Chiropractors have diverse views of practice, but the impact on their patient profiles and treatment approaches remains unclear. We assessed the association between chiropractors’ view of practice (unorthodox versus orthodox) and patient encounter-level characteristics among chiropractors who practice in Ontario, Canada.We conducted a cross-sectional study using Ontario Chiropractic Observation and Analysis STudy (O-COAST) data. In O-COAST, Ontario chiropractors were randomly recruited from a list of registered chiropractors in 2015 and recorded up to 100 consecutive patient encounters. We classified chiropractors’ response regarding their views of practice as unorthodox when viewing “vertebral subluxation as an encumbrance to health that is corrected to benefit overall well-being”; other views were considered orthodox. Patient encounter-level characteristics included: (1) non-musculoskeletal reason-for-encounter; (2) subluxation as diagnosis; (3) duration of encounter (log-transformed for modeling); (4) unimodal manipulative treatment; and (5) patient health characteristics . We conducted multilevel logistic regression to assess the association between view of practice and aforementioned characteristics, accounting for potential confounders and clustering of encounters within chiropractors. The multilevel models had two levels , with level 1 patient encounters nested within level 2 chiropractors.We included 40 chiropractors  and 3,378 chiropractor-patient encounters. The 2,332 unique patients identified had a mean age of 48.5 years (SD = 18.5). Chiropractors with unorthodox views had higher odds of having patients with a non-musculoskeletal reason-for-encounter (adjusted odds ratio (aOR) 16.5, 95% CI 3.2–84.0) and subluxation as diagnosis . Encounters of chiropractors with unorthodox views were 0.6 times shorter than those with orthodox views (95% CI 0.4–0.9). Chiropractor level explained 32%, 75%, and 49% of the variability in non-musculoskeletal reason-for-encounter, subluxation as diagnosis, and encounter duration, respectively. We observed no association between unorthodox view and unimodal manipulative treatment or patient health characteristics.Chiropractors’ unorthodox view of practice was associated with treating non-musculoskeletal conditions, subluxation as diagnosis, and shorter duration of encounter. Chiropractor level explained a high proportion of variability in these outcomes. Findings have implications for understanding chiropractic practice and informing interprofessional collaboration.The online version contains supplementary material available at 10.1186/s12998-021-00398-x. Chiropractic is a healthcare profession concerned with the management of musculoskeletal conditions, and a commonly used complementary and alternative medicine therapy –3. ChiroChiropractors have diverse views of practice related to the health conditions that they treat. Some chiropractors have an unorthodox view, considered as viewing spinal dysfunctions, termed ‘vertebral subluxations’, as an encumbrance to the expression of health that is corrected to benefit overall patient well-being . This viIt remains unclear to what extent the unorthodox view among some chiropractors influences their patient profiles and treatment approaches. McGregor et al. reported that an unorthodox view of practice among chiropractors was associated with non-evidence-based treatment choices, use of radiographs that was not consistent with evidence-based guidelines, and a negative attitude towards vaccination . PreviouTherefore, the objective of this study was to assess the association between chiropractors’ view of practice (unorthodox versus orthodox) and patient encounter-level characteristics among chiropractors who practice in Ontario, Canada.We conducted a cross-sectional study using data from the Ontario Chiropractic Observation and Analysis STudy (O-COAST) . We repoEligible for the study were chiropractor participants of O-COAST within the primary care setting in Ontario, Canada. We excluded chiropractor participants with missing data on view of practice (1 chiropractor) and those with data errors (1 chiropractor). In O-COAST, chiropractors were randomly recruited from a list of 3,978 chiropractors registered with the College of Chiropractors of Ontario in 2014. Chiropractors in active clinical practice  in Ontario were eligible to participate. Each chiropractor invited consecutive patients to participate until 100 encounters were recorded per chiropractor, or when four weeks of recording elapsed. A total of 135 randomly selected chiropractors were approached, of which 120 chiropractors were eligible, and 43 agreed to participate (36% response rate). One chiropractor withdrew due to personal reasons and 42 completed the study (98% follow-up rate). The 43 chiropractor participants provided information on 3,523 chiropractor-patient encounters between July 3, 2014 and July 15, 2015. No direct identifiers were collected from the chiropractor. Information on patients was kept confidential by the research team. Re-identification of individuals is not be possible, since only aggregate findings are reported. Additional details on O-COAST methods are available in the published study .Ontario is the largest province by population (~ 14.6 million in 2020) in Canada . A chiroWe defined chiropractors’ view of practice based on a single question with six statements that best describes their predominant view of the conditions treated . Informed by previous literature , the lisSubluxation as diagnosis (subluxation versus other)Duration of patient encounter (continuous variable in minutes)Unimodal manipulative treatment i.e., one intervention only Very good/satisfied health status Activity limitations In subset of data due to data availability:We estimated the direction and magnitude of the association between chiropractors’ view of practice and patient encounter-level characteristics. As informed by previous literature –16, 22, Very good/satisfied health status was defined as a composite measure of three variables, which were self-rated general health, quality of life, and satisfaction with health. Self-rated general health was based on the question, “In general, would you say your health is excellent, very good, good, fair, or poor?” (excellent/very good versus good/fair/poor). Self-rated quality of life was based on the question, “How would you rate your quality of life” (very good versus good/neither poor nor good/poor/very poor). Self-rated satisfaction with life was based on the question, “How satisfied are you with your health?” (very satisfied/satisfied versus neither satisfied nor unsatisfied/fairly dissatisfied/dissatisfied). Activity limitations was based on the question, “how many activities does your pain or discomfort prevent?” (none/a few versus some).We included the following patient encounter-level variables as covariates in the models: age (in years); sex ; number of comorbidities , and imaging undertaken . We identified other potential confounders using the model building approach outlined in the analysis section.We described the chiropractor participants with respect to sociodemographic, education, and practice-related factors using means [standard deviations (SD)] or medians [interquartile ranges (IQR)] and percentages for continuous and categorical variables, respectively, stratified by chiropractors’ view of practice. Similarly, we described the patient encounters with respect to sociodemographic, treatment- and health-related factors, stratified by chiropractors’ view of practice.We conducted multilevel logistic regression models to assess the association between chiropractors’ view of practice and the aforementioned patient encounter-level characteristics, accounting for clustering of encounters within chiropractors. The multilevel regression models had two levels , with level 1 patient encounters nested within level 2 chiropractors. The main independent variable, view of practice, is measured at the chiropractor level, while covariates and outcomes are measured at the patient encounter level. The variance explained by chiropractor level is that explained by both unorthodox/orthodox view of practice and the random intercept. We adjusted for potential confounders in the models, which included confounders determined conceptually and those selected using a model building strategy to assess for a 10% change in the exposure regression coefficient. Variables that led to a 10% change in the exposure regression coefficient were new patient encounter , extended health insurance as payment method , injury related to motor vehicle collision , and injury related to workers’ compensation ; therefore, these variables were included in the fully adjusted models. We used a link function in the analysis for duration of patient encounter (log-transformed) to account for skewed distributions. The analysis for this study was generated using SAS software, version 9.4. of the SAS System for Windows We conducted sensitivity analyses to explore the impact of potential misclassification of the chiropractors’ view of practice as described by McGregor et al. . First, The O-COAST data had a total of 42 chiropractor participants and 3,523 chiropractor-patient encounters; we excluded 1 chiropractor due to missing exposure data and 1 chiropractor due to data errors  and median 12.5 years in practice (IQR 6.0–24.0), with 33% women , with 58.4% female and 16.2% living in a rural area (Table A higher proportion of patients seeing chiropractors with an unorthodox view had subluxation as a diagnosis (74.4% versus 20.8%) and received unimodal manipulative treatment (12.4% versus 0.4%) than chiropractors with an orthodox view of practice  = 16.5, 95% CI 3.2–84.0) and subluxation as diagnosis   and non-guidelines-based radiograph use  [Findings have implications for understanding chiropractic practice and informing shared decision-making between patients and chiropractors. Our study more comprehensively describes the characteristics of patients treated by chiropractors with an unorthodox view and their associated diagnosis and encounter characteristics. This can help guide patient expectations when making informed decisions about their treatment options when seeking care from chiropractors. For example, understanding the type of care provided by chiropractors can facilitate shared decision-making with patients, including enabling patients to make reasoned informed choices and guiding communication on appropriate choices of treatment . AlthougOur study results can also inform interprofessional collaboration between chiropractors and healthcare providers. Previous studies reported that orthopedic surgeons and obstetricians in Canada considered diverse views of practice among chiropractors as a barrier to interprofessional collaboration –28. ThisThe study has a number of strengths. First, O-COAST data used a number of approaches to minimize measurement error. O-COAST used a valid and reliable method of recording patient encounters in the primary care setting . Data coThe study has limitations. First, despite inviting a random sample of chiropractors to participate, O-COAST had a 36% response rate and has potential selection bias. This response rate is higher than similar studies assessing patient encounters in general practice (27% response rate)  and chirChiropractors with an unorthodox view of practice were associated with treating a non-musculoskeletal reason for encounter, subluxation as diagnosis, and shorter duration of encounter. Chiropractor level explained a high proportion of the variability in these outcomes. Findings have implications for understanding chiropractic practice and informing interprofessional collaboration and future research. Describing the patient profiles and treatment approaches of chiropractors based on view of practice can help guide patient expectations and communication when making informed decisions about their treatment options through shared decision-making. Our findings highlight a potential area for education among chiropractors to guide evidence-based approaches for non-musculoskeletal conditions. In addition, communication related to diagnosis and types of conditions treated may facilitate collaboration between chiropractors and other healthcare providers to improve patient outcomes.Additional file 1. Question assessing chiropractors’ view of practice based on study by McGregor et al.Additional file 2. List of non-musculoskeletal conditions .Additional file 3.Additional file 3a. Characteristics of chiropractors participating in Ontario Chiropractic Observation and Analysis STudy (O-COAST) by view of chiropractic practice (with unorthodox view classified as predominantly treating subluxations) (n = 40)a. Additional File 3b. Characteristics of unique patients in encounters as recorded by participating chiropractors by view of practice (with unorthodox view classified as predominantly treating subluxations) (n = 2332)a. Additional File 3c. Effect estimates of the association between unorthodox view of practicea and encounter characteristics based on unadjusted, age and sex adjusted, and fully adjusted models in sensitivity analysis (n = 3378 encounters). Additional File 3d. Odds ratio of the association between unorthodox view of practicea and patient health characteristics based on unadjusted, age and sex adjusted, and fully adjusted models in sensitivity analysis (n = 1559).Additional file 4.Additional File 4a. Characteristics of chiropractors participating in Ontario Chiropractic Observation and Analysis STudy (O-COAST) by view of chiropractic practice (with unorthodox view classified as predominantly treating subluxation or lifestyle/wellness issues) (n = 40)a. Additional File 4b. Characteristics of unique patients in encounters as recorded by participating chiropractors by view of practice (with unorthodox view classified as predominantly treating subluxation or lifestyle/wellness issues) (n = 2332)a. Additional File 4c. Effect estimates of the association between unorthodox view of practicea and encounter characteristics based on unadjusted, age and sex adjusted, and fully adjusted models in sensitivity analysis (n = 3378 encounters). Additional File 4d. Odds ratio of the association between unorthodox view of practicea and patient health characteristics based on unadjusted, age and sex adjusted, and fully adjusted models in sensitivity analysis (n = 1559).


Human infections are reported worldwide, but vaccines have been approved only in China and Korea. Orthohantavirus vaccine development has been pursued with no sense of urgency due to the relative paucity of cases in countries outside China and Korea. However, the orthohantaviruses continuously evolve in hosts and thus the current vaccine may not work as well against some variants. Therefore, a more effective vaccine should be prepared against the orthohantaviruses. In this review, we discuss the issues caused by the orthohantavirus vaccine. Given the pros and cons of the orthohantavirus vaccine, we suggest strategies for the development of better vaccines in terms of pandemic preparedness.Zoonotic transmission of  Hantaan orthohantavirus (HTNV) is a member of the Hantaviridae family, which is characterized by a single-stranded negative-sense RNA genome consisting of three RNA segments designated as small (S), medium (M), and large (L). The nucleocapsid protein (N), membrane surface glycoproteins (GPs: Gn and Gc) and RNA-dependent RNA polymerase protein (RdRp) are expressed from the S, M, and L segments, respectively. HTNV expresses only N, GPs, and RdRp from the negative-sense RNA genome. In addition, some hantaviruses express non-structural protein (NSs) not from an ambisense strategy but from an open reading frame (ORF) within the N gene [Apodemus agrarius, in 1976 and isolated by inoculating A. agrarius with acute-phase patient sera in 1978 [hantavirus with a different mode of transmission and unique terminal genome sequence [e N gene . HTNV wa in 1978 ,3. HTNV sequence .orthohantaviruses have been identified [orthohantavirus species might increase as new species are discovered [orthohantaviruses, human pathogenicity is unknown [Orthohantaviruses associated with human disease have been isolated all from rodent species [orthohantaviruses have shown three main geographic distributions: HTNV and Seoul orthohantavirus (SEOV) in the Far East, Puumala orthohantavirus (PUUV) and Dobrava-Belgrade orthohantavirus (DOBV) in Europe and Russia, and Sin Nombre (SNV) and Andes orthohantavirus (ANDV) in the Americas [Rattus norvegicus) [So far, more than 20 species of entified . Each spentified . The numscovered . For mos unknown . Orthoha species . Human pAmericas . Human iAmericas . Howevervegicus) ,12,13.orthohantavirus infection is, in most cases, the result of zoonotic transmission through inhalation of the aerosolized viral particles from the excretions of infected rodents [orthohantavirus from patients with hemorrhagic fever with renal syndrome (HFRS), there have been reports of human-to-human transmission of ANDV from patients with hantavirus (cardio)-pulmonary syndrome (HPS or HCPS) through close contacts or nosocomial transmission most likely involving respiratory secretions, saliva, or both [Human  rodents . Althoug or both ,16,17,18 or both ,19,20.orthohantaviruses cause different symptoms with different case fatality rates. The so-called Old World orthohantaviruses HTNV, SEOV, PUUV, and DOBV cause HFRS with a case fatality rate from <1–15%. HTNV and DOBV are associated with severe, SEOV with moderate, and PUUV with mild cases. The New World orthohantaviruses SNV and ANDV cause HPS or HCPS with a case fatality rate up to 40% [orthohantavirus infects endothelial cells via avb3 integrins. The avb3 integrins regulate permeabilizing responses of vascular endothelial growth factor (VEGF) directed by VEGFR2 receptors. The accumulation of viral particles on the endothelial cell surface via interaction with avb3 integrins is associated with hyperpermeability of endothelial cells and platelet inactivation, leading to hemorrhage [orthohantavirus infection illness is likely also related to immune pathology [orthohantavirus infection in the acute phase [Orthohantavirus infection is asymptomatic and persistent in its natural rodent reservoirs [Different species of human pathogenic p to 40% ,9,15. Thmorrhage . Howeverathology ,22. Subote phase . Acute pte phase . Complemte phase , althougservoirs ,26,27.After gaining attention during the Korean War, Korean hemorrhagic fever (KHF), later designated as HFRS, had been an important military problem . Such urorthohantavirus, there are no approved antiviral drugs. Treatment with nucleoside analog-based classical broad-spectrum antiviral drugs such as ribavirin has been attempted, but efficacy has not been proven [In terms of therapeutic countermeasures against n proven ,32.orthohantavirus vaccine licensed for use in Korea. This vaccine is made with a formalin-inactivated HTNV strain (ROK 84-105) grown in the suckling mouse brain [50) were adopted as a criterion for neutralizing antibody (nAb) positivity. Immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) were also performed in all studies except for Sohn et al. (2001) [50 titer were determined. The results of these studies are summarized in Hantavax is an se brain . Four clse brain ,34,35,36Antibody response positivity was determined one month after intramuscular injection of Hantavax ,34 and aField studies to evaluate the protective effect of Hantavax against HFRS were conducted in the Army of the Republic of Korea (ROK Army) ,38. The 50  with a wide range of time from the last dose  and a VE50 . Althougorthohantavirus infections that there were at least strong anti-N antibody responses after Hantavax vaccination.As summarized in orthohantavirus-exposed people tens of years after natural infection [Anti-N antibodies do not have neutralizing activity . The neunfection ,41,42,44orthohantavirus infection is not well understood, and detection of virus or viral antigen in convalescent humans has never been reported [The immunological background of continuous IgG production in natural reported . It is iorthohantavirus membrane glycoproteins Gn and Gc [Orthohantavirus Gn and Gc form heterodimers which assemble into a lattice of (Gn/Gc)4 tetrameric spikes formed by intra-spike Gn:Gn contacts, and the surface lattice is formed by inter-spike Gc:Gc contacts  vaccine, but there could be fragmentations such as split virus (SV) vaccines. We considered these possibilities of intact IWV (iIWV) and semi-intact IWV (siIWV), respectively . A largeOur schematic analysis may oversimplify the actual immune responses upon Hantavax vaccination. However, we can see that anti-N antibodies might not be just non-nAb. During acute HTNV infection, strong N-specific T cell responses measured by interferon-g (IFN-g) enzyme-linked immunosorbent spot analysis were associated with mild or moderate HFRS . In the Interestingly, while mice immunized with DNA vaccine encoding the secreted form of N protein of PUUV showed not only N-specific antibody response but also N-specific lymphoproliferative response, mice immunized with purified N protein showed only high N-specific antibody response without lymphoproliferative response , which iOrthohantavirus infection under naïve conditions would generate antibody responses initially like those presented in orthohantavirus could be best as a vaccine target antigen in designing a broad-spectrum orthohantavirus vaccine? While broad cross-reactivity in antibody responses to N proteins was observed [orthohantavirus threats that may appear decades later.Designing a viral vaccine in preparation for the future necessitates broad-spectrum cross-reactivity that is effective for decades. Which of the proteins of observed ,70,71,72observed . Howeverobserved . What coobserved ,75, the observed . Althougobserved , it was observed ,77,78, borthohantavirus could be neutralized intracellularly by anti-N antibodies as seen in intracellular neutralization of rotavirus by anti-VP6 antibodies [orthohantavirus is not likely exposed in endosomes, where intracellular antibodies are known to function, unlike the nucleocapsid protein VP6 of non-enveloped rotavirus. Since anti-N antibodies were shown not to have a neutralizing activity in the in vitro infection assay [orthohantavirus in terms of being an enveloped virus containing a segmented single-stranded RNA genome [orthohantavirus and its specific vaccine types.We have discussed the potential role for non-nAb, N-specific antibodies in enhancing N-specific T cell responses, schematically analyzing existing studies. It is not known whether tibodies . The RNPon assay , a role on assay . In thison assay . CoincidA genome . HoweverBroad cross-reactivity in antibody responses to the N protein might be utilized for broad-spectrum vaccine development ,70,71,72orthohantavirus vaccine studies may be animal models in which orthohantavirus replication is not highly active. When the viral growth rate is low, it is easy to clear the infection. The issue is whether reported protective effects in such animals would be translatable to humans. Instead of using asymptomatic animals [The caveat of  animals  and surr animals  might beorthohantavirus vaccines. Strong anti-N antibody induction due to Hantavax vaccination encompasses the effect of rN vaccination in addition to the IWV and SV vaccination effects. The current study trend might be towards subunit vaccines using purified proteins or structural protein-expressing DNA vaccines, but whole virus vaccines or split vaccines, including Hantavax, also need to be better characterized. It appears that further analysis of the immune responses of Hantavax using animal models would enlighten us on many aspects. A Hantavax cross-reactivity study using the Syrian hamster model of ANDV infection would be valuable for determining the potential of Hantavax as one existing pandemic preparedness vaccine against diverse orthohantaviruses. Although Hantavax has shown only short-term efficacy, a pandemic vaccine might not necessarily need to be proven for long-term efficacy. In the event of an orthohantavirus pandemic, the priority may be proven safety and the potential to cut the chain of infection. Although not related to antigenicity, whether the Hantavax production method could meet pandemic capacity also needs to be examined to reformulate Hantavax as a pandemic vaccine.We have discussed the antigenicity of Hantavax and potential


Multi-target recognition and positioning using robots in orchards is a challenging task in modern precision agriculture owing to the presence of complex noise disturbance, including wind disturbance, changing illumination, and branch and leaf shading. To obtain the target information for a bud-cutting robotic operation, we employed a modified deep learning algorithm for the fast and precise recognition of banana fruits, inflorescence axes, and flower buds. Thus, the cutting point on the inflorescence axis was identified using an edge detection algorithm and geometric calculation. We proposed a modified YOLOv3 model based on clustering optimization and clarified the influence of front-lighting and backlighting on the model. Image segmentation and denoising were performed to obtain the edge images of the flower buds and inflorescence axes. The spatial geometry model was constructed on this basis. The center of symmetry and centroid were calculated for the edges of the flower buds. The equation for the position of the inflorescence axis was established, and the cutting point was determined. Experimental results showed that the modified YOLOv3 model based on clustering optimization showed excellent performance with good balance between speed and precision both under front-lighting and backlighting conditions. The total pixel positioning error between the calculated and manually determined optimal cutting point in the flower bud was 4 and 5 pixels under the front-lighting and backlighting conditions, respectively. The percentage of images that met the positioning requirements was 93 and 90%, respectively. The results indicate that the new method can satisfy the real-time operating requirements for the banana bud-cutting robot. Recent years have seen an unprecedented rise in the cost of human labor, with the increase reaching up to 12–15% in 2019 . At presIn the fruit industry, visual inspection and image processing for the recognition and positioning of fruits and flowers are among the most intensively studied topics . Visual Target fruit recognition and positioning in a field is quite challenging . The envThe object detector used for deep learning algorithms is mainly divided into two types: one is a two-stage detector based on candidate regions, and the other is a single-stage detector based on the regression method. The first type of two-stage detector divides object detection into stages, as in the case of the R-CNN series. This type of neural network has been widely applied to deep learning for fruit and flower detection in orchards. To rapidly classify and recognize the objects, researchers have put forward the second type of one-stage object detection algorithm. You only look once (YOLO) and single-shot multibox detector (SSD) series belong to this type . CompareFor edge detection and cutting point determination for targets in orchards, In banana orchards, machine vision and deep learning algorithms have been successfully applied to the classification and detection of a single target, such as fruits or stems. Addressing complex environmental noises, we modified the YOLOv3 algorithm for accurate and rapid multi-target classification and recognition, Some parameters in YOLOv3 are optimized, and on the basis of clustering algorithm, the cross entropy loss function is introduced into the confidence and classification error model. An image segmentation and denoising algorithm was used to obtain the images of banana buds and inflorescence axes. A spatial geometry model was thus established. The function for positioning the inflorescence axis was based on finding the centroid of the banana bud. The coordinate information of the cutting point was obtained.The highlights of the present study mainly include the following:(1)Images of banana buds and inflorescence axes were obtained by image segmentation and denoising. The center of symmetry and the centroid were calculated for the edges of the flower buds.(2)The cutting point was positioned using an edge image processing algorithm and the geometric method. Thus, the function for solving the inflorescence axis was established.The testing equipment had both hardware and software components. The hardware part of the testing equipment  includedThe sampling objects were banana fruits, flower buds, and inflorescence axes connecting the buds to the fruits . Each baThe experimental images were obtained in two batches. The photographs were taken on July 25, 2020, which was a sunny day, at the Lingnan Fresh Fruit Base of the Guangzhou Fruit World in Guangdong Province, China. The weather changed from sunny to cloudy from August 4, 2020 to August 5, 2020. The location was a banana orchard in Jiangmei, Guangdong Province. Multiple targets, including fruits, inflorescence axes and buds, were included in the photographs. Thus, the banana plants had multiple features that could be detected as targets in this study. During the sessions, 5,343 images were collected under different lighting conditions. Among them, 5,300 images were selected and subjected to annotation using an image annotation tool. A script was written for the automatic, random division of the samples into the training, validation, and testing sample sets. There were 4,800 images in the training set, 364 images in the validation set, and 685 images in the testing set, accounting for 90.60, 7, and 13%, respectively.Samples for the multi-target recognition of bananas are shown in For multi-target detection of bananas in which different features were recognized simultaneously, the prediction was done on multiple scales. The influence of resolution on the prediction is mainly determined by the resolution information, that is, the number of pixels . LogistiYOLOv3 utilized the feature extraction network part Darknet-53, which was composed of five residual blocks. This model borrowed from the residual neural network. The number of anchor boxes used in the algorithm changed from 5 in the original YOLOv2 model to 9. The size of the anchor boxes was calculated by applying k-means clustering to optimize the width and length of the actual target frame of the bananas .The separate detection of each feature of the banana plants (inflorescence axes and buds) was performed using multi-scale feature fusion maps. This method could enhance the detection performance for targets of varying sizes and also the shaded ones. Besides, connections were introduced between the layers to strengthen the convergence performance. The multi-target detection of bananas was conducted in a complex field environment where noise interference abounded. The parameters of the existing YOLOv3 architecture still needed to be modified. Here, the loss function was modified and improved. The network architecture is shown in The loss function of YOLOv3 can affect the model convergence. It also serves as the basis for penalizing incorrect detection. The Sigmoid function was designed and used for the activation of the final output. Subsequently, SSE was used to calculate the final loss. However, the Sigmoid function has a saturation problem. Once the input falls within the saturation region, it approaches zero. As a result, the gradient nearly vanishes. If the error value calculated by using the squared error is very small, the network parameters can hardly be trained effectively . One wayl_1box), classification error (l_2obj), and error of the confidence level (l_3cls). The squared error is considered as a function to reduce the cumulative error of the loss function and to mitigate the gradient vanishing when calculating the coordinate errors (location errors). If this error increases, the parameter gradient will increase. But when the error is very large, the parameter gradient decreases, leading to an uncertainty problem. Therefore, when calculating the confidence interval and the classification error, we introduced a cross-entropy loss function . Clustering was performed using the k-means clustering and training dataset. The number of target candidate frames, height, and width fit for the prediction were updated. The parameters of the multi-target candidate frame are shown in re), recall, F1-score and Matthews Correlation Coefficient (MCC) as precision measure of the binary classification model. The calculation formulae are shown in Eqs. (4)–(6):To assess the generalization ability of the deep learning network and optimize the model stepwise, we determined the precision . For each epoch, one group of precision and recall was obtained. By setting different thresholds of the modified YOLOv3 model, several groups of precision and recall were obtained, and a PR curve was drawn. The area under the curve was the average precision (AP).Geometrically, the flower buds are inverted cones with basic symmetry. The cutting point of the bud is located on the inflorescence axis. The midpoint of the bounding rectangle of the inflorescence axis can be easily found when the bud and the inflorescence axis point vertically downwards. However, some buds and inflorescence axes do not point vertically downwards but at an angle δ with respect to the vertical direction. In this case, finding the midpoint of the bounding rectangle of the inflorescence axis to position the cutting point may lead to mistakes.Without loss of generality, the cutting point should be calculated by determining the centerline when the bud is at an angle of δ. First, multi-target classification was done using the YOLOv3 model based on clustering optimization. The image edges of the buds and inflorescence axes were determined using the edge algorithm. The centroid and the center of geometric symmetry were solved based on the symmetry of the bud. Next, the cutting point in the inflorescence axis was calculated using the geometric method. This method first determined the distance from all the detected straight lines to the centroid. Then, the straight line with the shortest distance was chosen by imposing the minimum constraint on the distance from the point to the line. This is the line along which the inflorescence axis runs. The midpoint of this line is chosen based on the coordinates and treated as the cutting point. The spatial coordinates of this cutting point forms the basis for configuring the parameters of tool posture in the robot actuator. The schematic of the cutting point positioning algorithm is shown in Image segmentation for flower buds and inflorescence axes is the basis of the cutting point positioning algorithm. Multi-target classification and recognition of banana fruits, flower buds, and inflorescence axes were first performed for noise reduction. However, some bud images might contain green or dry leaves, fruit branches, and lighting noises. The bud color differs from the color of a few green or dry leaves. Here, the bud edges were extracted by extracting the color components and by Otsu’s binarization.Mathematical morphology is applicable to the denoising of complex images and image restoration due to its intuitiveness and suitability for processing geometrical structures. The opening operation can inhibit positive impulse noise in the bud signals, while the closing operation can inhibit the negative impulse noise. To remove the positive and negative noise from the signals simultaneously, we combined the opening and closing operations to form a morphology filter. Noise in the binary images of the buds were mainly composed of the surrounding noise blocks and the noise holes inside. The opening operation was adopted to remove the noise surrounding the buds, and the closing operation was for removing the noise holes inside. That is, set A was closed using the structure element B. Denoising was performed using the morphological method. However, the black noise within the contour of the bud became enhanced .After image segmentation, some small curled petals might be separated from the overall contour of the bud under certain conditions. However, these petals were of negligible size. To reduce the error in the computation of the image centroid, we proposed extracting the maximum connected region from the main contour of the bud. In this study, 50 sample images were used for cutting point positioning on the inflorescence axis. Given the large sample size, the conventional denoising approach usually has a low efficiency as denoising is performed for one image at a time. Here, denoising was performed using the batch processing method.After extracting the maximum connected region, a binary image was obtained for the bud region. The pixel value of this region was set to 1 (white), while the remaining was set at 0 (black). The centroid coordinates of the bud were estimated  using thci,yci are the centroid coordinates of the upper and lower parts of the bud, respectively; x,y are the pixel coordinates; f is the pixel value of the binary image at point .where xrc1 in the upper part and the centroid rc2 in the lower part were connected to find the centerline of the bud. The image centroid of the banana buds is shown in 0, = 0, the bud and the inflorescence axis point vertically downwards and are symmetric. When λ < 900, the bud and the inflorescence axis are at an angle of δ with respect to vertical direction, as shown in After binarization, the image in the H channel was divided into the upper and lower parts. The pixel location information was estimated for each target. It was determined whether the pixel point fell within the bud region. The pixel information was estimated and the centroids in the upper and lower parts were determined. The centroid lmax is the maximum width of the bud contour; s is the distance from the detected straight line to the centroid; T is the highest point on the bud edge; Qi1 and Qi2 are end points of the detected line segment; H_t is the height of our interest in the inflorescence axis; Qih is the cutting point in the inflorescence point, with a distance of H_Q. Based on the principles above, the image centroid and central axis were found for the buds, as shown in According to the growth features of the buds and inflorescence axes and the principles of geometric method, we constructed a schematic diagram for the calculation of the cutting point. First, the bounding rectangle of the inflorescence axis was found. Then, the inflorescence axis information and growth direction were extracted. Finally, the coordinates of the optimal cutting point were determined.Due to shading and color interference, it was difficult to extract the inflorescence axis. In The images where converted into the HSV coordinate system; the HSV channels were observed, and the V channel  showed tThe following assumption was made within the bounding rectangle of the inflorescence axis to eliminate the influence of the remaining irrelevant area and boundary irregularity of the inflorescence axis: The inflorescence axis had the largest area ratio. As to the bounding rectangle of the inflorescence axis, the boundaries were determined by finding the pixel pair. First, the boundary coordinates of the inflorescence axis were estimated and clustered. The outliers were eliminated by using the clustering-based denoising algorithm. The feature boundary line was fitted. The center of the inflorescence axis was found based on the mean diameter or pixels of the inflorescence axis.i1, Qi2 in the line segment of the inflorescence axis are  and , respectively. The two endpoints are connected by a straight line:The pixel coordinates of the two end pointsQmin(Si) for the distance from the point to the line was solved to find the straight line segment where the cutting point was located. The midpoint of this straight line segment Qih was considered the cutting point  was manually set up. The optimal cutting point was located along the centerline of the inflorescence axis (20 ± 15 pixels × 65 ± 30 pixels). The pixel positioning errors calculated for the 60 images are shown in As shown by the cutting point positioning errors for the buds and inflorescence axes, the error was kept below 15 pixels for 55 images. On these images, the cutting point was positioned along the edge of the inflorescence axis. The error was above 15 pixels for five images, where the cutting point was positioned outside the inflorescence axis. The positioning error along the Y-direction was significantly lower than that along the X-direction. This was probably because the mask generated by the morphological processing of shadows on the edge of the inflorescence axis shifted in the X-direction.When detecting the rectangular region associated with the inflorescence axis, the cutting point might be mistakenly positioned at the edge of the axis. However, the optimal cutting point must be located on the centerline of the inflorescence axis. The positioning error can be estimated as the radius of the fruit stem. We have developed a clamping and cutting mechanism for the end actuator based on fault tolerance analysis . This meThe previous algorithms can only get better results when facing fruits with simple outline shape, but cannot get better classification results when facing fruits with complex and irregular or fruits with complex growing environment (such as bananas). The algorithm proposed in this study can further calculate the detected target on the basis of deep learning, and has achieved high detection accuracy in both bright and backlight environments, and has high robustness in cooperation with the end effector based on fault-tolerant design developed by us.This study focused on the multi-target recognition of banana fruits, buds, and inflorescence axes in a complex orchard environment, and proposed YOLOv3 model and edge detection algorithm based on cluster optimization, and constructed a calculation model of flower bud cutting point. Experiments showed that the mAP and speed of modified YOLOv3 and YOLOv4 were satisfactory. The precision was 92.98 and 93.46%, respectively. The average time for field detection of each image was 0.24 and 0.2 s, respectively. The accuracy of the improved YOLOv3 is 97.90 and 91.11% under the conditions of front-lighting and backlighting conditions, respectively. The improved YOLOv3 had better performance and higher recall rate, and had achieved a good balance between speed and accuracy. Under the conditions of front-lighting and backlighting, the total pixel positioning errors between the calculated optimal cutting point and the manually determined optimal cutting point in flower bud were 4 and 5 pixels, respectively. The proportion of images meeting the positioning requirements was 93 and 90%, respectively. The experiments showed that the proposed algorithm could satisfy the requirements for recognition performance and comprehensive performance in the cutting point positioning process.Multi-target classification and recognition of bananas from images potentially offer data support for the yield estimation of bananas. In the present study, we performed multi-target detection of bananas using monocular vision and by calculating the cutting point on the xy-plane. We recommend stereoscopic vision to obtain the 3D spatial information required for the detection. Another important research task related to the smart banana orchard operation is the robotic recognition of spatial coordinates of the inflorescence axis based on stereoscopic vision and the robot obstacle avoidance and cutting behavior.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.JD designed the experiments. YY and SC carried out the experiments. PA and FW analyzed experimental results with improved algorithms. FW wrote the manuscript. JD and ZY supervised and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Borrelia burgdorferi s.l. in CSF of Swedish children with LNB. This study was performed retrospectively on CSF and serum samples collected from children evaluated for LNB (n = 233) and controls with other specific neurological disorders (n = 59) in a Swedish Lyme endemic area. For anti-Borrelia antibody index, the IDEIA Lyme Neuroborreliosis kit (Oxoid) was used. Two in-house real-time PCR assays targeting the 16S rRNA gene were evaluated (TaqMan® and LUX™). Among patients classified as LNB cases (n = 102), five children (5%) were Borrelia PCR-positive in CSF with the TaqMan® assay. In the Non-LNB group (n = 131), one patient was Borrelia PCR positive with the TaqMan® assay. Among controls (n = 59), all CSF samples were PCR negative. When amplifying and sequencing ospA, we found B. garinii (n = 2), B. afzelii (n = 2), B. bavariensis (n = 1), and one untypable (n = 1). With the LUX™ technology, all CSF samples were PCR negative. The TaqMan® assay could detect only few cases (n = 6) of B. burgdorferi s.l. in CSF among children with LNB and the sensitivity was very low (5%). However, using larger CSF volumes and centrifugation of samples, the PCR technique could still be useful as a complementary diagnostic method when evaluating LNB. Furthermore, detection of spirochete DNA in clinical matrices, including CSF, is the method of choice for studying epidemiological aspects of LNB, a tick-borne emerging disease.The aim of this study was to evaluate polymerase chain reaction (PCR) as a diagnostic method for the detection of  Borrelia (B.) burgdorferi sensu lato (s.l.) . Al. Al21]. Borrelia AI) were classified as Non-LNB . Additionally, another control group was included consisting of children with other specific neurological diagnoses . This control group is referred to as Other neurological disorders .Children not meeting the criteria for Definite LNB or Possible LNB   and in Other neurological disorders  were negative for anti-Borrelia AI. Children in control group 2 (n = 59) are used for determination of specificity for the PCR assays.All children in Non-LNB   [Borrelia antibodies  was defined as AI > 0.3. Pleocytosis in CSF was defined as total white blood cell count > 5 × 106/L in CSF [The serological method used for anti-ire, UK) . IntrathL in CSF . In a feL in CSF .16S rRNA gene of Borrelia genus, were evaluated for laboratory diagnosis of LNB in the present study. One PCR protocol included a real-time PCR assay (TaqMan® technology) targeting the 16S rRNA region for Borrelia species and B. burgdorferi s.l., respectively, followed by a conventional nested PCR designed to amplify the ospA gene fragment, which would determine different B. burgdorferi s.l. species as described by Ornstein et al. [Two different in-house real-time PCR assays, both targeting the n et al. , 37. Then et al. .DNA was extracted from 200 μL CSF using Bio Robot EZ-1 with DNA Tissue Kit  after treatment with Proteinase K at 56 °C for 30 min followed by heating at 95 °C for 15 min and addition of 0.01 μg calf thymus DNA (Sigma Aldrich). Elution volume was set to 50 μL. The amplification was carried out in a 25-μL reaction mix of SensiFAST Probe No-ROX Kit  with primers and probes as described elsewhere, and 5-μL template –39.Borrelia spp. were further determined by nucleotide sequencing the outer surface protein A gene (ospA) using a nested PCR approach as earlier described by Ornstein et al. [In the TaqMan® real-time PCR protocol, n et al. . PCR pron et al. .n = 292) were analyzed with the TaqMan® real-time PCR assay, but due to unforeseen technical issues, samples from only ninety-five patient samples (n = 95) were analyzed with the LUX™ real-time PCR assay.All samples in the study (n = 68), 15% as Possible LNB (n = 34), and 56% as Non-LNB . Clinical signs and symptoms and laboratory data in different groups are shown in Table n = 59) consisted of children with epilepsy, infantile spasm, high intracranial pressure, head trauma, papilledema, viral encephalitis, aseptic meningitis, varicella infection, myasthenia gravis, and the Guillain-Barre Syndrome. Clinical characteristics in all patient groups are shown in Table Out of 233 children being evaluated for LNB, 29% were classified as Definite LNB (n = 68) and Possible LNB (n = 34), only five children (5%) were Borrelia spp. PCR positive in CSF with the TaqMan® assay (Table n = 131), one patient was Borrelia PCR positive in CSF with the TaqMan® assay (Table B. garinii (n = 2), B. afzelii (n = 2), and B. bavariensis (n = 1), and in one sample, the Borrelia species was untypable (n = 1)  (see description of cases below). At the 2-month follow-up visit, all six Borrelia PCR-positive patients reported full clinical recovery, even the one patient classified as Non-LNB, who did not receive antibiotic treatment. Thus, the diagnostic performance for the TaqMan® protocol showed a clinical sensitivity of 6% in the Definite LNB group  and 3% in the Possible LNB group . The overall clinical sensitivity was 5% . The clinical specificity was 100% . The one patient with positive PCR in Non-LNB was a misdiagnosed LNB patient at the time of routine laboratory evaluation and therefore received no antibiotic treatment.Out of the six NB Table , accordiBorrelia PCR protocol, all of them were negative with the LUX™ Borrelia PCR assay (Table n = 24), Possible LNB (n = 18), Non-LNB (n = 41), and Other neurological disorders (n = 12).Among 95 patients analyzed with the LUX™ real-time ay Table . They weBorrelia PCR-positive children are shown in Table The six 6/L, out of which 366 were mononuclear cells), positive anti-Borrelia IgM antibodies in serum but negative anti-Borrelia AI. Data on CXCL13 in CSF was not available. He was diagnosed as Possible LNB and was treated with doxycycline perorally for 14 days. He reported as full recovered at the clinical follow-up. He was PCR positive for B. garinii in CSF so his diagnosis was re-defined as Definite LNB.The first case is an 11-year-old boy with headache, fatigue, neck pain, neck stiffness, and vertigo for 1–2 weeks, June 2011. He had observed a tick bite 2–4 weeks before, but no EM. He was living in Jönköping but had been in the County of Östergötland and on the island of Öland during the summer. He had pleocytosis in CSF  and positive anti-Borrelia AI. Data on CXCL13 in CSF was not available. She was treated with ceftriaxone intravenously for 10 days. She had not reported persisting problems, but did not come for the final follow-up. She was PCR positive for B. garinii in CSF. She was initially classified as Definite LNB and remained so.The second case is a 5-year-old girl with facial nerve palsy, fatigue, headache, fever, neck pain, and loss of appetite for 3–6 days in October 2011. She had not noticed any tick bites, but probably a redness of the earlobe (lymfocytoma) and also in the face (EM). She was living in the County of Östergötland (Linköping) but during summer had visited Stockholm (east cost of Sweden) as well as Gothenburg (west coast of Sweden). She had pleocytosis in CSF , a positive anti-Borrelia AI, and positive CXCL13 in CSF (710 pg/mL). He was treated with ceftriaxone intravenously for 10 days and was fully recovered at follow-up. He was PCR positive for B. afzelii in CSF. His diagnosis was initially and remained Definite LNB.The third case was a 4-year-old boy with facial nerve palsy, headache, and fatigue for 3–6 days. He was admitted in December 2012. He had noticed a tick bite 3–5 months before, but no EM. He was living in Norrköping in the County of Östergötland and had visited the island of Öland (east coast of Sweden) during summer. He had pleocytosis in CSF . She was initially treated with cortisone as in idiopathic facial nerve palsy, but due to positive anti-Borrelia IgM antibodies in serum, she was additionally treated with doxycycline perorally for 10 days. She was completely recovered at the clinical follow-up already 2 weeks later. Her diagnosis was initially idiopathic facial nerve palsy as part of the group Non-LNB. However, since she was PCR positive for B. afzelii in CSF; she was re-defined as Definite LNB, having a very early LNB with cranial nerve affection, but not yet pleocytosis in CSF.The forth case is a 12-year-old girl with facial nerve palsy, headache, and fatigue for 3–6 days. She was admitted in March 2012. She had noticed neither tick bite nor EM. She was living in Skövde in Skaraborg area  but had visited the west coast of Sweden during summer. She had no pleocytosis in CSF, negative anti-6/L, out of which 158 were mononuclear cells), positive anti-Borrelia AI, and highly positive CXCL13 in CSF . She was treated with ceftriaxone intravenously for 14 days. She recovered without any sequelae. She was PCR positive for B. bavariensis in CSF. Her diagnosis was initially and remained Definite LNB.This was a 6-year-old girl with facial nerve palsy for 1–2 days, headache, and fatigue since 1–2 weeks and who was admitted in July 2014. She had also pain in the jaws, neck, and scalp. Tick bites or an EM had not been observed. This girl was living in Falun in the County of Dalecarlia and had been visiting Stockholm and Uppsala (east part of Sweden). She had pleocytosis in CSF , negative anti-Borrelia AI, and positive CXCL13 in CSF (6060 pg/mL). In serum, she had elevated titers of anti-Borrelia IgM and IgG antibodies. She was diagnosed as Possible LNB and received ceftriaxone intravenously for 10 days with good clinical effect. Her symptoms had resolved already at the 3-week follow-up. She was PCR positive for Borrelia, but the species in this last case was untypable. Her diagnosis was re-defined as Definite LNB.The last case was a 6-year-old girl admitted to the outpatient pediatric department in Linköping in October 2011 because of facial nerve palsy for 1–2 days, headache, and fatigue for 3–6 days. No tick bite had been noticed and she had not developed any EM. She was living in the County of Östergötland and had not been visiting other parts of Sweden. She had pleocytosis in CSF  followed by a nested ospA PCR. The second 16S r RNA PCR assay was based on LUX technology. We had expected an acceptable sensitivity in children with short duration of symptoms , especially in children with a high clinical suspicion of LNB, pleocytosis in CSF but negative anti-Borrelia AI (Possible LNB). However, with the TaqMan® protocol, B. burgdorferi s.l. DNA could be detected only in six out of 233 samples, whereas with the LUX™ protocol, all 95 tested samples were negative. Thus, the clinical sensitivity for PCR in children with LNB remains low with an overall clinical sensitivity of 5% (in the Definite LNB and Possible LNB groups taken together). Our PCR results indicate that one patient in the Possible LNB group should be re-defined as Definite LNB and one patient in the Non-LNB should be re-defined as Definite LNB . This supports the usefulness of a PCR protocol (TaqMan® assay) as a complementary diagnostic method for detection of B. burgdorferi s.l. in CSF in pediatric LNB patients with short duration of symptoms. The specificity of the TaqMan® protocol was as high as expected (100%).In this study we have evaluated two in-house real-time PCR protocols, both based on amplification of the Borrelia PCR positivity of 46% in CSF of patients defined as Definite LNB and 50% in CSF of patients defined as Probable LNB  [Borrelia AI, and negative anti-Borrelia antibodies in serum) [Our findings of low sensitivity in CSF of patients with LNB are in concordance with previous studies , 40. Howr serum) . Howevern serum) . DuratioThe reason for discordance in sensitivity between different studies might reasonably be found in the primer/probe design of the different PCR assays. However, the two PCR protocols used in the present study  have previously been compared in another study and they have shown very similar results .B. burgdorferi s.l DNA since the number of Borrelia spirochetes in CSF is suspectedly very low [Borrelia PCR detection, whereas in the Norwegian study, CSF volumes between 0.5–1 mL were centrifuged into pellets and resuspended in 0.2 mL of CSF. Thus, this difference in CSF volume and procedure may be the major explanation for the discrepancy between PCR results. Consequently, larger volumes of CSF samples, improved procedure of centrifuging, lower elution volume (< 30 μL) or larger volumes of DNA templates (> 5 μL) should, if possible, be used when evaluating pediatric LNB patients, in order to obtain a higher proportion of Borrelia PCR-positive LNB patients.It has been suggested that the volume and procedure of collected CSF could play a role in the probability of detecting the very low . In our B. burgdorferi species have been detected in CSF of Swedish LNB patients: B. burgdorferi s.s., B. afzelii, and B. garinii [B. afzelii predominates, followed by B. garinii and B. burgdorferi s.s. [B. garinii was detected in CSF from 16 out of 35 LNB cases, whereas the rest could not be completely identified [Borrelia bavariensis has, to our knowledge, not previously been found in CSF in pediatric LNB patients. The clinical picture of our LNB patient with positive PCR for B. bavariensis is very much representative of a typical pediatric LNB case , symptoms were less specific with headache, fatigue, neck pain/stiffness, and vertigo. This finding suggests that detectable levels of Borrelia DNA in the CSF are associated with both specific and non-specific neurological symptoms in LNB, which may be relevant but challenging knowledge for the clinician. Furthermore, high levels of pleocytosis in CSF were found in the two B. garinii-infected LNB patients and in the one LNB patient with B. bavariensis. These two species are known to induce a prominent inflammatory response, whereas B. afzelii infections are associated with a lower level of inflammation in the CSF [Facial nerve palsy was the main specific neurological manifestation in five out of six  the CSF , 40.B. afzelii (Case 4), who was primarily classified as Non-LNB (due to absence of pleocytosis and negative anti-Borrelia AI), was, in our present study, re-defined as Definite LNB due to positive Borrelia PCR in CSF. This case was a very early case of LNB with neither pleocytosis nor intrathecal production of anti-Borrelia antibodies present, but with a positive serum IgM. This case shows us that in very early LNB cases, children might start producing Borrelia antibodies in serum before showing any sign of inflammation in CSF. Detection of Borrelia infection in the early phase by positive Borrelia PCR has been previously described [One patient with escribed , 41.n = 6) of B. burgdorferi s.l. in CSF among children with LNB, but the overall sensitivity was very low (5%). As methodological aspects could be improved, using larger CSF volumes and to centrifuge all CSF samples, the PCR technique could still be useful as a complementary diagnostic method, when evaluating pediatric LNB cases with short duration of neurological symptoms. Furthermore, detection of spirochete DNA in various clinical matrices, including CSF, is still the method of choice for studying epidemiological aspects of LNB, a tick-borne emerging disease.The TaqMan® protocol could detect a few cases (


In critically ill mechanically ventilated children, midazolam is used first line for sedation, however its exact sedative effects have been difficult to quantify. In this analysis, we use parametric time-to-event (PTTE) analysis to quantify the effects of midazolam in critically ill children.In the PTTE analysis, data was analyzed from a published study in mechanically ventilated children in which blinded midazolam or placebo infusions were administered during a sedation interruption phase until, based on COMFORT-B and NISS scores, patients became undersedated and unblinded midazolam was restarted. Using NONMEM® v.7.4.3., restart of unblinded midazolam was analysed as event. Patients in the trial were divided into internal and external validation cohorts prior to analysis.Data contained 138 events from 79 individuals . In the PTTE model, the baseline hazard was best described by a constant function. Midazolam reduced the hazard for restart of unblinded midazolam due to undersedation by 51%. In the blinded midazolam group, time to midazolam restart was 26 h versus 58 h in patients with low versus high disease severity upon admission (PRISM II < 10 versus > 21), respectively. For blinded placebo, these times were 14 h and 33 h, respectively. The model performed well in an external validation with 42 individuals.The PTTE analysis effectively quantified the effect of midazolam in prolonging sedation and also the influence of disease severity on sedation in mechanically ventilated critically ill children, and provides a valuable tool to quantify the effect of sedatives.https://www.trialregister.nl/trial/1913.Clinical trial number and registry URL: Netherlands Trial Register, Trial NL1913 (NTR2030), date registered 28 September 2009 The online version contains supplementary material available at 10.1007/s11095-021-03113-w. Sedation of critically ill children during mechanical ventilation in paediatric intensive care units (PICU) ensures patient comfort and reduces the incidence of adverse events like accidental self-extubation. Midazolam is used first line in sedating mechanically ventilated paediatric patients, however quantifying the effect of midazolam in adequately sedating critically ill children proved to be challenging , 2.To date, there has been little progress in the characterisation of midazolam pharmacodynamics in children, where large variability in response has been reported . InsteadA pharmacodynamic analysis of sedatives in critically ill children is complicated due to the multitude of patient intrinsic and extrinsic factors that can influence sedation and the removal of patients from studies due to discharge. Time to event (TTE) analyses have become increasingly popular for assessments of sedation efficacy . Rather In this study we therefore aimed to assess the effect of midazolam in maintaining adequate sedation in critically ill pediatric patients using a parametric time to event (PTTE) analysis. For this analysis we used data from the ‘daily sedation interruption in critically ill children’ trial  , a randoThe DSI study recruited a total of 129 participants from three different study centres, of whom 8 patients did not receive a trial infusion of either blinded midazolam or blinded placebo . From thDetails on the DSI study are described elsewhere , 12 and Patients were screened on a daily basis during their PICU stay, allowing for multiple occasions of sedation interruption. Up to three occasions of sedation interruption per patient were included in the analysis. Subsequent occasions were removed from the PTTE analysis due to the limited number of patients per occasion. In Fig. Generally, a PTTE analysis assesses the ‘survival’ of a population over a period of observation pertaining to the loss of individuals in the population due to death, relapse or any other defined event. In the current analysis, an event was described as the commencement of unblinded midazolam at the end of a sedation interruption phase due to undersedation  from the analysis  and eliminated if a selective exclusion from the final model did not worsen the model by significance of p > 0.01 (dOFV > 6.63 per parameter excluded). For the dropout model, covariate analysis was conducted by simultaneously incorporating each covariate on the event and dropout event. The covariate was only added to the model in case of a statistical improvement of p < 0.05 . Additional selection criteria for covariates included the overall impact of the estimated parameter on the event hazard or dropout event hazard, and relative standard errors of parameter estimates.Covariate analysis was first performed on the clinical event hazard model and then on both the clinical event hazard and dropout model. For the clinical event hazard model, covariates were added in a stepwise forward selection process for a statistical improvement of The influence of dichotomous categorical covariates (COV) such as reason for admission to PICU, treatment arm (blinded placebo or midazolam), sex, day vs. night, concomitant use of other sedatives and ordinal categorical covariates such as number of failing organs (0–5) and paediatric logistic organ dysfunction (PELOD) scores categorised as 0 (1–3), 1 (10–13), 2 (20–23) and 3 (30–33), were tested as a proportional difference  and paediatric index of mortality (PIM II), both logistic regression models estimating risk of mortality, were also assessed as continuous covariates. Equation 3 describes the influence (The effect of midazolam plasma concentrations (C(t)), cumulative midazolam area under the curve (AUC) from the first administration of midazolam until clinical event, midazolam AUC in the six hours prior to time t, daily midazolam AUC and midazolam AUC over the duration of the sedation interruption phase on clinical event hazard at time t were tested in a proportional or sigmoidal function . IndividEquation 4 describes the effect , using the graphical interface R-studio (v1.2.5019). Model development was performed on NONMEM v 7.4.3 using the Pirana workbench (v. 2.9.9) and the Perl-speaks-NONMEM (PsN v.4.9.0) library of modules.−1·h−1 (IQR 100–224 µg·kg−1·h−1)). Patient characteristics in both primary analysis and validation cohorts for each arm (placebo and midazolam) are reported in Table A total of 138 events in the 79 patients was available for the primary analysis cohort with all patients having at least one occasion of sedation interruption (blinded midazolam or placebo), 41 patients having a second occasion, and 18 patients having a third occasion. The validation cohort consisted of 75 observations in 42 patients who had at least one occasion, with 21 having a second occasion, and 12 having a third occasion of blinded infusion. Viral respiratory insufficiency and pneumonia were the leading causes for admission to PICU, accounting for 64% of all cases. Patient PRISM II scores ranged from 0 to 44, and the paediatric index of mortality ranged from 0.1 to 45%. Midazolam continuous infusions were administered at a similar rate in both arms prior to the first blinded infusion . This finding is illustrated in Fig. In the covariate analysis, randomisation to the blinded midazolam treatment arm during sedation interruption resulted in a reduction of 51% in the hazard of requiring a restart of the unblinded midazolam infusion compared to patients randomised to the blinded placebo arm , indicating a reducing risk of requiring a restart of midazolam with increasing severity of disease. The negative relationship between PRISM II scores and dosing requirements for midazolam was also reflected in reduced infusion rates of unblinded midazolam administered throughout the study when compared using a Kruskal–Wallis test between the categories of PRISM II scores . The relationship of PRISM II scores with the clinical event hazard is illustrated in Fig. As a second covariate, patient PRISM II scores, incorporated as a continuous covariate into the hazard model reduced the hazard of midazolam restart per unit of PRISM II score increase  are well described for the placebo arm, as the observed events fall within the 95% confidence interval band on the Kaplan–Meier curve. For the midazolam arm, duration of the sedation interruption phase for 50% of the observed population in the external validation cohort was lower than that of the primary analysis cohort dataset , which was not captured within the 95% confidence interval of the model based simulations .In this study, we applied PTTE modelling to analyse the efficacy of midazolam in maintaining adequate sedation in critically ill children. To date, few studies have evaluated the pharmacodynamics of midazolam, none being in critically ill children. In this analysis, we utilized data a randomized clinical trial assessing daily sedation interruption in critically ill children that compared blinded midazolam and placebo during the daily sedation interruption phase. The PTTE analysis enabled us to observe a relationship through the estimation of the hazard for experiencing undersedation, in the form of the event of requiring a restart of an unblinded infusion of midazolam after the blinded infusion sedation interruption phase. Two covariates of significance were found to affect time to undersedation in this analysis. The first covariate was the administration of midazolam as part of the blinded infusion, which halved the hazard for requiring a restart of unblinded midazolam and the second, an inverse relationship, between increasing PRISM II scores and event hazard.Parametric time to event analyses offer an advantageous tool over conventional linear and logistic regression analyses, given their ability to handle the influence of time over the course of the observation period. Time is assessed as part of estimating the baseline hazard for an event through various functions and also in the capacity to handle time varying covariates, such as drug exposure and also clinical pathology results. Furthermore, PTTE analyses also have the capacity to assess the influence of dropout on event hazard, which is of interest in a population prone to dropout during a clinical trial such as the critically ill in PICU.Using this approach, we showed that patients randomised to (blinded) midazolam had a longer duration of adequate sedation during the interruption phase compared to blinded placebo. In our analysis, the longer durations of blinded infusions in the midazolam arm were reflected as a reduction in the baseline hazard for requiring a restart of unblinded midazolam by 51%. The difference in the duration of blinded infusion between the two arms is distinctly observable in the Kaplan–Meier VPC Fig.  where meAlthough the DSI study highlighted a difference in duration of blinded infusions between the placebo and midazolam arms, a notable outcome of the study was the large proportion of patients on blinded midazolam, who still required the restart of unblinded midazolam, suggesting undersedation is not entirely mitigated by the administration of midazolam. The restart of unblinded midazolam despite infusion of blinded midazolam may lead us to speculate on a lack of efficacy of the applied dosages of midazolam for sedation of critically ill children.To date, only few studies have linked midazolam concentrations directly to sedation, fewer still in critically ill patients. Studies in adult critically ill patients, have typically correlated midazolam plasma concentrations with increasing ordinal categorical sedation scores, such as the Ramsay sedation scale, albeit with large inter-individual variability , 17. SimThe hazard for restarting unblinded midazolam was not only influenced by an extrinsic factor (use of midazolam), but also by the intrinsic factor of disease severity, estimated as a PRISM II score upon PICU admission. We were able to conclude on this covariate because in the DSI study, midazolam was infused as per protocolized sedation  which liOur work is predicated on the assumption that COMFORT-B scores accurately quantify sedation, a subjective endpoint, in our population. Theoretically, the decreased midazolam requirements with increased disease severity could be attributed to difference in the performance of the COMFORT-B measurements with increased disease severity. However, it has been shown that this scale can detect treatment-related changes in pain or distress intensity in critically ill children . MoreoveSome limitations may apply for this study. First, despite the fact that PTTE modelling allows for an investigation into the exposure–response relationship of midazolam, we could not identify an influence of midazolam exposure or concentration on the hazard for restart of unblinded midazolam infusion. For this exploration we had access to the midazolam concentrations and exposure over time in 79 of 121 patients of the DSI study that was published separated using a population PK modelling approach . InsteadIn conclusion, the PTTE analysis confirmed a clear effect of midazolam in adequately sedating mechanically-ventilated critically ill children, and also demonstrated a substantial influence of disease severity. By improving our understanding of midazolam requirements, this analysis provides the basis for improved dosing guidelines which in turn will result in a better understanding of the PK–PD relationship of midazolam in critically ill children. The PTTE analysis approach we propose here may also be used to determine the effect of other sedatives and analgesics in vulnerable populations like critically ill children.Supplementary file1 (DOCX 90 kb)Below is the link to the electronic supplementary material.


In the ‘can’t intubate can’t oxygenate’ scenario, techniques to achieve front of neck access to the airway have been described in the literature but there is a lack of guidance on the optimal method for securing the tracheal tube (TT) placed during this procedure. The aim of this study was to compare three different methods of securing a TT to prevent extubation following a surgical cricothyroidotomy.A randomised controlled trial was undertaken. The population studied were emergency physicians (EPs) attending a cadaveric airway course. The intervention was securing a TT placed via a surgical cricothyroidotomy by suture. The comparison was securing the TT using fabric tape with two different tying techniques. The primary outcome was the force required to extubate the trachea. The trial was registered with ANZCTR.org.au (ACTRN12621000320853).p < 0.001).17 emergency physicians completed intubations using all three of the securing methods on 12 cadavers for a total of 51 experiments. The mean extubation force was 6.54 KG (95 % CI 5.54–7.55) in the suture group compared with 2.28 KG (95 % CI 1.91–2.64) in the ‘Wilko tie’ group and 2.12 KG (95 % CI 1.63–2.60) in the ‘Lark’s foot tie’ group; The mean difference between the suture and fabric tie techniques was significant (Following a surgical cricothyroidotomy in cadavers, EPs were able to effectively secure a TT using a suture technique, and this method was superior to tying the TT using fabric tape. The final step in the ‘can’t intubate can’t oxygenate’ (CICO) scenario is front of neck access (FONA) and whilIn the emergency department (ED), TTs placed during orotracheal intubation may be secured using fabric tape tied using a ‘Lark’s foot’ knot Fig. 2] or al or al2] Our aim is to provide clarity on an optimal method for securing a surgical airway which can be taught alongside FONA techniques. In this study we have chosen to compare the force required to extubate a fresh-frozen cadaver where the surgical cricothyroidotomy TT has been secured by either a Lark’s foot knot, using fabric tape, a ‘Wilko tie’, again using fabric tape, and a ‘drain stitch’, using a silk suture.This randomised controlled trial was approved by the University of Melbourne research ethics committee . The trial was registered before clinician enrolment at Australian New Zealand Clinical Trials Registry (ACTRN12621000320853). Twelve fresh-frozen cadavers were provided by the University of Melbourne’s department of human anatomy. Seventeen emergency physicians (EP) were recruited from The Airway Course, a one-day cadaveric airway management course for critical care clinicians.https://www.randomizer.org).Prior to the experiment, participants received instruction on the securing methods as part of The Airway Course. Each EP performed all three techniques on three different cadavers amounting to 51 experiments, the order of which was randomised in advance by computer randomisation to minimise learning effects .After the TT was deemed secure by the participant, the cadaver was draped such that the investigator was unable to determine the securing method used.The force required to extubate the cadaver, the primary outcome, was estimated using a digital force gauge  attached to the TT. The same investigator, blinded to the securing method, performed all measurements by applying slow gradual upwards traction on the meter, until the TT cuff was completely through the surgical wound, and recording the maximum value. Assuming Force = Mass x Acceleration, and given the slow traction technique, where acceleration was considered negligible (and consistent across experiments), the weight measurement of the gauge was taken to be a surrogate for the extubation force.After performing each technique the participant was asked to mark a visual analogue scale (VAS) to indicate how easy or hard the technique had been to undertake following minimal instruction.A minimum of 10 clinicians had been determined by a power analysis based on an alpha of 0.05, a power of 80 % and a small difference in extubation force (200 g) between procedures. A sample size of 15 participants was then recruited to ensure complete data for the analysis.P values < 0.05 were considered statistically significant.Symmetrical numerical data has been summarised using the mean (SD); skewed numerical data has been summarised using the median (IQR); and categorical data has been summarised using frequencies (%). The statistical significance of these measures of association were tested using paired statistical testing procedures, i.e., Repeated Measures Analysis of Variance (ANOVA). The planned sample size was achieved with 17 clinicians completing the three techniques on 12 cadavers for a total of 51 experiments. Characteristics of the participants are presented in Table Extubation force Fig.  and VAS p < 0.001), as seen in Table p < 0.001) and Lark’s foot technique . There was no significant difference identified between the Wilko Tie and Lark’s foot techniques.The mean extubation force was highest for the suture technique, followed by the Wilko Tie and then Lark’s foot technique, which had the lowest force required. The difference between these mean values was significant using a repeated measures ANOVA (p = 0.016), as seen in Table p = 0.012). There was no significant difference identified between the other techniques.The mean VAS was highest for the Wilko Tie technique, followed by the suture technique and then the Lark’s foot technique, which was reported as the easiest of the techniques. The difference between these mean values was significant using a repeated measures ANOVA (This randomised controlled trial compared the force required to extubate a TT placed via a surgical cricothyroidotomy, using three securing techniques, and found that a suture ‘drain stitch’ method was able to resist a greater extubation force than two fabric tape tying methods. The suture technique was also not reported to be more difficult to perform than the fabric tape tie techniques.One of the concerns with the fabric tape tie techniques is that the tape must be passed around the neck with the associated risk of occluding venous return which may affect intracranial pressure, particularly in head injured patients. The tecSurgical airways are performed rarely in the ED and as sWe have demonstrated that EPs with a brief training intervention are able to successfully secure a TT placed via a surgical cricothyroidotomy using a silk suture, which is able to resist a mean extubation force equivalent to at least 6 KG.in vivo, which can involve sudden application of force in any direction. Lastly, the study’s participants were a relatively experienced group of EPs who had chosen to undertake a cadaveric airway course, and this may also limit the generalisability of the findings.There are limitations to this study. Firstly, we used cadavers rather than anaesthetised ED patients and so the results cannot be automatically extrapolated to this patient group. Fresh frozen cadavers represent the best existing alternative model for this type of research, however, particularly when compared with manikin models. Secondly, the force applied to the TT in this experiment may not be representative of accidental extubations Following a surgical cricothyroidotomy in fresh frozen cadavers, EPs were able to effectively secure a TT using a simple suture technique and this method was superior to tying the TT using fabric tape.


A range of modern density functional theory (DFT) functionals have been benchmarked against experimentally determined metal hydride bond strengths for three first-row TM hydride complexes. Geometries were found to be produced sufficiently accurately with RI-BP86-D3(PCM)/def2-SVP and further single-point calculations with PBE0-D3(PCM)/def2-TZVP were found to reproduce the experimental hydricity accurately, with a mean absolute deviation of 1.4 kcal/mol for the complexes studied. At the forefront of modern chemistry is sustainability, with a drive towards ‘greener’ processes and development. Catalysis has always been a tool used to reduce energetic costs and to promote specific reactions, improving selectivity and the efficacy of various transformations. Traditionally, homogeneous catalysts have been based upon expensive and unsustainable metals such as platinum, palladium and rhodium  and whilFirst-row TM catalysts have undergone huge development in the past few years and are being optimised to ultimately become competitive against their unsustainable heavy metal congeners. Iron and manganese are attractive due to their low toxicity and high abundance, and work on catalytic hydrogenation reactions using these metals is very topical see . For a rDensity functional theory is a powerful tool for the elucidation of reaction mechanisms and for the rational design of these catalytic systems. To accurately study such systems, the DFT functional used must be suitable for the system, accurately predicting bond energies that are crucial to the functionality of the molecule. 3d TMs are notoriously tricky to study with DFT, especially bare TMs where the electronic structure is not always predicted correctly ,8. For iA number of papers have used DFT computations to examine the reactivity of these catalytic systems and a variety of methodology has been employed . The choAn alternate approach was adopted by Ge et al. , where tWe now present a validation study on the performance of these and other functionals for the calculation of hydricity for a few select complexes. Hydricity is the heterolytic bond dissociation energy of a metal hydride to a metal cation and hydride (Equation ), prediccf. upper arrow in A sizeable amount of periment , the ultGeometry optimisations were performed primarily at the GGA level of DFT using the BP86 functional, employing a variety of basis sets from the redefinitions of the Ahlrichs family of bases ,41,42,43nsidered . The nate; B3LYP ,53,54, Be; B3LYP ,55,56,57e; B3LYP , BP86 +. In contrast, the two Co complexes have large ligands attached to each metal centre and are far more sterically crowded MeCN), with a large triple-To quantitatively predict the thermodynamic hydricity of a complex, the ability of the functional used to produce accurate energies is of prime importance. Single-point energy calculations have been performed with a range of functionals, on geometry optimised structures at the level of RI-BP86-D3 and Clearly, the Minnesota functionals of M06-2X and M06-HF, both highly parameterised for main group chemistry, are poor in their description of these 3d TM complexes. The lowest MAEs were obtained from RI-BP86, PBE0 and revPBE0 . The percentage of HF exchange has been shown to be important by Moltved et al.  and thisBased purely upon the MAEs obtained here, revPBE0 would be the functional of choice; however, we recommend to use PBE0 /def2-TZVP, but with CPU times for the frequency calculation, at the level of geometry optimisation, for Co(dppe)2H of approximately 12.5 days compared to 2 days for def2-TZVP and def2-SVP basis sets, respectively). Additionally, there was little change in the optimised geometry between the use of def2-SVP and def2-TZVP, with central metal ligand bond distances reproduced to within 0.006 Å /def2-TZVP//RI-BP86-D3(PCM)/def2-SVP for use on systems involving 3d TM hydride complexes.


Background: Cerebrospinal fluid (CSF) samples from patients with non-inflammatory neurological diseases are used for control groups in biomarker studies. Since large amounts of CSF are withdrawn, patients with idiopathic intracranial hypertension (IIH) or normal pressure hydrocephalus (NPH) are especially suitable. The serially taken CSF portions are usually collected in different tubes. We aimed to investigate whether the later random choice of one of these tubes for CSF investigations might harbor the risk of different CSF protein findings due to the so-called ventriculo-lumbar CSF gradient. Methods: Patients with IIH (9) and NPH (7) were included. CSF was serially taken and collected in six tubes of 5 mL each. Concentrations and CSF-serum quotients of immunoglobulins, albumin and the virus-specific antibody index (AI) were determined in the first, fourth and sixth CSF fraction. Results: CSF immunoglobulin and albumin concentrations and CSF-serum protein quotients were significantly lower in the fourth and sixth CSF fraction compared with the first CSF fraction. Virus-specific AI did not significantly differ in the different CSF fractions. Conclusions: CSF protein analytics should be performed in the first CSF fraction in order to avoid different measurement results and achieve comparability within a control group and between different control and patient groups. The choice of an appropriate control group is essential in studies investigating biomarkers in cerebrospinal fluid (CSF) . PatientThe total volume of the CSF compartment in adults compromises approximately 140 mL, and many CSF proteins are not homogenously distributed within the CSF compartment ,9,10. CoWhen large amounts of CSF are taken during one LP, usually several serially fractions of CSF are collected in different sample tubes. When these CSF samples are stored for later use in control groups, there is usually no mention of which portion of the CSF aliquot was used . We hypoIn order to evaluate the influence of the ventriculo-lumbar CSF gradient, we determined the CSF protein concentration, the CSF–blood-barrier function, the intrathecal synthesis of immunoglobulins and the virus-specific intrathecal antibody synthesis in serially taken CSF samples in a cohort of patients with IIH and NPH.n = 9) and NPH (n = 7) who were treated in Hannover Medical School between 2018 and 2019. All patients received LP for standard diagnostic or treatment purpose. After the spinal needle was inserted between the lumbar vertebrae L3/L4, L4/L5 or L5/S1, 6 CSF fractions, each with 5 mL CSF, were serially taken during the LP . LP was performed in sitting position in patients with NPH and in lying position in patients with IIH. A blood sample was taken instantly after LP from each patient.CSF and serum samples were obtained from patients with IIH . CSF samples with elevated cell count were not included. CSF protein measurements were performed in the first, fourth and sixth CSF fraction. CSF total protein (cutoff = 500 mg/L) was measured by using a Bradford dye-binding procedure . ConcentThe intrathecally produced antibodies against the viruses measles, rubella, varicella zoster (VZV) and herpes simplex virus (HSV) were determined by calculating the virus-specific antibody index (AI) according to the following formula: (CSF virus-IgG/serum virus-IgG)/. In the case of a detectable intrathecal total-IgG production by the method of Reiber–Felgenhauer, the calculation of a corrected AI according to Reiber and Lange is generally recommended ,18,19. AVirus-IgG was measured in CSF and serum by ELISA kits from Virion/Serion . To improve sensitivity, the detection antibody from the ELISA kit was exchanged for a polyclonal rabbit anti-human IgG-horseradish peroxidase (HRP) from Agilent . CSF and serum sample concentrations of virus-IgG were determined in reference to the standard curve .The Neurochemistry Laboratory of the Department of Neurology participates regularly in the external INSTAND survey program for analytic methods quality control .x-axis) representing the independent value and albumin as well as immunoglobulin CSF concentrations (y-axis) being the dependent values. The p-values < 0.05 were determined to be statistically significant.For statistical analysis, GraphPad Prism  was used. Values in the main text are presented as median and range. Values in figures and Of the 16 patients included, nine patients were diagnosed with IIH  and seven patients were diagnosed with NPH .p-values are displayed in p-values for the comparison of CSF-serum concentration quotients are displayed in p-values between 0.0763 and 0.1218). The median IgG index also did not differ significantly among the different fractions , as depicted in p-values between 0.3531 and 0.6545; An intrathecal immunoglobulin synthesis calculated with Reiber’s diagram could not be detected in any of the CSF samples.p = 0.3182). The one patient with elevated virus-specific AI  was diagnosed with secondary progressive multiple sclerosis and NPH. Virus-specific AI were lower in the fourth and sixth CSF fraction in this patient. Details are depicted in Virus-specific AIs were below the cutoff in all but one patient in the first (medians between 0.9 and 1.0) CSF fraction, and values did not significantly differ in the fourth (medians between 0.9 and 1.0) and sixth (medians between 0.9 and 1.0) CSF fractions , significant differences concerning the CSF albumin concentrations or the age-dependent Q Albumin were not observed [Another observation of our study is that CSF concentrations and quotients of albumin and immunoglobulins in the first CSF fraction were not significantly different between IIH and NPH patients. Although NPH patients were found to be significantly older than IIH patients  CSF fraction for analytics to improve diagnostic validity. If the first CSF fraction is not used, then we recommend to state which CSF fraction was used.