have been implemented to help slow wetland loss and conserve remaining vernal pool biodiversity, with particular emphasis on conservation of vernal pool crustaceans. Conservation banks and habitat conservation plans are two important tools that establish permanent, managed habitat protections for vernal pools. These land ownership plans are designed to offset small-scale habitat loss from development and facilitate the protection of strategically-located, large, unfragmented habitats. A central part of these agreements are provisions for repeated, long-term monitoring of all listed species to help ensure their persistence within protected lands. Repeated monitoring ensures that the habitat bank perpetually supports the target, but comes at the cost of human impact on lands that are often pristine and delicate. Monitoring tools that minimize repeated impact would be useful for long-term monitoring of vernal pool conservation banks.Currently, vernal pool branchiopods (Arthropoda: Crustacea: Branchiopoda) are primarily monitored during the wet season using dip-net surveys. Surveyors wade into vernal pools and move a fine mesh net through the water column to disrupt the substrate and capture benthic invertebrates. Once captured, adult vernal pool branchiopods are identified visually in-field or in the lab under a microscope. Drawbacks to this labor-intensive survey method include difficulty identifying morphologically-similar branchiopod species in-field, habitat disturbance, and death of specimens used for laboratory identification. Additionally, no detection rates have been established for dip-net surveys of adult vernal pool branchiopods, and juveniles in common target genera cannot be identified to species visually. Long-term monitoring plans would benefit from a survey method that was repeatable, with a known detection rate, that minimized the sacrifice of protected species while providing highly accurate results. Environmental DNA (eDNA) provides a viable alternative for sampling many habitats, including vernal pools, and monitoring species of conservation concern.Branchinecta lynchi (BRLY), the Midvalley Fairy Shrimp Branchinecta mesovallensis (BRME), and the Conservancy Fairy Shrimp Branchinecta conservatio (BRCO), as well as for the endangered Vernal Pool Tadpole Shrimp Lepidurus packardi (LEPA). We designed four species-specific qPCR assays to detect the presence of each of these four large branchiopods, which are commonly targeted and monitored in Central Valley dip-net surveys. We tested these protocols on tissue-derived DNA for species discrimination and using eDNA samples collected from existing conservation lands alongside dip-net surveys to determine their viability as an alternative monitoring method.Here, we developed DNA species determination and eDNA monitoring methods for three morphologically-similar vernal pool species of conservation concern: The Vernal Pool Fairy Shrimp Materials and Methods). Using individuals from multiple geographic sites allowed us to account for inter-population, intraspecific single-nucleotide polymorphisms (SNPs) which might impact the assay efficiency. We determined the best gene and assay independently for each species. We developed assays on the 12S Ribosomal RNA Gene for BRLY and BRME, the 16S Ribosomal RNA Gene for LEPA and Cytochrome Oxidase I for BRCO  for each species  or 20 cycles  of qPCR. These results apply exclusively to the relatively high concentrations of tissue-derived DNA. In order to ensure no confusion between true positives and cross-amplification, we recommend that when these assays are used on high-concentration, tissue-derived DNA that they be run in conjunction with a high quality positive control. We also recommend off-target controls in any assay application that is differentiating between target Branchinecta species.To ensure that our assays were specific to our target species, we tested them using a panel of off-target, tissue-derived DNA that included every congeneric species known to occupy the same pools as any of our target species plus a frequent co-occurring branchiopod from the genus To determine the sensitivity of our assays, we used gBlock synthetic double-stranded oligos (IDT) matching the target amplicon of each assay at a series of known concentrations. We produced a standard curve for each assay using five gBlock concentrations ranging from 1 copy/reaction to 100 copies/reaction with eight replicates at each concentration and eight no-template-controls per assay. We used the equation of each standard curve to calculate the limit of detection (LOD) and limit of quantitation (LOQ) for each assay. We found that the BRME assay was the least sensitive, with a calculated LOD of 3.27 copies/reaction and a calculated LOQ of 24 cycles/reaction, while our most sensitive assay, BRCO, had a calculated LOD of 1 copy/reaction and a calculated LOQ of 11 copies/reaction see . A singlMaterials and Methods). Overall, we found high levels of agreement between our eDNA assays and dip-net survey results , average water volume filtered (across replicates), pool area, and filtration protocol (in-field or in-lab). We produced graphs representing the probability of detecting a target in at least one replicate water sample using the R package ggplot2 [To estimate the detection rates of each assay, we carried out occupancy modelling for each target species using the side-by-side dip-net and eDNA survey results. We used the Unmarked  to produ ggplot2 . For theIn contrast to BRME and BRLY, our LEPA assay was sensitive to filtration protocols. Our comparative model fitting suggests that for LEPA filtration protocol (laboratory vs. field) was an important covariate of detection. Laboratory filtration was consistently superior to field filtration. Materials and Methods). Our comparative model fitting suggests that for BRCO, average volume of water filtered and filtration protocol were the important covariates of detection. Our models did not suggest that the change in filter type or pore size affected detection rates. The BRCO assay detection rate was positively correlated with average water volume filtered, with very low detection rates for samples of 50 mL or less and much higher success rates with 1 L samples. Surprisingly, the filtration protocol seems to strongly favors in-field filtration. If samples are lab-filtered and water samples are <1 L, there is no number of replicate water samples that ensures a >95% detection rate. Because BRCO is a relatively rare species, we added an extra year of sampling in 2018 at three vernal pools to produce additional data. We used a larger-pore filter which allowed us to filter an increased volume of water, as pools at this site have high turbidity  and 4.25 x 10−5 (BRCO). We conclude that the BRCO assay has a negligible estimated false positive rate, but that the LEPA assay may have a false positive rate of 9.1%, assuming every possible false positive was indeed false.To determine the false positive rate, we compared our dip-net and eDNA survey results for each species and investigated cases where the dip-nets did not detect our target, but the eDNA assay returned a positive response. We found few false positives in our data. There were no instances of positive eDNA survey results when dip-net results were negative in our BRLY or BRME datasets. There were three instances where the LEPA assay detected the presence of the target species but dip-nets did not find them. We examined these three sampling events in depth and found that in all three of them, the species was found in that pool either later in the same year or in other years. This leads us to believe that the assay is very sensitive and may out-perform dip-net surveys, but would benefit from expanded testing at sites known to be historically negative for LEPA. The BRCO assay had three sampling events where the eDNA assay detected BRCO but the dip-nets did not. All three of these sampling events were from three pools sampled in early 2018. The species was independently confirmed to be present in all three of these pools later in the year. These pools are large and turbid, and these sampling events represent cases where the eDNA assay appears to be more sensitive than dip-net methods. In order to model these potential false positives, we used Branchinecta lynchi (BRLY), Branchinecta conservatio (BRCO), Branchinecta mesovallensis (BRME) and Lepidurus packardi (LEPA) because they are common targets of dip-net monitoring surveys and are protected species, or, in the case of BRME, coexist with and closely resemble protected species. Despite their protected status, few resources have been developed to support the long-term conservation of these species, and this is the first time that specific molecular markers have been published for any of these organisms. We designed these assays to maximize their utility and provide diverse applications in the field, but particularly aimed to support the long-term survey efforts required by conservation plans established for conservation banks and protected lands.We designed qPCR assays that reliably assess the presence of four California vernal pool branchiopod species using either tissue or environmental DNA samples. We targeted Branchinecta species are phenotypically plastic and morphologically similar. Accurate visual identification can be difficult and likely varies with surveyor experience. DNA-based identification offers an objective method of differentiation that may improve survey data quality. Additionally, immature Branchinecta spp. are not visually distinguishable even under a microscope, which restricts dip-net surveys to the brief period when adult shrimp are occupying the pools. By applying these assays to preserved immature fairy shrimp specimens, it may be possible to expand dip-net surveys earlier in the field season. Alternatively, eDNA applications of these assays may reduce the need for dip-net surveys altogether.Our assays can be used with tissue-derived DNA for species-level identification of specimens collected by dip-net surveyors. Though visual identification error rates have not been established, A method that is non-lethal, repeatable and objective may be of particular value for the long-term monitoring of vernal pool habitats. Our method can be used without causing injury or mortality to protected species, and minimizes the destruction of the sensitive, high quality vernal habitat typically preserved by land-based conservation strategies. This study also provides the only quantitative detection rate for any vernal pool branchiopod survey method. Although dip-net survey results are considered to be “perfect” by regulatory and monitoring bodies, the detection and error rates of dip-net surveys for branchiopods are unknown. Each of our assays achieved greater than a 95% detection rate using optimized eDNA sampling protocols. This suggests that these assays can be used as part of a monitoring plan to reliably and repeatably detect our targets.To achieve our goal of providing accurate detection data, we included false positive rate estimations where appropriate and found that our LEPA assay may potentially have a false positive rate of nearly 10%. This rate can be taken into consideration when developing eDNA-based monitoring plans, for instance by requiring more positive replicates before determining the positive presence of LEPA at the site. As an example, if using laboratory filtration, taking three water replicates and requiring at least two positive results in order to consider a site positive for LEPA would provide a 97.2% (95% CI: 93.5–99.4%) detection rate while reducing the false positive rate to 0.0081 . For our other assays, the false positive rate was zero or functionally zero.These assays contribute to a growing library of resources to support the long-term maintenance of protected vernal pool species. Recent work has used eDNA metabarcoding methods to investigate branchiopod communities in Southern California  and to sVernal pool habitats continue to decline and the USFWS Recovery Plan calls for long-term, repeated monitoring of currently-conserved habitat alongside the perpetual conservation of at least 80% of suitable habitat for target species . RepeataWe used both archived and fresh specimens when developing our assays. Archived tissue was sourced from the Bohart Museum of Entomology in Davis, CA and from the USFWS Sacramento Regional Office in Sacramento, CA. We extracted DNA from ethanol-archived specimens using the DNEasy Blood and Tissue Kit (Qiagen) following a modified protocol from the manufacturer. Our first modification was to rinse each specimen in DI water and air-dry it for 10 minutes on a Kimwipe to remove excess ethanol. Following the rinse and dry step, we removed the intestines and mature egg sacs with scalpels and forceps. All equipment was sterilized using bleach and DI water between specimens. Our second modification was to incubate specimens at 55 C on a rotisserie for 12 hours or overnight after the addition of Qiagen Buffer ATL and proteinase K. Extraction continued as recommended by the manufacturer, using the optional second elution step for a total of two 60 μL elutions.We Sanger sequenced our extracted DNA using previously-published primers provided in 2, slope, and intercept. We report the LOD and LOQ values obtained for a single replicate, these values improve with more replicates.To calculate the Limit of Detection (LOD) and Limit of Quantitation (LOQ), we used gBlock double-stranded gene fragments (IDT) that matched the target amplicon of each of our target species as the input for each assay. We produced a standard curve with concentrations that ranged from 100 copies/reaction to 1 copy/reaction. The LOD is defined as the lowest concentration of analyte that can be reliably differentiated from a blank at 95% confidence, while the LOQ is defined as the lowest concentration that can be accurately quantified. To calculate the LOD and LOQ of our assays, we used an R script developed by Klymus et al. 2020 . This scJMH performed dip-net surveys under permit FWSSFWO-16 from the U.S. Fish and Wildlife Service. SRK performed dip-net surveys under blanket permit FWSSFWO-16 and individual permit TE28101C-0, both from the U.S. Fish and Wildlife Service, and Scientific Collecting Permit SC-013798 from the California Department of Fish and Wildlife.We sampled 89 vernal pools for branchiopod presence during the 2017 wet season and an additional three vernal pools during the 2018 wet season. To collect environmental samples from a vernal pool, we collected water into a sterile 1L Nalgene bottle. We took three replicate water samples at each vernal pool and each water sample was collected from a different area of the pool. After eDNA sampling we immediately dip-net surveyed the pool and collected pool area measurements by walking the perimeter of the pool using a GPS device with an area tracking feature (Garmin). We followed the USFWS guidelines for large branchiopod surveys and collected voucher specimens in 95% ethanol. When possible, voucher specimens were independently identified to species.Filtration of eDNA samples was carried out by pouring the water from the screw-top Nalgene over a single-use cellulose nitrate filter  housed in a single-use plastic filter housing. The filter and housing were attached to a 1 L vacuum flask which was attached via rubber tubing to a peristaltic pump. Water was continually poured into the filter housing. Water volume filtered was not varied intentionally, but filtration was ended when the filter clogged or 500 mL was filtered (up to 1000 mL in 2018).To determine if immediate, in-field filtration of our water samples increased our detection rates, we tested both in-field and in-laboratory filtration protocols. In the field, filtration took place immediately after environmental sample collection at the side of the vernal pool, before dip-net surveys were carried out. To detect any possible contamination when filtering in the field, a “field negative” control consisting of 500 mL sterile nanopure water was carried into the field in a closed screw-top Nalgene and filtered immediately following the filtration of the three replicate water samples. The negative control was included in extraction and qPCR analysis identically. Sampling events where the negative control amplified for any species were thrown out. Laboratory-based filtration took place immediately after returning from the field. After environmental sample collection, the screw-top Nalgenes containing our water samples were placed in individual sealed 1-gallon zip-top bags. Bags were placed on ice in a cooler and returned to the filtration laboratory within 4 hours, at which time they were immediately placed into a 4°C refrigerator and filtered within 12 hours following the same protocol as above. An “equipment negative” identical to a field negative was included for lab filtration.In 2018, we tested a larger-pore glass fiber filter on three pools at Jepson Prairie Preserve to increase the number of BRCO samples in our data. We visited three vernal pools three times each for nine total samples (27 replicate water samples) and dip-net surveyed each pool after each visit. We performed all filtration in the laboratory and filtered up to 1 L of water, and no voucher specimens were collected. Filter storage and processing occurred exactly as in 2017.To extract DNA from our filtered eDNA samples, we used the Qiagen DNEasy Blood and Tissue Kit using the following modifications. 1) Because filtered samples were stored in 180 uL Buffer ATL, Proteinase K was added directly to this tube. 2) Samples were incubated on a rotisserie for 12 hours or overnight. 3) When samples were transferred to columns, care was taken to avoid transferring any remaining filter, as filters did not disintegrate fully. 4) Instead of a single elution, two elutions of 60 mL each were performed, and 5) a 15-minute incubation of the eluent at 55°C (rather than one minute at room temperature) was performed during each elution step. Finally, to prevent PCR inhibition, after extraction, all samples were proactively treated for inhibitor removal using the Zymo One-Step PCR Inhibitor Removal Kit. We tested a subset of 66 samples for inhibition by adding 1 uL low-concentration, tissue-derived target DNA four replicates of each sample and found no evidence of inhibition in our samples. For all applications, all consumables including microcentrifuge tubes, forceps, pipette tips and qPCR plates and water were sterilized before use in a UV crosslinker for 10 minutes.Field replicate samples were assayed separately. Each sample and replicate was run in quadruplicate and each qPCR plate included two low-concentration gBlock positive controls and a no-template control, each also run in quadruplicate. Each qPCR plate tested for only one target species using the optimized thermocycling conditions for 50 cycles. If one qPCR replicate of the four amplified, the sample was re-run. If two or more amplified, the sample was considered positive. If zero amplified, the sample was considered negative. If one replicate of four amplified after being re-run a second time, it was considered positive.We attempted to control for contamination at every step in the process. During environmental sample collection, collectors wore single-use nitrile gloves when handling the bottles and the filtration mechanisms, which were changed between pools. Collectors wore sterile, single-use boot covers when they had to wade into the pools. The 1 L Nalgene collection bottles were sterilized between uses by soaking in 20% bleach for 30 minutes, followed by triple rinsing and 15 minutes sterilization under a UV hood. All reusable filtration equipment was sterilized between uses following the same method. Boots and nets were cleaned between sites following USFWS decontamination procedures. We maximized our single-use equipment, choosing single-use filter housings, filter storage tubes, zip-top bags and forceps. Filtration was carried out in a designated laboratory space that contained no PCR product or extracted DNA, but was not our clean laboratory (as field gear and filtration supplies would contaminate a clean laboratory).All post-filtration steps were carried out in a clean laboratory, where no PCR product or tissue-derived DNA was permitted. Personnel were not permitted to move between the clean laboratory and any laboratory containing crustacean tissue, tissue-derived DNA or PCR product without first changing clothes and shoes. No reagents, equipment or consumables were permitted into the clean laboratory from any laboratory containing crustacean tissue, tissue-derived DNA or PCR product. Within the clean laboratory, all work took place in a UV hood.During DNA extraction an “extraction blank”, a tube containing only nanopure water, was carried through the entire extraction process alongside our samples. These blanks were tested with all four assays to ensure the extraction process did not introduce contaminants. We used filter-tip pipette tips for all work. All consumables  were UV sterilized for 10 minutes in a UV crosslinker before use, along with all water used in DNA extraction or qPCR. Reusable equipment, such as tube racks, forceps, and beakers, were bleach sterilized by soaking for 20 minutes in 20% bleach and triple rinsing in DI water, followed by 10 minutes in the UV crosslinker. After setting up qPCR reactions, plates were sealed and removed to another, non-clean space still free of tissue-derived or amplified crustacean DNA, where qPCR was carried out on a Bio-Rad CFX96.Unmarked. We treated each sampling event (consisting of three replicate water samples and a dip-net survey) as an independent event. We produced models for every combination of our four covariates of detection  and used AIC-based model selection to select the best model for each species. We used the MacKenzie and Bailey goodness-of-fit test [Unmarked’s “predict” function to estimate detection rates at specific relevant parameters. For BRCO and LEPA, we used the occuFP function to determine false positive rates using type 3 data. We considered samples with corresponding positive dip-net detections as “certain positives” and all other positives as “uncertain positives”. We did not attempt to fit any covariates of false positives, as the assay would be expected to produce false positives at the same rate regardless of variables like pool area or filtration method.We developed our models using the R package fit test  on each S1 TableColumns include a unique collection ID, vernal pool ID, the property sampled, the filter type, filtration protocol, sampling date, pool area in square meters, water volume sampled (per water replicate) in milliliters, and the latitude and longitude of the pool in decimal degrees.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Non-invasive brain stimulation can reduce the severity of tinnitus phantom sounds beyond the time of stimulation by inducing regional neuroplastic changes. However, there are no good clinical predictors for treatment outcome. We used machine learning to investigate whether brain anatomy can predict therapeutic outcome. Sixty-one chronic tinnitus patients received repetitive transcranial magnetic stimulation of left dorsolateral prefrontal and temporal cortex. Before repetitive transcranial magnetic stimulation, a structural magnetic resonance image was obtained from all patients. To predict individual treatment response in new subjects, we employed a support vector machine ensemble for individual out-of-sample prediction. In the cross-validation, the support vector machine ensemble based on stratified sub-sampling and feature selection yielded an area under the curve of 0.87 for prediction of therapy success in new, previously unseen subjects. This corresponded to a balanced accuracy of 83.5%, sensitivity of 77.2% and specificity of 87.2%. Investigating the most selected features showed the involvement of the auditory cortex but also revealed a network of non-auditory brain areas. These findings suggest that idiosyncratic brain patterns accurately predict individual responses to repetitive transcranial magnetic stimulation treatment for tinnitus. Our findings may hence pave the way for future investigations into the precision treatment of tinnitus, involving automatic identification of the appropriate treatment method for the individual patient. Poeppl et al. applied machine-learning algorithms to structural MRI for prediction of response to non-invasive brain stimulation in patients with tinnitus phantom sounds. The identified classifier predicted therapy success with an accuracy of 83.5%. The findings may hence pave the way for future investigations into precision treatment of tinnitus.  That is, while previous findings may provide insight into mechanisms underlying therapeutic response, they unfortunately do not represent a predictive biomarker.Complicating matters, no useful demographic or clinical predictors of rTMS treatment outcome have been identified so far.,Here, we used multivariate pattern analysis to test whether brain anatomy as assessed by sMRI allows to predict therapeutic outcome in individual patients that were not part of the training set. To this end, we obtained standardized images from tinnitus patients before application of rTMS based on a protocol yielding significantly more treatment responders than sham-stimulation and previous stimulation protocols.,We recruited a cohort of rTMS-naïve subjects with chronic subjective tinnitus. All patients provided written informed consent to participate in the study. The study protocol was approved by the local ethics committee. MRI was performed immediately before the first of all 10 rTMS sessions that subjects underwent on 10 consecutive working days. No subject received concomitant psychotropic medication. Treatment response was defined according to current standards on the basis of previously calculated minimal clinically important difference.After determination of the resting motor threshold , in eachAfter visual inspection for quality assurance, T1-weighted MRI scans were processed using the Computational Anatomy Toolbox . One chaP < 0.05.To investigate mass-univariate differences in brain structure between responders and non-responders, (parcel-wise) GMVs were compared by means of an ANOVA accounting for age and sex. Inference was performed by non-parametric, permutation-based thresholding at a FWE corrected In order to predict treatment effects, we employed an SVM ensemble. Model performance was assessed using leave-one-out cross-validation, i.e., based on the classification accuracy in patients that have not been seen by the algorithm during model training. More specifically, among the training set, we randomly sampled the same number of responding and non-responding patients. Sampling was performed without replacement at 95% of the smaller group within the training set. That is, if the training set contained 39 patients that did not respond and 21 that did, we randomly sampled 20 patients from either group. Among the sampled subjects, we then performed feature selection, only retaining the top 5% based on the univariate difference in the selected subset of the training data. Based on the hereby selected features, we then fitted an SVM model using the LIBSVM libraryThe data that support the findings of this study are available from the corresponding author, upon reasonable request.P < 0.05, FWE). At an uncorrected threshold of P < 0.05, widespread differences were observed. Responders exhibited more GMV in the left and right superior temporal cortex, left fusiform cortex, left occipital cortex, the left superior parietal and bilateral inferior parietal lobules, left insula, right operculum, bilateral premotor cortex, left ventrolateral and right dorsomedial prefrontal cortex. In contrast, non-responders had more GMV in the left dorsolateral prefrontal cortex, left cerebellum, left (para-)hippocampus and left superior parietal lobule hippocampus and dorsolateral prefrontal cortex were characterized by a grey matter decrease.In addition, we fitted a supervised pattern extraction algorithm based on sociodemographic and clinical characteristics hippocampus and dorsolateral prefrontal cortex before treatment may mediate subsequent tinnitus alleviation induced by rTMS to the left dorsolateral prefrontal and temporal cortex. This neuroanatomical baseline variance in tinnitus patients hence seems to represent a critical biological factor determining the efficacy of rTMS in improving phantom sounds.,,Intriguingly, the mass-univariate approach was not able to discriminate responders from non-responders before rTMS treatment with statistical significance but our multivariate pattern learning algorithm could detect a brain configuration predicting individual response to rTMS with high accuracy. This is in line with the observation that brain patterns predicting response to rTMS are highly complex and vary between other diseases such as depression and schizophrenia.,Notably, this anatomical grey matter pattern overlaps with regions that show rTMS-induced neuroplastic changes underlying therapeutic response in tinnitus patients.,Still, the detailed link between the classifier’s mesoscopic structural brain pattern and the putative underlying mechanistic surrogate remains incomplete. Parallel investigations using diacritic MRI protocols may specify contributions of histopathological properties and thus reveal the microscopic mechanisms underlying the observed mesoscopic brain pattern, which determines the individual capability to respond to rTMS. However, positron emission tomography studies showed that high-frequency rTMS of the left dorsolateral prefrontal cortex modulates dopamine release in other prefrontal regions.,Given response rates between 30–40% and costs of ≈2000 USD per patient for a course of rTMS,In conclusion, we showed that individual responses to rTMS treatment for tinnitus may be accurately predicted using biomarkers based on structural neuroimaging. The results provide a robust basis for the development of personalized rTMS interventions taking into account individual neurobiology. Our findings may hence pave the way for future investigations into precision tinnitus treatment involving automatic identification of the appropriate treatment method for the individual patient suffering from this multifaceted phenomenon.Brain Communications online.S.B.E. was supported by the Deutsche Forschungsgemeinschaft , the National Institute of Mental Health (R01-MH074457), the Helmholtz Portfolio Theme ‘Supercomputing and Modeling for the Human Brain’ and the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 945539 (HBP SGA3).The authors report no competing interests.fcab115_Supplementary_DataClick here for additional data file.


A Letter fluency and a Picture naming task were performed prior and following transcranial direct current stimulation, to assess modulatory effects on language. Behavioural frontotemporal dementia patients were impaired in all evaluation tasks at baseline compared with healthy controls. Computational finite element method (FEM) models of cortical field distribution corroborated expected impacts of left-anodal and right-cathodal transcranial direct current stimulation over the dorsolateral prefrontal cortex and showed lower radial field strength in case of atrophy. However, none of the two tasks showed statistically significant evidence of language improvement caused by active transcranial direct current stimulation compared with sham. Our findings do not argue in favour of pre-therapeutic effects and suggest that stimulation strategies evaluating the modulatory role of transcranial direct current stimulation in the behavioural variant of frontotemporal dementia must carefully weigh the influence of symptom severity and cortical atrophy affecting prefrontal regions to ensure clinical success.The behavioural variant of frontotemporal dementia is a neurodegenerative disease characterized by bilateral atrophy of the prefrontal cortex, gradual deterioration of behavioural and executive capacities, a breakdown of language initiation and impaired search mechanisms in the lexicon. To date, only a few studies have analysed the modulation of language deficits in the behavioural variant of frontotemporal dementia patients with transcranial direct current stimulation, yet with inconsistent results. Our goal was to assess the impact on language performance of a single session of transcranial direct current stimulation on patients with the behavioural variant of frontotemporal dementia. Using a sham-controlled double-blind crossover design in a cohort of behavioural frontotemporal dementia patients ( et al. report no language improvement in frontotemporal dementia after applying transcranial direct current stimulation over dorsolateral prefrontal cortices in a double-blind sham-controlled study. Authors discuss alternative stimulation strategies and patient-customized approaches integrating individual anatomical and functional/clinical features.Sanches   Additionally, executive capacities and language production are severely impaired.7–10 Behavioural FTD patients present compromised communication abilities, caused by a progressive breakdown of language initiation/activation and word search mechanisms in the mental lexicon. These disorders are reflected as difficulties in naming and verbal fluency tasks,11 which are in part sustained by left dorsolateral prefrontal systems.13 To date, pharmacological trials in bv-FTD have failed to demonstrate significant benefits, e.g.15 and no effective treatment for cognitive impairment is currently validated. Speech therapy has shown benefit for language dysfunction, but with no proof-of generalization to untrained items or long-lasting improvements.16According to the revised diagnosis criteria by Rascovsky 17–19 Transcranial DCS uses a mild electric field conveyed between two surface electrodes to electrically polarize regions of the cerebral cortex and modulate the membrane potential of exposed neurons, rendering them more or less prone to discharge action potentials and engage in synaptic transmission.21 Neurodegenerative diseases encompassing large cortical areas such as bv-FTD might prove particularly suited to the spatially broad action of tDCS compared with the higher focality characterizing TMS applications.22 Moreover, thanks to its low cost, excellent safety profile and portability, this technique holds promise for increasing activity in the atrophic cortical regions and enhancing cognitive function in neurodegenerative diseases.With the increase of the elderly population and consequently higher prevalence of neurodegenerative diseases, a crucial need for non-pharmacological neuromodulation therapies is emerging. Non-invasive brain stimulation technologies, such as transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) have shown the ability to modulate brain systems and improve cognitive functions such as attention, language, decision making or memory in healthy individuals.24 Moreover, delivered to the dorsolateral prefrontal cortex (DLPFC), tDCS has been shown to induce facilitation of verbal fluency, e.g.12 and improvement of lexical access.25 In several neurodegenerative diseases, tDCS over the DLPFC has boosted lexical access, such as in Primary Progressive Aphasia,27 Alzheimer’s disease,28 Parkinson’s disease29 and Progressive Supranuclear Palsy.13 However, to date, only a few studies have investigated the effects of tDCS on language impairments in bv-FTD, with inconsistent outcomes.31 All in all, the low number of patients tested and the marginal level of evidence encourage further investigations.Transcranial DCS has already generated beneficial outcomes in several diseases affecting language networks. In post-stroke aphasia, the stimulation of left hemisphere language-related regions boosted the recovery of naming abilities.32 upon which transcallosal connections mediate rivalrous inhibitory interactions of cortical activity between the two hemispheres. In such framework, we tested and compared across three independent stimulation sessions: (i) anodal stimulation over the left DLPFC aiming to re-boost language processes implemented by language-related prefrontal regions; (ii) cathodal stimulation over the right DLPFC to reduce the inhibition that right hemisphere systems exert on the left dominant language network and (iii) sham tDCS. Letter fluency and Picture naming tasks were used to evaluate tDCS effects on lexical access and language initiation/activation, whereas a non-verbal executive control task was used to tease apart language-specific effects from an impact on executive processes.The present study evaluated the modulatory effects of a single tDCS session applied over the left and right DLPFC on language initiation/activation in bv-FTD patients. We capitalized on interhemispheric inhibition principles,7 including progressive deterioration of behaviour and executive capacities characterized by at least three of the following six criteria: disinhibition, loss of empathy, apathy, perseverative or compulsive behaviours, hyperorality and executive dysfunction, including low verbal fluency. The diagnosis was also supported by neuroimaging (MRI) signs such as the atrophy of prefrontal regions.7 None of the patients were under medication interfering with central nervous system activity during their participation in the study.Twelve bv-FTD patients were recruited at the National Reference Center for ‘Rare Dementias’ of the Pitié-Salpêtrière Hospital in Paris (France). All participants were native French speakers. The diagnosis was established by expert clinicians following international diagnostic criteria for bv-FTD,33 or major cognitive disorders {[mini-mental state examination (MMSE)] < 1534; frontal assessment battery (FAB) < 1035}. Fifteen healthy controls, with similar characteristics as patients for handedness, sex, age and years of education , were used to determine normative performance levels in our evaluation tasks. The study received approval from the local Ethics Committee  and written informed consent was obtained from all the participants. Demographic data are summarized in Exclusion criteria were (i) psychiatric disorders or neurologic diseases other than bv-FTD; (ii) contra-indications to MRI or tDCS, such as the presence of intracranial ferromagnetic devices, scalp or skull lesions or epilepsy and (iii) major depression ,We applied a double-blind, sham-controlled crossover design in which each bv-FTD patient of our cohort underwent three independent tDCS sessions in 3 separate weeks: anodal tDCS over the left DLPFC, cathodal tDCS over the right DLPFC and sham tDCS over the left DLPFC. Each stimulation session was preceded and immediately followed by a series of computer-implemented evaluation tasks to determine the impact of each stimulation condition tested. The order of the three stimulation sessions was counterbalanced across patients to avoid order biases . Stimulation sessions were set a week apart to prevent unlikely carry-over effects and ensure independency of interventions. Before the inclusion of the first patient of the study, a computer-generated randomization list was created, and 12 sequentially numbered sealed envelopes, each one containing one of the 12 possible order sets for the three types of stimulation sessions, were stored in a locked research file box. Following the inclusion of each patient, the researcher responsible for delivering the stimulation opened the corresponding envelope and accessed the session order, without revealing such information to any other person involved in the study.In contrast with TMS protocols, the lack of any accompanying tactile scalp sensations or auditory patterns characterizing tDCS made patients totally unaware of the stimulation condition  delivered on each session. The double-blind character of our design was warranted by asking two different investigators to be in charge either of tDCS application or the application of the evaluation tasks exploring language and executive function.36; or the right and left DLPFC in Progressive Supranuclear Palsy patients13). In short, electrode placement on the scalp of each patient was guided by means of an MRI-guided stereotaxic frameless neuronavigation system , minimizing the Euclidean distance from the skin surface to a well-defined cortical target. Left-anodal and right-cathodal tDCS over the DLPFC regions targeted Montreal Neurological Institute coordinates  and ,28 respectively. A contralateral supraorbital electrode  acted as a return electrode for each montage. The location of active electrodes corresponded to ∼F3  and ∼F4  sites of the 10–20 EEG reference system, whereas the contralateral supraorbital return electrode was placed either on AF8 or AF7. Stimulation was delivered through round sponge electrodes  at an intensity of 1.59 mA (to achieve a current density of 0.06 mA/cm2). The current was ramped up for 30 seconds and kept on at 1.59 mA during 20 min before being ramped down for 30 s. Emulating the same transient skin itching sensations characterizing the first and last 10–20 s of active stimulation, the current intensity in the sham tDCS condition was ramped up and down during 30 s at the initial and final phase of the 20-min stimulation blocks.We followed the same tDCS procedures previously described in different populations of neurodegenerative patients  or outward  field. The two tDCS conditions applied in this study were modelled. Further details on tDCS modelling procedures implementing identical parameters  can be found in Valero-Cabré et al.13 The impact of prefrontal cortical atrophy on electric field current density levels impacting the left and right DLPFC was specifically addressed by modelling a cortical thinning (0.8 mm atrophy) of these areas ]se areas .34 the FAB,35 the trail making test A and B,39 an evaluation of aphasia severity ,40 a picture naming test (D08041) and a verbal fluency test comprising phonemic and category fluency.42 Healthy controls were tested with the MMSE and the FAB. Group average test scores for bv-FTD patients and healthy control groups are summarized in Assessment with standardized tests contributed to the diagnosis of bv-FTD and to the constitution of a relatively homogenous cohort of patients. The general cognitive assessment included the MMSE,Letter fluency task was used to assess the initiation/activation of language and lexical access. Participants were asked to generate orally in 1 min as many words as possible beginning with a given letter [‘C’ (version 1) or ‘P’ (version 2)], displayed on the computer screen and provided orally by the examiner  within a set of 15 items arranged in a triangular configuration  Spatial sequence generation task and (iii) Picture naming task.To control for potential biases linked to the modulation of executive dysfunction, the impact of tDCS on a guration . They weSpatial sequence generation task were displayed on a touch-sensitive screen tablet (HP Envy 8 x2). During the task, patients sat in front of the computer, which automatically recorded their responses in the presence of an examiner. The cumulative duration of the three tasks was consistent with the period during which the effects of tDCS are thought to remain active46 and significant (∼20 min).The two language tasks were programmed with the E-Prime software  and were presented on a laptop computer . The stimuli for the n = 12 bv-FTD patients were compared with those of the n = 15 healthy controls, to specify the language impairment in the patient cohort. A non-parametric Wilcoxon signed-rank test was used due to the non-normality of data distribution.First, performance levels [behavioural scores and reaction times (RTs)] at baseline for the cohort of To verify the absence of an across-session learning effect, baseline performances for the three sessions were compared using the non-parametric Friedman test. Regarding stimulation effects, the results obtained in the different tasks (performance scores and RTs) before and immediately after tDCS were compared using the Wilcoxon signed-rank test for each stimulation modality: left-anodal, right-cathodal and sham. The total change in each task (post-stimulation performance minus pre-stimulation performance) was compared across the three stimulation conditions using the Friedman test.Anonymized data, statistical methods and experimental material not entirely published within the article will be shared upon request from any qualified investigator.46 The presence of mixed polarities between the two stimulation electrodes suggests that stimulation polarity  depended on the orientation of the electric field relative to the cortical surface. A model simulating atrophy of the left and right DLPFC revealed a decrease in radial electric field strength compared with intact brain models  were predicted to differentially modulate activity in the lateral and rostral aspects of the targeted DLPFC. The direction of the current flow also indicated opposite modulatory effects.n models .P-values < 0.001)  .χ2(2) = 0.706, P = 0.703], ruling out across-session learning biases. Nonetheless, the difference between the number of words produced in the Letter fluency task immediately before versus after stimulation did not reach statistical significance, regardless of stimulation modality . Similarly, no statistically significant differences in post- versus pre-stimulation levels were found between the three tDCS conditions   = 3.619, P = 0.164; RT: χ2(2) = 4.167, P = 0.125). Likewise, no statistically significant differences were found between pre- and post-stimulation performance for any stimulation modality for scores neither for RT . Our analyses also failed to reveal statistically significant differences in post-minus pre-tDCS performance across the three stimulation modalities   = 3.244, P = 0.197], suggesting, as for our two language tasks, the lack of across-session learning effects and independency of the three stimulation sessions.For the Z = −0.257, P = 0.797; right-cathodal: Z = −0.920, P = 0.358; sham: Z = −0.154, P = 0.877). No statistically significant differences were found regarding post- versus pre-tDCS differences comparing the three stimulation modalities   calls for a detailed discussion of this discrepancy.A systematic review of studies using non-invasive transcranial stimulation technologies aimed at improving cognitive impairments in neurodegenerative diseases showed inconsistent outcomes49 Accordingly, significant improvements in verbal fluency have been reported following anodal tDCS over the DLPFC in Parkinson’s disease,50 Primary Progressive Aphasia52 and bv-FTD,31 when delivered daily for 10 consecutive stimulation sessions. Similar to our outcomes, highlighting the influence played by the number of accrued stimulation sessions on the significance or the magnitude of the derived clinical outcomes, Huey et al.30 also failed to find significant improvements in verbal fluency after a single anodal tDCS session over the DLPFC in patients with advanced bv-FTD.Two explanations seem particularly important to explain the lack of significant language improvements in bv-FTD patients outcomes revealed by our study. First and foremost, the induction of cortical modulatory effects, which following the delivery of a single tDCS session, might have been too weak to supersede a minimal threshold of change in focal activity, hence, to significantly modulate language performance. Supporting this hypothesis, it has been suggested that cumulative effects obtained with periodical multi-day stimulation regimes give rise to the higher magnitude and longer-lasting modulations, linked to the induction of cerebral plasticity phenomena.et al.29 reported improvements in phonemic fluency after a single anodal tDCS session over the DLPFC in patients with Parkinson’s disease for whom baseline performance was relatively preserved (17 words/minute) compared with our cohort (7 words/minute). A study evaluating the effects of TMS in a semantic task (word–picture association) in Alzheimer’s disease suggested that patients in the early stages displayed higher improvements than patients at later stages of this condition.53 Nonetheless, our limited sample size and the relative homogeneity of baseline scores of our cohort  following a single session of anodal tDCS over the left DLPFC. However, Progressive Supranuclear Palsy is characterized by important damage to subcortical structures and relatively mild levels of atrophy in the DLPFC, leaving spared prefrontal neural resources that can be more efficiently boosted by tDCS, than in bv-FTD patients with severely atrophic prefrontal cortices. Strengthening the plausibility of this explanation, a tDCS resting-state functional MRI study in Parkinson’s disease patients characterized by predominant subcortical damage, reported improvements in verbal fluency following anodal stimulation over the left DLPFC along with increased functional connectivity between frontal and inferior parietal regions.29 Taken together, these outcomes suggest that anatomical factors such as the severity of cortical atrophy  may influence the efficiency of stimulation regimes.A related factor that may limit tDCS efficiency is the degree of cortical atrophy affecting the area targeted by tDCS, thus the spared neuronal resources available for modulation. Valero-Cabré 55 Kim et al.56 modelled individually the current density predicted by tDCS over the left DLPFC in a cohort of healthy adults and reported improvement in a working memory task which correlated with the magnitude of the predicted current density at the cortical target. These findings suggest that inconsistent behavioural outcomes of non-invasive brain stimulation approaches (either tDCS or TMS) might be importantly influenced by interindividual differences of head and brain features, which might become even more variable in patient populations.57–59 On this basis, we hypothesize that the high degree of DLPFC atrophy shown by bv-FTD patients at the time of diagnosis may be one of the main causes that limit the beneficial impact of tDCS. It follows that more effective therapeutic approaches, based on a different rationale for the choice of electrode montages, should identify and target relatively spared cortical areas within language networks involved in lexical/semantic access and verbal fluency, rather than focusing on often highly damaged DLPFC areas with scarce viable resources to modulate.In the same vein, it has been shown that the volume of the cerebrospinal fluid in the subdural space, gyral depth and the distance between the surface tDCS electrode (anode or cathode) and the cortical target accounted for up to 50% of the spatial variability in electric field strength.61 More specifically, in bv-FTD, the temporal–parietal junction and the anterior temporal cortex, involved, respectively, in lexical62 and semantic representations,63 remain relatively spared, hence should be alternatively considered as more viable targets. Emphasizing the importance to combine adequate cortical targets and suitable stimulation strategies in order to optimally influence cognitive networks, a published study used bilateral anodal stimulation over fronto-parietal regions in bv-FTD patients, which improved scores of the neuropsychiatric inventory and visual RTs.64Indeed, language-processing related cortical sites in prefrontal, parietal and temporal regions revealed by neuroimaging studies which might stay undamaged on a given disease could provide alternate targets for neuromodulation.36 we here aimed to achieve brief lasting improvements of language impairments in bv-FTD patients with a single tDCS session targeting the DLPFC. Our study failed to provide such proof-of-concept. However, together with prior investigations, our results highlight the need to factor in the magnitude of language impairments at baseline and consider the degree of cortical atrophy impacting cortical target regions; therefore, to identify in larger bv-FTD populations the optimal window of severity at which patients might be more responsive to stimulation, and consider the application of tDCS to relatively undamaged regions of the language network. Individual patient approaches might call for a selection of candidates to specific tDCS therapeutic protocols according to their individual anatomical parameters and clinical profile of disease severity. On a very similar basis, a ‘personalized precision medicine’ framework using individually profiled therapeutic approaches based on multiple variables (such as symptom severity and degree of atrophy) may gain ground in brain stimulation. In parallel, biophysically inspired computational models of tDCS generated current fields, individualized to each patients’ head and brain structure, may help tailor the electrode montages and stimulation parameters most suited to optimize outcomes.66The use of well-designed double-blind sham-controlled pre-therapeutic studies is paramount to obtain proof-of-concept proving beneficial effects of transcranial stimulation in neurological diseases and, on such basis, justify more costly, larger-scale clinical trials in search of longer-lasting therapeutic outcomes. As successfully achieved for other neurodegenerative diseases,fcac050_Supplementary_DataClick here for additional data file.


Coats' disease is an idiopathic non-hereditary condition first described by Coats in 1908 as a congenital retinal telangiectatic and aneurysmal disease associated with retinal exudation. Its presentation is classically in early childhood. We report a rare case of Coats' disease that first presented during adulthood in a 35-year-old male. The patient presented with visual loss in the left eye for two months. His visual acuity was counting fingers in the left eye and fundus examination revealed extensive lipid exudation in the macula with telangiectatic vessels and microaneurysms with vascular malformation in the inferotemporal quadrant. Fluorescein angiography showed leakage from the telangiectatic vessels, and optic coherence tomography showed significant macular edema. A provisional diagnosis of adult-onset Coats’ disease was made. The patient responded well to intravitreal ranibizumab injections for macular edema and sectoral argon laser photocoagulation for peripheral vascular abnormalities. This case is unusual in adulthood onset and the first presentation was during adulthood in the third decade of life in contrast to the typical age of onset which is younger than five years. Coats' disease, also known as retinal telangiectasia, is an idiopathic non-hereditary condition. It was first described by Coats in 1908 as a congenital retinal telangiectatic and aneurysmal disease associated with retinal exudation that may progress to massive exudative retinal detachment -6. SimilA 35-year-old male patient presented to the outpatient clinic complaining of decreased vision in his left eye for a duration of two months. There was no associated pain, photophobia, or redness, no history of trauma, no history suggestive of any previous episode of ocular inflammation, and no previous laser treatment. The patient had no prior history of any systemic diseases such as diabetes, hypertension, and hypercholesterolemia. On eye examination, his best-corrected visual acuity (BCVA) at presentation in the right eye (OD) was 20/20 while the left eye (OS) was limited to counting fingers at a distance of 50 cm. In addition, anterior segments were normal in both eyes (OU). Fundus examination (OD) was unremarkable, and (OS) showed extensive lipid exudates in the macular area involving the fovea, forming a plaque-like exudate that measured around two-disc diameter vertically, two and a half-disc diameter horizontally with areas of microaneurysms and telangiectatic vessels. Peripheral inferotemporal vascular malformation with adjacent retinal hemorrhage was also seen Figure .However, there were no signs of inflammation or vasculitis, drusen, retinal pigment epithelial changes, or evidence of healed choroiditis in both eyes. Optical coherence tomography (OCT) (OS) showed significant macular edema, while (OD) was unremarkable Figure .Fundus fluorescein angiography (FFA) (OS) showed a hypofluorescent area in the macula due to blockage of the background fluorescence by macular lipid exudates and leakage from telangiectatic vessels. There was also a well-defined inferotemporal hyperfluorescent area corresponding to abnormal vasculature with leakage, and FFA (OD) was normal Figure . Based oAs a result, intravitreal ranibizumab was repeated in the left eye. The second follow up four weeks later, his BCVA improved to 20/60, and OCT showed complete resolution of macular edema. The patient received four more ranibizumab injections at a monthly interval, but no further response was seen on the subsequent visits. Long term follow-up after three years showed that his BCVA remained stable at 20/60 (OS), fundus examination showed scattered lipid exudates, and OCT showed clear macula.Coats' disease is an idiopathic, unilateral exudative retinal vascular abnormality manifested as telangiectasia and aneurysmal vascular dilations. It has variable severity and can range from isolated vascular abnormalities without lipid exudation to massive intra- and subretinal lipid exudates deposition . Coats' Adult-onset Coats’ disease is characterized by retinal vascular abnormalities which are similar to those seen in younger patients. In the pediatric group, the disease is usually progressive occurring in boys with a peak incidence between 6-8 years of age, while the adult form usually presents over the age of 35 years . Rishi eIn line with the published case series by Bottini et al., they described two cases of adult-onset Coats’ disease with predominant arterial macroaneurysms with hemorrhages and exudation and minimal telangiectatic changes . In compCoats' disease is primarily a disease of childhood though we should consider its diagnosis in adult patients presenting with extensive retinal lipid exudation and prominent vasculopathy. The natural course of adult-onset Coats' disease is better than that of children. However, treatments should not be delayed if the disease is macular threatening to halt its progression and further deterioration of vision.


Myocardial perfusion cardiovascular magnetic resonance (CMR) using first-pass contrast-enhanced imaging is an established non-invasive test for the detection of myocardial ischaemia. Current practice involves visual interpretation of a series of dynamic images and relies on experienced reporters to identify perfusion defects. The acquired data can be used to derive quantitative maps of myocardial blood flow (MBF). Potential advantages over visual reading include removal of operator dependence, simpler and faster analysis, and the ability to detect disease with global rather than regional reduction of MBF. Recent developments allow semi-automated or fully automated in-line calculation of MBF. Although these methods remain mostly in the research domain, they are on the threshold of becoming integrated into routine clinical care. This ‘How to’ article gives a brief practical overview of the steps involved in generating quantitative MBF maps and suggests how these may be used in clinical practice. This article is not intended as an exhaustive review of the principles or clinical evidence, which have been summarised elsewhere.3, achieved by fast imaging techniques such as fast gradient echo imaging or steady-state free precession.Myocardial perfusion CMR acquires a dynamic series of images immediately after injection of a T1-shortening gadolinium-based contrast agent (GBCA). At least three myocardial short-axis slices are acquired, every 1–2 heartbeat(s) typically over 40–60 s using a T1-weighted dynamic pulse sequence. The in-plane spatial resolution should be at least 2.5 × 2.5 × 10 mmTable 1 demonstrates steps to performing QP CMR.From these images, signal intensity profiles are taken from the left ventricular (LV) blood pool [to provide the arterial input function (AIF)] and the LV myocardium (which provides the tissue response). After conversion of dynamic MR signal changes to gadolinium contrast agent concentrations for the AIF and myocardium, quantitative perfusion (QP) assessment can be performed using a number of different models, providing MBF values in units of millilitres of blood per minute per gram of tissue. For visual interpretation of myocardial perfusion CMR images, a dose of 0.05–0.1 mmol/kg GBCA is generally recommended to optimise signal changes in the myocardium. QP CMR additionally requires measurement of signal in the LV blood pool, where the concentration of GBCA is severalfold higher than in the myocardium and no longer linearly related to signal intensity. Uncorrected, this leads to miscalculation of MBF. To overcome this problem, in the ‘dual bolus’ method, a dilute bolus of contrast agent  is injected for LV blood pool analysis, followed by a full concentration bolus for myocardial analysis. In the ‘dual sequence’ method, images of the LV blood pool are acquired interleaved with the myocardial images following a single contrast bolus, but using a separate, less T1-sensitive pulse sequence, which avoids signal saturation. Once available outside of research settings, the dual sequence method is likely to integrate better into clinical workflow than the dual bolus method.In order to allow reliable modelling of QP and comparison between myocardial segments, raw signal intensity data needs to be corrected for baseline signal and surface coil inhomogeneities. This is typically achieved using proton density-weighted images or myocardial T1 mapping preceding the acquisition of perfusion data.Reliable QP analysis mandates elimination of bulk cardiac motion, usually achieved by breath-holding. However, long breath-holds can cause involuntary diaphragmatic drift and changes in heart rate. Furthermore, during pharmacological stress, patients may be unable to hold their breath reliably. These problems can be overcome by free-breathing acquisition methods with correction of bulk cardiac motion (rigid models) or more complex non-rigid deformation methods. As all motion correction methods can introduce artefacts, clinicians should always review both raw and motion-corrected images.After the dynamic images are corrected for baseline signal and respiratory motion, signal intensity profiles are derived for the AIF and the myocardium using manual or automatic contouring. The AIF is typically taken from the basal LV but other sampling locations such as the ascending aorta have been proposed.Endocardial and epicardial contours define the myocardium and further analysis can be on a global, segmental, or pixel basis. AIF and myocardial signal intensity profiles are converted to GBCA concentration time curves. Several mathematical models such as the Fermi function have been proposed for the final step of quantification of MBF, each with specific advantages and limitations that are beyond the scope of this article but have been reviewed elsewhere.The results of MBF quantification can be displayed at a segmental or pixel level with colour coding representing the magnitude of MBF. Rest and stress myocardial perfusion data are analysed separately to derive both rest and stress MBF. The ratio of stress/rest MBF defines the myocardial perfusion reserve (MPR), which can be displayed as a further polar plot. Further outputs from the analysis may include the AIF, myocardial signal intensity profiles, and other data that can be used for quality assurance.A reduction in stress MBF or MPR implies either coronary artery disease (CAD) or coronary microvascular disease (CMD), but can also be caused by inadequate stress. Verification of adequate stress is typically achieved by reviewing the patient’s symptoms , heart rate response (rise of ≥10 bpm), systolic blood pressure  during the study, and splenic switch off on the acquired stress-images.Dark rim artefacts affect the diagnostic accuracy of CMR perfusion and can mimic subendocardial perfusion defects leading to false-positive diagnosis of CAD. Using QP CMR, MBF is generally lower in true perfusion defects compared to dark rim artefacts, but may remain a source of error.Registration, segmentation, and other errors may lead to erroneous MBF maps and automated and manual quality checks should be a routine part of quantitative myocardial perfusion imaging interpretation.No current guidelines exist on how to integrate quantitative myocardial perfusion CMR into clinical reporting and on how to combine it with visual analysis. As the bulk of the existing evidence for myocardial perfusion CMR is for visual analysis, this should continue to form the principal analysis strategy. QP maps may supplement visual interpretation in several ways:Confirmation of visual read: Successive or simultaneous visual and quantitative analysis may enhance diagnostic certainty where the two strategies agree and, when results are discrepant, may alert the reader to the presence of artefacts.Adequacy of haemodynamic response: When inadequate haemodynamic response is suspected based on a lack of clinical response or absence of splenic switch off, review of QP maps can help confirm (low-stress MBF and/or MPR) or refute (high-stress MBF and/or MPR) this suspicion.Suspected CMD: Visual interpretation of myocardial perfusion CMR has limited ability to detect CMD. In patients with no regional visual perfusion defects and adequate haemodynamic response, but low-stress MBF or MPR on quantitative myocardial perfusion mapping, CMD is a likely diagnosis. Thresholds for diagnosing CMD with quantitative myocardial perfusion CMR have been proposed in small studies but have not been widely validated and may not be applicable across different acquisition methods.Disease extent: Visual read of myocardial perfusion CMR compares signal changes between different myocardial regions and is thus adjusted for the lowest perfused area in an image, potentially masking less severe defects elsewhere. Quantitative analysis provides objective absolute blood flow values for each region. This may be advantageous in multi-vessel CAD, where quantitative myocardial perfusion CMR may better identify disease extent than visual read.Follow-up studies and research: Quantitative myocardial perfusion CMR provides absolute numbers of MBF, which can help assess treatment effects.Historically, calculation of QP has been laborious, requiring manual contouring of hundreds of images, exporting data to external computers and manual analysis of the data with locally developed post-processing tools. This has prevented the use of quantitative CMR perfusion in routine clinical care. Recently, fully automatic in-line methods that include motion correction, automatic detection of the AIF, segmentation of the myocardium, and pixel-wise calculation of MBF have been proposed.Large, multicentre prospective randomised-controlled studies are needed to further explore the prognostic value of quantitative myocardial perfusion CMR and its ability to guide revascularisation decisions. The most effective integration of QP into clinical pathways needs to be defined. Despite emerging consensus on the methodology, a number of different approaches to quantitative myocardial perfusion CMR are currently in use and international standardisation is needed.Conflict of interest: J.Schwitter receives research support from Bayer Schweiz (AG). All other authors declared no conflict of interest.


Point-of-care ultrasonography (POCUS) is increasingly recognized as a safe, efficacious, and cost-effective diagnostic and procedural tool used by many medical disciplines. Although standardized POCUS curricula are lacking, simulation represents an effective modality to teach the fundamentals of POCUS in medical education. We sought to characterize POCUS simulation cases available within MedEdPORTAL, the primary repository of learning resources for health professions, to highlight areas for future curricular development and study.This systematic review was performed based on a comprehensive search of MedEdPORTAL. Identified simulations were categorized and contrasted with respect to their target audiences, settings, pathologies, required materials and equipment, and POCUS techniques tested.A total of eight curricula were identified. The majority (6/8) were targeted at trainees in acute care specialties. Pathologies included in most simulations involved cardiac or pericardial disease, although obstetric and medical diseases were also tested in isolated cases. While half (4/8) of the identified simulation curricula incorporated diagnostic POCUS interpretation, only a few (2/8) allowed for high-fidelity ultrasound simulation. While self-reported learner satisfaction appeared to be generally high, most (7/8) identified curricula did not include objective assessments of learning outcomes.A small number of simulation-based POCUS curricula have been published within MedEdPORTAL. The widespread use of simulation for POCUS may be limited by the financial costs of high-fidelity training equipment. While simulation provides a highly promising solution to the need for greater instruction in POCUS, there is a need for comprehensive, standardized, and cost-effective curricula that can be adapted to varied educational environments. Point-of-care ultrasound (POCUS) has assumed an increasingly prominent role in the delivery of medical care with the introduction of more affordable, portable, and versatile ultrasound devices. New diagnostic applications for ultrasound technologies, which are being developed and taught across a range of specialties, can offer immediate answers to diagnostic questions while avoiding harmful radiation exposure and reducing healthcare costs across clinical contexts and practice settings .While demand for and applications of POCUS have grown, ultrasound education has not been consistently provided or standardized across medical training programs. This lack of a unified curriculum presents a significant barrier to the widespread adoption of ultrasound as a diagnostic modality, as it has been repeatedly demonstrated that operator experience is the primary limiting factor in the diagnostic and procedural utility of the technology . There iGiven the demonstrated versatility and effectiveness of simulation education in developing clinical problem-solving and technical skills, the availability of standardized simulation exercises for POCUS training holds great promise. Furthermore, the literature suggests that, as the use of ultrasound across various fields expands, enthusiasm for ultrasound education among medical educators and learners continues to grow . ResearcHere, we review the available ultrasound simulation cases and associated learning materials contained in the MedEdPORTAL® database, a peer-reviewed repository of instructional materials and medical education literature maintained by the Association of American Medical Colleges (AAMC). MedEdPORTAL® was selected as the ideal database for this search because of its reputation as a one-of-a-kind repository for free, open-access, and peer-reviewed curricula designed by and for medical educators . The eduMethodsSearch Strategyhttps://www.mededportal.org/). Combinatorial search keywords included “simulation,” “curriculum,” “sonography,” “echocardiography,” “bedside ultrasound,” “POCUS,” and “ultrasound education.”To address our study questions, we performed a comprehensive search of publications available online through MedEdPORTAL®  aimed at least in part at improving learners’ POCUS skills and (2) based on one or more simulated cases. Search results were excluded if the identified curricula did not explicitly involve simulation-based instruction, did not include sufficient materials to reproduce the simulated case(s) described, or involved simulations that did not include diagnostic or procedural POCUS techniques. For the purposes of this review, we also excluded applications of ultrasound not traditionally included within POCUS curricula, such as transvaginal ultrasonography, as well as curricula that described only the preparation of ultrasound simulation materials  without hands-on POCUS training components. Our complete search methodology is summarized in Figure  aimed atData Collection and AnalysisAfter target publications were selected, each curricular report and its accompanying materials were parsed by featured pathologies, target audiences, settings, required materials and equipment, and sonographic techniques taught. We also reviewed each publication for reported learner outcomes, particularly for validated and significant improvements in diagnostic or procedural POCUS skill, and for key limitations to the effectiveness or reproduction of each simulation curriculum. All authors of this review have significant experience in point-of-care ultrasound, and the supervisors of this study (EAB and MGC) have successfully completed the Examination of Special Competence in Critical Care Echocardiography (CCEeXAM) by the National Board of Echocardiography (NBE).ResultsSearch ResultsAfter reviewing 47 reports returned during an initial search, we identified a total of eight eligible simulation curricula targeted toward teaching POCUS. These curricula are summarized in Table Underlying Pathologies in Simulation CurriculaThe majority (5/8) of the identified simulation curricula centered on the sonographic diagnosis and management of cardiovascular diseases. A diverse curriculum published by Kobayashi et al. created a package of five different ultrasound simulation cases, including right ventricular strain with pulseless electrical activity, pericardial tamponade, hemoperitoneum after trauma, ruptured ectopic pregnancy, and ruptured abdominal aortic aneurysm . In addiTarget Curricular Audiences and Training EnvironmentsAll the identified curricula were tailored at least partially toward physician trainees enrolled in graduate medical education programs, and most (6/8) were aimed at acute care audiences. Kobayashi et al. developed their simulation package for emergency medicine trainees within their institution, although the authors stated that the simulations could be more broadly utilized for training undergraduate and graduate medical trainees, staff, nurses, and paramedics. Their simulation materials consequently appear to be most applicable for training clinicians practicing in emergency, intensive care, or perioperative settings . The simTypes of Simulation Equipment UsedThe simulation exercises incorporated into the identified curricula commonly contained three elements: a patient model or mannequin, a mock or high-fidelity ultrasound transducer, and a means of rendering or displaying simulated ultrasound images. However, the specific materials used varied considerably. The simulations in the curricular package developed by Kobayashi et al. involve the use of a high-fidelity simulation mannequin with live vital sign monitors coordinated by a technician to reflect dynamic responses to clinical decisions made by the trainee . The curIncorporation of Diagnostic Ultrasound Into Simulation CasesHalf (4/8) of the identified simulations aimed to teach diagnostic POCUS interpretation as a skill distinct from procedural ultrasound applications, and only two of these allowed for realistic ultrasound simulation practice. For instance, Alkhalifah et al. constructed a simulation using a high-fidelity simulator that allowed the performance of hands-on bedside ultrasound imaging. However, trainees were not explicitly instructed to perform POCUS to diagnose cardiac tamponade; rather, the exercise allowed learners to discover the utility and importance of performing hands-on bedside ultrasound on their own, as participants that incorporated ultrasound imaging were more successful in managing the standardized patient . The curIncorporation of Procedural Ultrasound Into SimulationsThe use of ultrasound to facilitate the performance of procedures was integrated into four of the identified curricula. Alkhalifah et al. assembled a low-cost pericardiocentesis trainer that allowed dynamic ultrasound-guided procedural practice . During Additional Materials Required to Conduct SimulationsIn addition to the ultrasound-specific equipment detailed previously, the curricular packages found on MedEdPORTAL® also included simulation scripts, patient data, and didactic modules with evaluative components. The curriculum by Kobayashi et al. provides scripts for the simulation role-play of the patient’s complaints and corresponding response to various potential interventions . Their cAlkhalifah et al. designed their case for use with a high-fidelity ultrasound simulator, but imaging and ultrasound video footage are also provided in Microsoft PowerPoint form for use in settings without the availability of such simulation equipment . The curIn addition to a case simulation, the curriculum by Sall et al. provides a pre- and post-simulation quiz, pre-simulation didactic videos, and PowerPoint slides in their case appendices . This isReported Learner OutcomesIdeally, curricula that are made available for learning POCUS will also include evidence of their educational efficacy. While learner satisfaction was generally very high when reported alongside the educational intervention, the majority (7/8) of the identified curricula did not present objective data with respect to learner competency. Only Sall et al. reported that 100% of the learners enrolled achieved a standard of minimum acceptable performance after completing their curriculum, which was determined based on a priori agreement by expert raters. Learners completing this curriculum could repeat the simulated paracentesis as many times as necessary until this standard was reached, and all learners studied were able to meet the standard for successful completion within one hour . The stuDiscussionThe use of simulation to teach POCUS is gradually gaining acceptance and momentum, as demonstrated by the variety of open-access curricula published in MedEdPORTAL®. At present, MedEdPORTAL contains only a small number of ultrasound simulation curricula that are directed toward specific patient pathologies, with a preponderance of curricula focusing specifically on cardiovascular pathologies as summarized in Table Ultrasound-guided procedures may also be practiced using commercial simulators, although few published curricula are available within the MedEdPORTAL® database. For example, Northwestern University’s internal medicine and emergency medicine residency programs utilize Simulab’s CentraLineMan® trainer to teach the insertion of internal jugular and subclavian central lines. Their research suggests that simulation-based teaching can significantly reduce central line complications such as arterial puncture while improving learner confidence . As in oMany of the currently existing POCUS-related simulation curricula available in MedEdPORTAL® are more appropriate for learners at the graduate medical education level, at which point some experience with basic ultrasound views has often been acquired. For early medical school students, a workshop format that pairs trainees with experienced instructors and standardized patients may be more appropriate for basic skill acquisition. For example, Blackstock and Carmody published a lecture to teach the basics of ultrasound physics and identify common abdominal structures to medical students . This leMajor Drawbacks of Simulation-Based POCUS CurriculaBased on our review, the most identifiable factor limiting the widespread adoption of the identified curricula is the availability of portable, high-fidelity ultrasound simulation systems, which can be prohibitively expensive for many training institutions. Numerous inventive workarounds have been devised, although these alternatives sacrifice a degree of fidelity for convenience and low cost. For example, Damjanovic et al. developed an easy-to-build, low-budget, POCUS simulator that incorporated a radiofrequency identification (RFID) antenna concealed within a mock ultrasound probe . Such anAs concluded in a prior systematic review, the quality and extent of the data supporting the utility of simulation-based POCUS training remain poor . Within Study LimitationsAlthough this systematic review uniquely identifies and assesses existing POCUS-related simulation curricula, it is limited to a single educational database. This narrow search strategy was deliberate and intended to maximize the utility of our findings for medical educators, particularly those seeking readily accessible curricular materials that have been utilized in live learning environments previously. Due to limitations in study availability and outcomes reporting, we were unable to directly compare the effectiveness between the identified curricula. It would be informative to examine the comparative effectiveness of POCUS-related simulation curricula in the future when a larger body of curricula becomes available. Point-of-care ultrasound is rapidly emerging as a rapid, accurate, and efficient modality for bedside diagnosis. Simulation-based learning has been demonstrated to improve competency in sonographic image acquisition, interpretation, and integration of this information into medical decision-making. Further rigorous study in the domain of simulation-based ultrasound education remains needed. However, ample opportunities exist for the development and incorporation of more expansive and advanced POCUS simulations into medical school and graduate medical education curricula - particularly as educational supplements to live bedside ultrasound practice - to meet the growing need for structured and standardized ultrasound training.


Background: There are sparse data addressing whether standard risk factors for febrile neutropenia (FN) are relevant in patients receiving myelosuppressive chemotherapy and primary prophylaxis for FN, which would have implications for variables to consider during real-world comparative analyses of FN incidence.Objective: To assess the impact of baseline patient-specific risk factors and regimen risk on the incidence of FN in patients receiving pegfilgrastim primary prophylaxis.Methods: This was a retrospective observational study in patients with breast cancer (BC) who received myelosuppressive chemotherapy and prophylactic pegfilgrastim identified January 1, 2017-May 31, 2018 from MarketScan® research databases. The outcomes were defined as incidence of FN in the first cycle and among all cycles of chemotherapy using three different definitions for FN. Logistic regression and generalized estimating equations models were used to compare outcomes among patients with and without patient-specific risk factors and among those receiving regimens categorized as high-, intermediate-, or other-risk for FN .Results: A total of 4460 patients were identified. In the first cycle of therapy, patients receiving intermediate-risk regimens were at up to 2 times higher risk for FN across all definitions than those receiving high-risk regimens (P<0.01). The odds ratio for main FN among patients with ≥4 versus 0 risk factors was 15.8 . Patients with ≥3 FN risk factors had significantly greater risks for FN across all cycles of treatment than those with no risk factors; this was true for all FN definitions.Discussion: The choice of FN definition significantly changed the impact of risk factors on the FN outcomes in our study, demonstrating the importance of evaluating all proxies for true FN events in a database study. This is particularly important during real-world study planning where potential missteps may lead to bias or confounding effects that render a study meaningless.Conclusions: In patients with BC receiving chemotherapy with pegfilgrastim prophylaxis, patient-specific risk factors and regimen risk levels are determinants of FN risk. In real-world studies evaluating FN incidence, it is imperative to consider and control for these risk factors when conducting comparative analyses. Febrile neutropenia (FN) is associated with increased morbidity and mortality in patients receiving myelosuppressive chemotherapy.Clinical practice guidelines published by the National Comprehensive Cancer Network (NCCN) stratify chemotherapy regimens into three FN risk categories as high risk (>20%), intermediate risk (10%–20%), and low risk (<10%) based on the agents, dose, and patient-specific risk factors. Employing these categories, they recommend the use of granulocyte colony-stimulating factors (G-CSFs), including short-acting  and long-acting  formulations, as primary FN prophylaxis in all patients receiving high-risk regimens and for patients receiving intermediate-risk regimens who have at least one additional risk factor.An FN risk model by Lyman et al demonstrated strong association between risk factors and the incidence of FN.Because of their influence on FN incidence, patient-specific risk factors and regimen risk level are imperative to consider as confounding variables in real-world comparative effectiveness analyses. With the introduction of six Food and Drug Administration (FDA)-approved biosimilar filgrastim and pegfilgrastim products, real-world data analyses will play an important role in provider confidence in treatment patterns or effectiveness in this multisource environment.In order to understand how FN risk factors could potentially confound outcomes in real-world data analysis of myeloid growth factors, this study assessed the impact of baseline FN patient-specific risk factors and regimen risk levels on the incidence of FN in patients receiving pegfilgrastim as primary prophylaxis.This was a retrospective, observational, database study in patients with breast cancer (BC) who received myelosuppressive chemotherapy and primary prophylactic use of pegfilgrastim. The IBM MarketScan Commercial Claims and Encounters database and IBM MarketScan Medicare Supplemental database (IBM MarketScan research databases) were utilized. These de-identified national claims databases include over 150 large employers and health insurance plans containing more than 200 million subjects. In addition to the Medicare population, this dataset represents >15% of the employer-sponsored, and privately-insured United States (US) population who are aged <65 years old. Medical data, pharmacy data, and enrollment information were collected.The study period was from January 1, 2012 to December 31, 2018; adult commercial and Medicare health plan members who were diagnosed with BC and received chemotherapy with pegfilgrastim prophylaxis from January 1, 2017 to May 31, 2018 were identified. The first use of pegfilgrastim prophylaxis was defined as the index date; pegfilgrastim prophylaxis was defined as ≥1 claim for either medical or pharmacy pegfilgrastim ≤5 days after the chemotherapy cycle start date or cycle end date, whichever came first. Patients were required to be continuously enrolled at least 6 months prior to (baseline period) and following the index date (follow-up period). Additionally, patients had to be chemotherapy-free for 5 years prior to the first chemotherapy administration identified during the index period , which eEvidence of BC was defined as ≥1 diagnosis of cancer prior to the index date, via either inpatient or outpatient services. Chemotherapy was defined as ≥1 claim for a systemic cancer agent identified from medical outpatient services during the index period. The chemotherapy start date (cycle 1) was defined as the date of the first claim for a cancer treatment after the cancer diagnosis date but during the study period; the regimen was composed of all treatments filled or infused within the first 5 days after the start of the regimen; the end date was the earliest of the following: day before next medication administration (start of next cycle), start of a new regimen (end date considered the day prior), discontinuation of all agents in a regimen (runout date considered the last date of administration plus 29 days for infused drugs and the days’ supply minus 1 for filled drugs), end of study period, disenrollment from the health plan, or surgery or radiation (end date considered the day prior for both). Patients who received pegfilgrastim via the on-body injector (OBI) method of administration were assigned a day of pegfilgrastim administration 1 day later than the billing claim date due to the intended delivery of pegfilgrastim by the OBI device approximately 27 hours after application to the patient’s skin.Patients were excluded if they had evidence of other malignancies; were male, pregnant, participating in a clinical trial, or HIV-positive; had a prior history of hematopoietic stem cell transplant; or had missing values of baseline and clinical characteristics.Supplemental Appendix. Of note, patients who received dose-dense regimens were identified by the time period between cycles (14-day regimens were considered dose-dense).The main objective was to assess the impact of baseline patient-specific FN risk factors and chemotherapy regimen risk on the incidence of FN. Risk factors for FN were identified via medical claims in the baseline period and included metastatic disease to the bone, baseline radiation, recent surgery, baseline liver dysfunction, baseline renal dysfunction, history of persistent neutropenia, and age ≥65 during the index year. Chemotherapy regimen risk levels for FN were categorized as high, intermediate, and other (including low and undefinable) utilizing the NCCN guidelines. Regimens and their associated FN risk utilized in this study are listed in the FN events were captured in each chemotherapy cycle from both medical inpatient and outpatient services by proxy using diagnosis codes for neutropenia, fever, and/or infection, as previously validated by Weycker et al.Descriptive statistics were provided for all study variables. To compare FN incidence among the first chemotherapy cycle, logistic regression models were developed, adjusting for geographic region, chemotherapy regimen FN risk category , Quan-Charlson comorbidity scores, days between chemotherapy and pegfilgrastim administration, and the number of patient-specific risk factors.To compare the FN incidence among all chemotherapy cycles, generalized estimating equations (GEE) models with binomial distribution, log-link function, and exchangeable correlation structure were developed to account for the fact that the probability of experiencing an FN event during one chemotherapy cycle was dependent on previous chemotherapy cycles . The GEE models estimated the average FN incidence across all chemotherapy cycles and controlled for the same covariates as the logistic regression model.A total of 4460 patients with BC were identified . The medP<0.01).The results of the logistic regressions showed that patients receiving intermediate-risk regimens had a significantly higher risk for first cycle FN across all FN definitions. The odds ratios (ORs) of first cycle FN for patients receiving intermediate- versus high-risk chemotherapy regimens ranged from 1.7  with the main FN definition, to 2.1  with the sensitive FN definition  were not significantly associated with risk of FN using any definition. Patients in the Northeast versus South region had a significantly greater FN incidence using the main definition only .P<0.01). The ORs when comparing >=3 versus 0 risk factors were 6.1  for the specific definition, 6.4  for the sensitive definition, and 10.1  for the main definition. This pattern was consistent when comparing patients with >=3 versus 2 risk factors across all FN definitions.Patients with increasing numbers of risk factors for FN had significantly higher probabilities of FN compared to those with no risk factors across all FN definitions . The ORsP<0.05). Patients residing in the Northeast and North central regions versus South region had a higher incidence of FN across all chemotherapy cycles, although not statistically different. Patients with ≥3 FN risk factors had a greater risk for FN across all cycles than those with no risk factors; the ORs were 3.3  for the specific definition, 3.3  for the sensitive definition, and 3.4  for the main definition, P<0.05 for both the specific and sensitive definitions  with the main definition to 1.8  with the sensitive definition  and the results may be driven by this small sample where bias would have a larger impact. Second, there may be factors that are unobserved in claims data that contribute to the observed difference but would be unaccounted for in our model. For example, providers may empirically dose reduce patients receiving high-risk regimens, decreasing their risk of FN. Last, we analyzed patient-specific risk factors as categorical variables, when in reality, they are more likely to be continuous variables. For example, we reported if patients had prior radiation, when in fact, the amount of prior radiation may also influence the risk of FN.The potential impact of these results is still important to further assess as this notion is imperative for clinicians in practice. Clinical practice guidelines traditionally recommended primary prophylaxis for patients receiving intermediate-risk regimens if they have at least one risk factor for FN.In our study, patients with increasing numbers of risk factors were significantly more likely to experience FN across all definitions in the first cycle and among all cycles of chemotherapy. This is aligned with previously published literature regarding FN incidence in patients receiving myelosuppressive chemotherapy.As with other real-world discussions, the economic impact of therapies must be considered due to the resource-restricted US health-care landscape. Recently, the Centers for Medicare & Medicaid Services requested information to further advance coordinated, high-quality, value-based care through the Oncology Care First model.Importantly, the previously discussed studies and the majority of published literature regarding patient-specific FN risk factors were developed by studying patients who were not receiving primary prophylaxis.Our study has several limitations to note. First, there are inherent limitations in database studies including misclassifications resulting in inaccurate or missing data. We excluded patients with missing clinical or baseline data, rigorously reviewed identified patients and chemotherapy regimens, and included all baseline variables in our statistical models to try to reduce confounding effects. Additionally, there is a risk of selection bias for patients receiving certain regimens, such as dose-dense chemotherapy. Patients selected for these regimens may be younger, fitter, and more likely to receive aggressive dosing; we attempted to control for this bias by including these baseline factors in our models.Next, it is difficult to accurately identify patients experiencing FN. We utilized previously validated definitions of FN, but this highlights the importance of recognizing and acknowledging shortcomings in database studies to ensure that the data presented are an accurate representation of practice as previously discussed. This study is descriptive in nature, and our use of multiple definitions of FN provides more information for future researchers using claims data to consider when designing their study.Because clinical practice guidelines do not routinely recommend primary pegfilgrastim prophylaxis in patients receiving intermediate-risk regimens without FN risk factors, our results are only valid for patients receiving primary prophylaxis with intermediate-risk regimens and are not externally valid for all patients receiving intermediate-risk regimens. Recent updates to clinical practice guidelines due to COVID-19 recommend primary prophylaxis with intermediate-risk regimens to reduce exposures to health-care settings; updated analyses using a dataset during the COVID-19 pandemic may yield more valid results for all patients receiving intermediate-risk regimens.Finally, we evaluated the effects of FN risk factors primarily identified by the Lyman model and recommended by NCCN for use in clinical practice. However, there may be additional significant risk factors unacknowledged in our model or confounders influencing the presence of risk factors that may be missing from this model.For patients with BC receiving chemotherapy with pegfilgrastim prophylaxis, geographic region, chemotherapy regimen risk of FN and the number of risk factors may affect FN incidence. For real-world studies attempting to compare the impact of pegfilgrastim products on FN incidence, this study demonstrates that it is critical to properly control for variables such as patient-specific risk factors and regimen-specific risk to estimate real differences in FN incidence among cohorts.