Knowledge graphs are a common form of knowledge representation in biomedicine and many other fields. We developed an open biomedical knowledge graph–based system termed Reasoning Over Biomedical Objects linked in Knowledge Oriented Pathways (ROBOKOP). ROBOKOP consists of both a front-end user interface and a back-end knowledge graph. The ROBOKOP user interface allows users to posit questions and explore answer subgraphs. Users can also posit questions through direct Cypher query of the underlying knowledge graph, which currently contains roughly 6 million nodes or biomedical entities and 140 million edges or predicates describing the relationship between nodes, drawn from over 30 curated data sources.We aimed to apply ROBOKOP to survey data on workplace exposures and immune-mediated diseases from the Environmental Polymorphisms Registry (EPR) within the National Institute of Environmental Health Sciences.P<.05 after false discovery rate correction. We then used ROBOKOP to (1) validate the associations by determining whether plausible connections exist within the ROBOKOP knowledge graph and (2) propose biological mechanisms that might explain them and serve as hypotheses for subsequent testing. We highlight the following three exemplar associations: carbon monoxide-multiple sclerosis, ammonia-asthma, and isopropanol-allergic disease.We analyzed EPR survey data and identified 45 associations between workplace chemical exposures and immune-mediated diseases, as self-reported by study participants (n= 4574), with 20 associations significant at ROBOKOP successfully returned answer sets for three queries that were posed in the context of the driving examples. The answer sets included potential intermediary genes, as well as supporting evidence that might explain the observed associations.We demonstrate real-world application of ROBOKOP to generate mechanistic hypotheses for associations between workplace chemical exposures and immune-mediated diseases. We expect that ROBOKOP will find broad application across many biomedical fields and other scientific disciplines due to its generalizability, speed to discovery and generation of mechanistic hypotheses, and open nature. The curated knowledge sources that populate a biomedical KG include both databases, such as DrugBank [if gene X is connected to both chemical exposure Y and disease Z, then the protein product of gene X may represent a potential drug target. Indeed, successful applications of KGs include drug repurposing [“Knowledge graphs” (KGs) have become a common approach for knowledge representation across scientific disciplines, including biomedicine . In a KGDrugBank  and CompDrugBank , and ontDrugBank  and HumaDrugBank . VariousDrugBank , thus alurposing  and the urposing .what genes are associated with recovery from COVID-19 infection? why is imatinib effective in the treatment of asthma? what biological pathways are associated with stroke-related morbidity? Note that these questions imply complex mechanistic relationships between terms in the query such as drug and disease. The ROBOKOP KG is designed to provide answers in the form of putative mechanistic pathways connecting the query terms.While KGs, such as Monarch, are openly available, many of the more sophisticated KGs remain proprietary. Perhaps the most well-known proprietary KG is the Freebase-derived Google KG that powers Google’s web search capability . As partHerein, we provide an overview of ROBOKOP and its real-world application to data derived from the Environmental Polymorphisms Registry (EPR) within the National Institute of Environmental Health Sciences. Specifically, we focused on an EPR study that aimed to explore the impact of workplace exposures on immune-mediated diseases (IMDs) such as asthma, allergy, multiple sclerosis, rheumatoid arthritis, and ulcerative colitis. We first conducted an exploratory analysis of self-reported exposures and IMD symptoms in order to identify significant associations between workplace chemical exposures and IMD. We then used ROBOKOP to (1) validate statistically significant associations by determining whether plausible connections exist within the ROBOKOP KG and (2) propose biological mechanisms that might explain the associations and serve as hypotheses for subsequent testing.is associated with, causes, and increases expression of. The ROBOKOP KG is derived from over 30 curated biomedical data sources  and a back-end KG, both of which are openly available -15. The  sources  that hav sources . The curThe ROBOKOP UI allows users to posit queries and quickly explore ranked-and-scored answer sets or subgraphs. ROBOKOP queries are meta-graphs -19 or qudisease X interacts with geneNPC1) with greater importance than more generic assertions .A given query will frequently produce many answers or subgraphs, especially for queries with little specification regarding nodes and edges or with multiple nodes and edges. As such, the ranking of subgraphs by relevance to the query and the strength of the supporting evidence are critical for user exploration of results. The ROBOKOP answer-ranking algorithm  weighs eThe ROBOKOP KG can also be queried directly, independent of the UI, using the Cypher query language  to find The EPR is a study of nearly 20,000 current participants that aims to better understand interactions among environmental exposures and genetic determinants of health and disease . The regAn IMD was defined as a self-reported allergic reaction ; asthma condition; or autoimmune disorder . EPR survey data were extracted in December 2018. The overall sample size was 4574 participants.heavy metals you have ever been exposed to for 15 minutes a week or more in any job you have held (CHOOSE ALL THAT APPLY).” Associations between workplace chemical exposures and individual IMDs were examined using chi-square analysis or the Fisher exact test, when sample sizes were small due to missing data or few positive cases. A false discovery rate correction was applied to the association tests. Chemicals were examined individually and also by chemical class. Odds ratios (ORs) with lower and upper bounds were calculated per convention and were not adjusted for small sample sizes. As this was an exploratory analysis, the significance level was set at α=.05 or .10, and we did not control for potential covariates such as age, sex, and race.An exploratory analysis was conducted to examine associations between each IMD and specific workplace chemicals classified into one of 18 classes . The surP<.10, with 20 associations significant at P<.05 after false discovery rate correction. In all cases, workplace chemical exposures were associated with increased odds of self-reported IMD  carbon monoxide-multiple sclerosis, (2) ammonia-asthma, and (3) isopropanol-allergic rhinitis, hay fever, or seasonal allergies. An overview of ROBOKOP results for each of these examples is provided below, with various functionalities of the interactive UI highlighted in the first example.We highlight the ROBOKOP application using three exemplar associations that were chosen because they were significant at P=.006) and chemical class level .The association between workplace exposure to carbon monoxide and multiple sclerosis was significant at both the chemical level , BDNF (brain-derived neurotrophic factor), IL10 (interleukin-10), NGF (nerve growth factor), IRF8 (interferon regulatory factor 8), KCNMA1 , and CASP8 (caspase 8).We posed the following question to ROBOKOP, but structured as a machine question: TNF, and 44 PubMed publications, again contributed by OmniCorp, supporting the association between carbon monoxide and TNF  and chemical class level .The association between workplace exposure to ammonia and asthma was significant at both the chemical level , PRKG1 (protein kinase cGMP-dependent 1), S100B , TNF, MPO (myeloperoxidase), IL6 (interleukin-6), IL1B (interleukin-1 beta), PDE4A (phosphodiesterase 4A), and PARP1 (poly(ADP-ribose) polymerase 1).The query that was posed to ROBOKOP aimed to identify potential genes that might mediate the association between ammonia and asthma, and it was structured similarly to the query in ADA as the intermediary gene. The metadata associated with ADA indicated that ADA metabolizes adenosine by converting it to inosine and ammonia. For this answer subgraph, the relationship between asthma and ammonia was supported by 93 PubMed publications, identified by OmniCorp. An ADA-asthma relationship was established using Monarch, with 32 supporting PubMed publications contributed by OmniCorp. Many of the supporting publications suggested a relationship between ADA mutations and asthma, allergy, and immune function [The lowest-ranked answer subgraph identified function .P=.02) and chemical class level .The association between workplace exposure to isopropanol and allergic rhinitis, hay fever, or seasonal allergies was significant at the chemical level . The intermediary gene in the second top-ranked answer set was IL6. An isopropanol-IL6 relationship was established by CTD, with one supporting publication provided by CTD and two additional supporting PubMed publications contributed by OmniCorp. An IL6-allergic disease relationship was established by Pharos, with 79 supporting PubMed publications contributed by OmniCorp. Many of those publications [ROBOKOP identified the following three intermediary genes: ications  suggesteications .P<.05 and largely unexpected a priori. Statistical evidence for an association, while important, does not establish a causal relationship or provide any insights into underlying mechanisms. Thus, we applied the open ROBOKOP KG system to validate the observed associations by demonstrating plausible connections between exposures and IMD conditions, and we provide mechanistic insights or hypotheses to explain them. We highlighted the following three use case applications: carbon monoxide-multiple sclerosis, ammonia-asthma, and isopropanol-allergic disease.We identified 45 significant associations between workplace chemical exposures and IMD conditions in an EPR cohort, with 20 of those associations significant at TNF, as one of several potential mediating genes. ROBOKOP metadata and publication support suggested that heme oxygenase-1, which oxidizes heme to bilirubin and carbon monoxide, plays an intermediary role [via carbon monoxide, has a homeostatic or anti-inflammatory role that might protect against multiple sclerosis or suppress disease exacerbation. However, more recent evidence suggests that a chronic heme oxygenase-1 response in glial cells may promote neurodegeneration and thereby exacerbate multiple sclerosis and other neurodegenerative diseases [ROBOKOP identified plausible answer subgraphs to a query structured as carbon monoxide–gene–multiple sclerosis, thus supporting the statistical association between workplace exposure to carbon monoxide and multiple sclerosis. ROBOKOP further identified the gene that encodes TNF, ary role . Levels ary role , and exoary role . These rdiseases .ADA, was identified by ROBOKOP as one of several potential intermediates in this relationship. ROBOKOP metadata indicated that ADA metabolizes adenosine by converting it to inosine and ammonia. ROBOKOP identified publications suggesting an association between ADA mutations and asthma, allergy, and immune function, including one publication suggesting an association with aspirin-intolerant asthma [ADA mutations and pulmonary complications, as well as an association between ammonia and pulmonary complications in persons with established respiratory disease.The statistical association between workplace exposure to ammonia and asthma was supported by multiple answer subgraphs that were returned by ROBOKOP. The gene that encodes ADA, t asthma . A recent asthma . Severalt asthma , and a mt asthma . These rIL6, was identified by ROBOKOP as one of several potential intermediary genes, with publication support suggesting the involvement of the innate immune response [ROBOKOP provided several answer subgraphs to support the statistical association between workplace exposure to isopropanol and allergic disease. The gene encoding IL6, response . In addiresponse . While Iresponse ,37. ThesROBOKOP has several limitations that should be considered when interpreting the results here or using the application. First, many of the associations between workplace chemical exposures and IMD conditions involved complex chemical mixtures such as “toner,” “transmission fluid,” and “motor oil.” ROBOKOP currently maps these entities to Medical Subject Heading terms, but the application does not have an approach in place for mapping such mixtures to the individual chemicals that constitute a given mixture. We are considering approaches to overcome this limitation. Second, ROBOKOP, like all KG-based applications, is limited by the challenge of KG completion. For example, if an exposure-gene relationship has not been established by one of the curated data sources underlying the ROBOKOP KG, then this relationship will not be identified. We are developing algorithms to overcome this limitation by inferring edges in the KG, but for now, this remains a limitation. Third, evidence for ROBOKOP assertions is derived from the following two main sources: (1) the curated knowledge sources used to create the KG and (2) the co-occurrence of terms in PubMed abstracts. The ranking and scoring algorithm that is used to rank answer subgraphs is based on these two sources of evidence, with the first source treated with greater importance than the second. Other relevant factors, such as date of publication and number of studies on a topic (versus publications), are not considered at present, but may be incorporated into future versions of the application. Fourth, as a prototype system, ROBOKOP does not yet support natural language processing capabilities or other sophisticated approaches to aid users. We encourage users to contact the developers and/or post GitHub issues should they encounter any challenges when generating queries and/or evaluating answer subgraphs. Fifth, while not a limitation of ROBOKOP itself, the workplace chemical exposure–IMD associations reported here were derived from survey data  and were not confirmed by clinical record review or expert judgement. As such, the potential exists for misclassification and/or bias in both the reported exposures and the reported IMDs. Moreover, the timing between workplace exposure and the onset of IMD cannot be determined due to limitations of the survey design and participant recollection at the time the survey was administered. Finally, as this was an exploratory analysis, we did not adjust the ORs for small sample sizes or control for potential covariates such as age, sex, and race.ROBOKOP demonstrated potential in its use to support real-world observations and generate mechanistic hypotheses. In this paper, we focused on significant associations between workplace chemical exposures and IMDs, identified as part of a larger EPR study. We note, however, that ROBOKOP has other applications. Indeed, one key feature of ROBOKOP is its generalizability across biomedical domains as a general question-answering system, with capabilities to support a variety of machine questions. For instance, we are using ROBOKOP to explore associations between medications and clinical outcomes, including adverse events, using data derived from electronic health records. We are also testing whether ROBOKOP can be used to support human reasoning on Medical College Admission Tests, and we have promising preliminary results . While tA second key feature of ROBOKOP is its ability to support speed to discovery. For instance, the example use cases presented here took little time to construct and execute, and the interactive UI allows even novice users to posit questions and explore answer subgraphs. The mechanistic insights that were gleaned from the ROBOKOP answer subgraphs were quickly realized, thus allowing for a rapid first-pass analysis of the results and evaluation of the supporting evidence. Moreover, ROBOKOP revealed all potential genes that might mediate the observed exposure-IMD associations via a single query; in effect, the user did not need to spend hours, days, or longer reading through the available literature. This speed to discovery afforded by ROBOKOP also allows investigators to quickly refute associations that may be nothing more than spurious findings . In the examples highlighted herein, we focus on three example exposure-IMD associations. While this may not seem like many, a full literature review to identify potentially novel insights, eliminate spurious findings, and explore supporting evidence would take abundantly more time than was needed to conduct the initial first-pass analysis using ROBOKOP. We plan to leverage ROBOKOP’s speed to discovery in a large-scale analysis of associations between single nucleotide polymorphisms and phenotypes as part of a broader EPR effort and in several other studies.A third key feature of ROBOKOP is its open nature. Indeed, access to ROBOKOP, whether via the UI or by direct Cypher query of the underlying KG, does not require login authentication or an account; rather, anyone with the URL can access the system. Moreover, the ROBOKOP KG can be downloaded independently of the application . The ope


N = 18, 15 boys, mean age 10.0 years; Controls: N = 62, 40 boys, mean age 13.9 years). Finally, we examined how methylation rates at those sites relate to the degree of local structural alterations where significant differences were observed between cases and controls. As a result, we identified 61 candidate DNA methylation sites involved in ADHD development in two pairs of discordant MZ twins, among which elevated methylation at a site in the sortilin-related Vps10p domain containing receptor 2 (SorCS2) gene was replicated in the case-control study. We also observed that the ADHD group had significantly reduced gray matter volume (GMV) in the precentral and posterior orbital gyri compared to the control group and that this volume reduction was positively associated with SorCS2 methylation. Furthermore, the reduced GMV regions in children with ADHD are involved in language processing and emotional control, while SorCS2 methylation is also negatively associated with emotional behavioral problems in children. These results indicate that SorCS2 methylation might mediate a reduced GMV in the precentral and posterior orbital gyri and therefore influence the pathology of children with ADHD.The present study examined the relationship between DNA methylation differences and variations in brain structures involved in the development of attention-deficit hyperactivity disorder (ADHD). First, we used monozygotic (MZ) twins discordant  for ADHD to identify candidate DNA methylation sites involved in the development of ADHD. Next, we tried to replicate these candidates in a case-control study (ADHD:  Attention-deficit hyperactivity disorder (ADHD) is one of the most common mental disorders in childhood, characterized by inattention, hyperactivity, and impulsivity, according to the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5)  and ofteSLC6A4, DRD4, COMT, BDNF, and NGFR, but also neurotransmitter release or neurite outgrowth including ERC2 and CREB5 and associated with the symptoms and severity of ADHD  recruited from the local community in our previous study were used as controls for the case-control study (1). All children had normal or corrected vision and normal hearing. All children, with the exception of the non-ADHD twins, were assessed using the Wechsler Intelligence Scale for Children-Fourth Edition  and 16-year-old females (pair 2). Eighteen children with ADHD  were also recruited at the department for the case-control study. The diagnosis of ADHD was assessed by licensed child and adolescent psychiatrists according to DSM-5 criteria . ParticiWISC-IV;  or the WWISC-IV;  and exclWISC-IV; , and witWISC-IV; .®•L2P reagent (DNA Genotek Inc.) and quantified using the Qubit™ dsDNA HS Assay Kit .Saliva samples were collected using the Oragene Discover OGR-500 kit . DNA was extracted using prepITP > 0.001 or those based on <3 beads were set to missing as were probes cross-hybridizing between autosomes and sex chromosomes  and (2) ADHD cases (N = 18)–controls (N = 62) study since (1) those samples were scanned within the same chip and the row positional balance was identical , and (2) batches were completely confounded with case-control group status . In such a case, it is not possible to remove technical signals when batches are confounded with variables of interest, even by employing tools such as ComBat  MZ twins discordant for ADHD and (2) the ADHD case-control study, respectively. We confirmed whether pair 1 and pair 2 were MZ twins using 59 “rs” probes on the EPIC chip using the R package ewastool (Genomic DNA (500 ng) was bisulfite-treated for cytosine-to-thymine conversion using the EZ DNA Methylation-Gold kit . The DNA was then whole-genome amplified, fragmented, and hybridized to the Human MethylationEPIC BeadChip . BeadChips were scanned using iSCAN (Illumina Inc.), and the methylation level  was calculated for each queried CpG locus using the GenomeStudio Methylation Module software, followed by the Psychiatric Genomics Consortium-Epigenome-Wide Association Studies quality control pipeline . Using Comosomes . CpG sitomosomes . Normaliomosomes  after bas ComBat . As suggewastool , and fouewastool  to estim2  implemented in MATLAB 2020b . T1-weighted images were segmented coarsely into gray matter (GM), white matter, cerebrospinal fluid, and skull/scalp compartments using tissue probability maps. The Diffeomorphic Anatomical Registration through Exponentiated Lie Algebra algorithm was applied to the segmented brain tissues to generate a study-specific template and to achieve an accurate inter-subject registration with improved realignment of smaller inner structures  was performed using a GE Signa PET/MR 3-Tesla scanner with an 8-channel head coil . A T1-weighted anatomical dataset was obtained using a fast spoiled-gradient recalled imaging sequence . Image acquisition for the other 28 controls in Cohort 1 in the case-control study was performed using a GE Discovery MR 750 3-Tesla scanner with a 32-channel head coil . A T1-weighted anatomical dataset was obtained from each subject by a fast-spoiled gradient recalled imaging sequence . VBM was performed as a global analytic approach using the Statistical Parametric Mapping version 12 softwareructures . The segFirst, to clarify epigenetic associations between proband and non-proband ADHD discordant MZ twins from methylation array data, multiple regression analysis was performed using CpGassoc . In thisP < 5.0E-05. DNA methylation at each CpG probe was entered as a dependent variable, and each group (case or control) as an independent variable. Age, sex, FSIQ, the proportion of epithelial cells, chip, and row for batch effect adjustments as explained previously, were entered as covariates, and results were threshold at false discovery rate (FDR) <0.05 by Benjamini-Hochberg.Second, to confirm the reproducibility of the probes from the MZ twin discordant pair analysis in the case-control analysis, we examined the subset probes threshold set at t-test models. Potential confounding effects of age, sex, FSIQ, scanner, and total GMV were modeled, and their attributed variances excluded from further analysis. Total GMV was calculated from the GM images obtained from pre-processing segmentation using the “Tissue Volumes” utility from the batching system in SPM12. A GM majority optimal threshold mask, created based on a study-specific sample, was applied to the analyses to eliminate voxels of non-GM for GMV-analyses  correction for multiple comparisons. The anatomical localization of significant clusters was investigated using automated anatomical labeling and Brodmann area atlases implemented in the MRIcron software package  between groups were analyzed in SPM 12 using two-sample analyses . The res package .P < 0.05. All statistical analyses were performed with R 3.6.3  controlled by control variables was performed. To this end, the adjusted eigenvariates, representing linearly transformed estimates of GMV, were extracted from the identified cluster. The significance level was set at  R 3.6.3 , SPM 12,3.The functional properties of structural regions with alterations between groups were decoded using a large-scale database-informed meta-analytic approach as implemented in NeuroSynth . A meta-The clinical status of the MZ twin discordant pairs is shown in P < 9.0E-08) (P < 5.0E-05 (−log(P) = 4.3) by visual inspection of the Q-Q plot because the top probes over the threshold had residuals that steeply deviated from the expected line (No CpG probes were detected above the genome-wide significance level under the number of EPIC array probes (9.0E-08)  as a natted line . Sixty-oted line , which wT(78) = 5.88, P < 0.001). A two-sample t-test was used to compare the total FSIQ and CBCL total scores between groups. Compared to the control group, the ADHD group showed lower FSIQ (T(78) = 3.05, P = 0.003) and higher levels of ADHD-related emotional and behavioral problems  = −8.90, P < 0.001), although data for CBCL was only available from Cohort 1. In addition, in the control group of Cohort 2, SDQ total scores were not significantly different from the mean of standard Japanese children using one-sample t-test  = 0.47, P = 0.641), suggesting no notable emotional and behavioral problems. Multiple regression analysis was also performed to examine the effect of age on the differences in FSIQ and CBCL total score between groups, with each variable as the dependent variable; the results showed that the effect of group was significant  while that of age was not . Regarding ADHD symptoms, the inattention score of SNAP-IV in the ADHD group showed clinically mild symptoms on average, and the hyperactivity/impulsivity and opposition/defiance scores did not reach clinically significant levels. Since the ratio of epithelial cells in saliva samples affects the estimated methylation level , we analyzed for the difference between groups finding no significant difference between the ADHD and control groups. We also estimated GMV, WMV, and total brain volume in the ADHD and control groups, respectively, but found no differences between groups in any of these parameters , transmembrane protein 240 (TMEM240), and sortilin-related Vps10p domain containing receptor 2 (SorCS2) genes were significantly associated with ADHD  in the coiled-coil domain containing 86 . Compared with the control group, the ADHD group showed reduced GMV in the left precentral gyrus  identified using a web tool based on human samples (P = 0.09).Sortilin-related Vps10p domain containing receptor 2 methylation was both positively correlated with GMV within a cluster in the precentral gyrus and the posterior orbital gyrus . This re samples  and alsoSorCS2 methylation and local GMV alterations, and clinical symptoms  associated with ADHD. As a result, a significant negative correlation was confirmed between SorCS2 methylation and CBCL total score , while no significant correlation was found with SNAP total score ; these scores also showed no correlation with both local GMV alterations. These results suggest that SorCS2 methylation may be involved in regulating emotional behavioral problems in children rather than ADHD-specific core symptoms.We performed a correlation analysis investigate the association between the neurobiological basis of both SorCS2 gene body region. Finally, we observed that the ADHD group had significantly reduced GMV in the precentral gyrus and posterior orbital gyrus compared to the control group and that this volume reduction was positively associated with SorCS2 methylation. In addition, the reduced GMV regions in children with ADHD are involved in language processing and emotional control, and SorCS2 methylation is also negatively associated with emotional behavioral problems in children. These results indicate that SorCS2 methylation might mediate a reduced GMV in the precentral and posterior orbital gyri and therefore influence the pathology of children with ADHD.This study investigated the relationship between DNA methylation differences based on array data and brain structure involved variations in the development of ADHD. First, we investigated two pairs of MZ twins discordant for ADHD and identified 61 candidates for DNA methylation sites involved in the development of ADHD. Next, using these candidates in a case-control study we found that children with ADHD had elevated methylation in the SorCS2 methylation is involved in either children or adults with ADHD  receptor trafficking in hippocampal neurons . Other data that support the results of this study are included in the article/The DNA methylation microarray data of monozygotic twins discordant samples have been deposited in the Gene Expression Omnibus database (GEO) with the primary accession code The study protocol was approved by the Research Ethics Committee of the University of Fukui, Japan, and all procedures were conducted in accordance with the Declaration of Helsinki and the Ethical Guidelines for Clinical Studies of the Ministry of Health, Labour, and Welfare of Japan. All parents provided written informed consent for participation in the study.TF, SN, and AT contributed to the conception and design of the study, performed the experiments and statistical data analysis, and wrote the first draft of the manuscript. KM, AY, ST, SH, KS, HO, and HM contributed substantially to performing the experiments and collecting the data, revised the manuscript critically for intellectual content, and approved the submitted version. 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.


Bartonella infection in dogs is lacking. This represents a critical limitation for the development and evaluation of new diagnostic tests, as well as for the diagnosis of, and research on, bartonellosis in dogs. This retrospective observational study aims to compare the results of commonly performed and newly-reported Bartonella spp. diagnostic tests in banked clinical specimens from 90 dogs with hemangiosarcoma (HSA) using composite reference standard (CRS) and random effects latent class analysis (RE-LCA) techniques. Samples from each dog were tested using six serological or molecular diagnostic assays, including indirect fluorescent antibody (IFA) and Western blot (WB) for the detection of antibodies in serum, and qPCR and droplet digital PCR (ddPCR) in blood and fresh frozen tissue biopsy samples . Bartonella infection prevalence was estimated to be 78% based on the CRS , and 64% based on the RE-LCA model. The assay with the highest diagnostic accuracy was qPCR performed on fresh frozen tissue biopsy samples . When comparing newly-reported to traditional Bartonella diagnostic assays, ddPCR was more sensitive for the detection of Bartonella DNA than qPCR when testing blood samples . Dogs that were positive on serological assays alone with negative molecular assays were highly unlikely (<3%) to be classified as infected by the RE-LCA model. These data indicate that Bartonella spp. DNA can be PCR amplified from fresh frozen tissues from a majority of dogs with HSA using both qPCR and ddPCR, supporting the use of these methods for future controlled studies comparing the prevalence of Bartonella spp. DNA in the tissue of dogs with HSA to that of unaffected controls.Currently, a gold standard diagnostic test for  Bartonella spp., seventeen of which have been associated with disease in dogs or humans, continues to present challenges to diagnosticians, clinicians, and patients in both human and veterinary medicine ). This assay also had the highest estimate specificity (100% [95% CI 71–100%]). For each individual assay, Bartonella diagnostic assays, ddPCR was more sensitive than qPCR for detection of Bartonella DNA when testing blood samples , though it was not when testing tissue samples . Based on the kappa value, there was moderate agreement between qPCR and ddPCR when both assays were performed on tissue biopsy samples . A dog was considered molecular assay positive if Bartonella spp. DNA was PCR amplified from one or more blood or tissue sample by qPCR, ddPCR, or both. A dog was considered serologically positive if it was IFA seroreactive and/or WB positive. When comparing overall Bartonella spp. molecular results with serologic results, there was slight to no agreement  were seroreactive. Conversely, 19% of the 32 seroreactive dogs did not have DNA amplified from blood or tissue. Based on the RE-LCA model, dogs that were positive on serology alone  with negative molecular assays were highly unlikely (<3%) to be classified as infected.When comparing combined molecular results with combined serologic results, molecular testing was more sensitive for detection of Bartonella spp. DNA . A dog was considered tissue molecular test positive if Bartonella spp. DNA was PCR amplified from one or more tissue biopsy samples using qPCR, ddPCR, or both (each dog had two tissue biopsy samples tested). A dog was considered blood molecular test positive if Bartonella spp. DNA was PCR amplified from its blood sample using qPCR, ddPCR, or both. Based on the kappa value, there was fair agreement between the molecular assay results when performed on blood samples compared to tissue samples  also had Bartonella DNA PCR amplified from blood samples. The combined result from three assays taken in parallel  had 100% sensitivity when compared to the inclusive reference standard.When comparing combined molecular assay results between blood and tissue samples, tissue molecular testing was more sensitive for the detection of Bartonella species could be identified with Sanger sequencing of PCR products from qPCR. Since no dog had Bartonella spp. DNA amplified from a blood sample by qPCR, Bartonella spp. identity could only be determined from tissue biopsy samples. In these tissue biopsies from dogs with HSA, B. henselae DNA was most often amplified. Homologies ranged from 99.3% to 100%  with B. henselae CAL1 and SA2 . Of the 56 Bartonella qPCR positive tissues, 52 (93%) contained B. henselae DNA, including 1 dog that had both B. henselae and B. koehlerae DNA PCR amplified from tissue. One dog had only B. koehlerae DNA PCR amplified from tissue . In two dogs, the Bartonella species could not be determined for the DNA sequences amplified; for these two samples, homology ranged from 95% to 98% with either B. henselae CAL1 or B. henselae SA2.Bartonella spp. DNA ddPCR amplified from both biopsy samples and 38 that had Bartonella spp. DNA ddPCR amplified from one of the two biopsy samples. There were 15 dogs that had Bartonella spp. DNA qPCR amplified from both biopsy samples , and 41 that had Bartonella spp. DNA qPCR amplified from one of the two biopsy samples.Two biopsy samples were tested with each molecular assay. There were 12 dogs that had Bartonella spp. diagnostic assays, 62% of dogs with histopathologically confirmed HSA and multiple Bartonella spp. assays performed were classified as infected with >99% probability based on latent class analysis. Bartonella qPCR performed on fresh frozen tissue biopsy samples had the highest estimated sensitivity and specificity (94% and 100% respectively) of any single assay. A dog with a positive result on tissue qPCR had a >99% probability of being classified as infected, regardless of the other assay results (positive or negative).In this study estimating sensitivity and specificity of Bartonella diagnostic assays, ddPCR was not more sensitive for detection of Bartonella DNA than qPCR when testing tissue samples (78% vs. 94% sensitivity). Though 70 dogs in this study had molecular and/or serological evidence of exposure to or infection with a Bartonella spp. when tested with all six different assays, not a single dog had Bartonella spp. DNA amplified from blood by qPCR, and only 8% were seroreactive by IFA. Based on RE-LCA, four of the six IFA positive dogs had >94% probability of being classified as not infected, demonstrating that while IFA is more specific than sensitive, the specificity is unlikely to be perfect. In contrast, half of the dogs positive by WB were classified as not infected–these included mainly dogs that were positive by WB alone or by WB and IFA .When comparing newly-reported to traditional Bartonella DNA from any dog’s blood sample, ddPCR proved a more sensitive DNA detection method from the blood. However, even ddPCR performed on blood samples had an estimated sensitivity of only 38%. There were 39 dogs in this study that had Bartonella DNA amplified from their tissue samples, but both ddPCR and qPCR testing of blood were negative. Previous studies in dogs and humans have shown very good analytical accuracy and high specificity of well-designed PCR assays [Bartonella spp., despite viable Bartonella spp. being isolated from tissues (bone marrow and lung) collected at the time of post-mortem examination [B. henselae DNA PCR amplified from biopsies of vasculitis lesions and normal skin, but Bartonella DNA was not PCR amplified, even after BAPGM enrichment culture, from multiple sequential blood samples [While qPCR did not amplify R assays ,46,64. DR assays ,48,49,51 samples .Bartonella spp. bacteremia, as well as the stochastic and dilutional phenomena associated with small quantities of Bartonella spp. DNA in patient blood samples. DdPCR was specifically designed to detect low-copy-number DNA on a background of abundant host DNA, and there were 25 dogs in this study that had qPCR negative but ddPCR positive blood samples. This supports the theory that small quantities of Bartonella spp. DNA in blood samples limits the clinical sensitivity of qPCR; ddPCR appears to improve upon this limitation. Testing multiple blood samples per dog (similar to the “triple draw” technique recommended for blood sampling for Bartonella PCR/BAPGM in humans) [Bartonella spp. DNA was amplified by qPCR from both tissue biopsy samples.Proposed explanations for lower sensitivity of both qPCR and ddPCR in blood compared to tissue include both the variable nature of  humans)  might fu humans) ,46, but Bartonella bacteremia, as indicated by PCR positive blood samples, Bartonella spp. bacteremia can also be fairly common in apparently healthy dogs. One study of healthy volunteer dogs in North Carolina found that approximately 10–20% had evidence of Bartonella spp. bacteremia based on BAPGM enrichment blood culture/PCR [Bartonella DNA is not cultured or PCR amplified from blood samples from dogs with Bartonella DNA present in tissue could include intermittent bacteremia, rapid clearance of bacteremia by the dogs’ immune system, a transient bacteremic phase of infection prior to a chronic tissue phase, or lower concentrations of bacterial DNA in blood compared to tissue. Regardless of the mechanism(s), Bartonella PCR performed on a single blood sample does not effectively predict whether a dog has Bartonella spp. DNA in its tissues.Adding to the difficulty interpreting the clinical significance of ture/PCR . The reaBartonella spp. ddPCR on fresh frozen compared to FFPE tissues have not been reported, based both on the mechanism by which formalin fixation decreases qPCR sensitivity, as well as studies of other infectious diseases, it is reasonable to assume that ddPCR sensitivity is also decreased when performed on FFPE tissue samples compared to fresh frozen samples [It is important to emphasize that fresh frozen rather than formalin fixed paraffin embedded (FFPE) tissues were used for the molecular assays performed in this study. Formalin fixation decreases PCR sensitivity by making DNA extraction more difficult, mainly because of chemical modification, as well as DNA trapping and potentially DNA fragmentation . Though  samples . Thus, iBartonella testing. However, it is notable that many of the tissues included in this study were skin biopsies from normal-appearing skin in dogs with pathology in distant organs. Skin biopsies can in fact often be obtained at substantially less risk and expense than biopsies of other potentially infected organs . The distribution of Bartonella in tissues is not likely uniform, but given the ability for Bartonella to induce pathology systemically via secreted factors such as BafA, and to infect vascular endothelial cells which are distributed throughout the body, it is possible that Bartonella infection in the skin may ultimately be shown to correlate with systemic illness [Bartonella infection in any given organ is poorly characterized at this time [While we were fortunate to have a set of samples for this study that included two biopsy samples of tissue as well as blood and serum, obtaining tissues is an invasive procedure that carries risks for complications and is generally accompanied by substantial sampling expense. These practical constraints may limit the widespread clinical utility of tissue biopsies for routine  illness ,71. Howehis time .Bartonella serology, when both WB and IFA results were considered in parallel Bartonella spp. serology was positive in less than half (36%) of HSA dogs that had Bartonella spp. DNA amplified from blood or tissue. Based on the RE-LCA model, dogs that were positive on serology alone  with negative molecular assays were highly unlikely (<3%) to be classified as infected. Previous studies documenting poor agreement between Bartonella spp. IFA seroreactivity and Bartonella spp. bacteremia based on PCR/BAPGM in dogs have been reported [Bartonella spp. exposure in dogs [Bartonella spp. IFA sensitivity at approximately 40% when compared to documentation of active infection by BAPGM enrichment blood culture/PCR [Bartonella spp. antigens, again compared to positive BAPGM enrichment blood culture/PCR results [Bartonella spp. DNA amplified from blood samples using qPCR, we cannot evaluate the sensitivity/specificity of IFA in dogs positive only by qPCR performed on blood samples.In regard to reported ,46,72. I in dogs ,74,75. I results . PossiblB. henselae antibodies (36% vs. 4%), but also lower specificity and a higher false positive rate. The five dogs positive solely by WB had a 97% probability of being classified as not infected based on the RE-LCA model . Because it is not possible to distinguish whether the antibodies detected by WB or IFA are indicative of active infection or previous exposure with effective clearance of bacteria, it is possible that seroreactive dogs were previously exposed, but not actively infected with a Bartonella spp. Conversely, for the 38 dogs that had Bartonella spp. DNA PCR amplified from blood and/or tissue, but were nonseroreactive, it is possible that the bacterial strategies used to evade the immune system in intracellular infection contribute to a lack of detectable antibodies by either IFA or WB.WB had higher sensitivity compared to IFA for detecting Bartonella-negative dogs with which to evaluate test specificity in similar manner to the traditional method using the inclusive reference standard to estimate sensitivity, and therefore ground our RE-LCA model in observed data. Using RE-LCA precludes the need for “known” disease status, however, and allows estimation of both sensitivity and specificity without a control population. Previous studies attempting to identify populations of dogs that had never been exposed to, or infected with, Bartonella spp. to serve as controls have often encountered considerable difficulty. For example, in an early study attempting to develop an experimental infection model of B. henselae and B. vinsonii subsp. berkhoffii, the two dogs obtained from a university breeding facility were infected with B. koehlerae prior to initiation of the study [Bartonella infection or exposure in dogs expected to be unexposed/uninfected, illustrate the difficulty of finding a truly negative dog population with which to evaluate test specificity [Bartonella spp. assays should use the same number of specimens for each sample type, and consider evaluating multiple blood samples to determine the degree to which repeated sampling improves molecular assay sensitivity in this sample type. In addition, future studies could consider using Bayesian techniques to evaluate diagnostic accuracy of Bartonella spp. assays. LCA using a Bayesian framework has been successfully performed for decades, and allows for greater flexibility in model creation as well as the use of informed priors that could be based on this and other studies [This study was designed to be primarily descriptive, and as such has multiple limitations. Importantly, there was no control group consisting of confirmed he study . This, acificity ,67. In a studies ,77,78.Bartonella spp., demonstration of live/viable bacteria is possible with culture or BAPGM-ePCR; unfortunately the samples in this study were not collected sterilely, precluding BAPGM-ePCR. Finally, because this was a retrospective study using a previously obtained set of samples from dogs with hemangiosarcoma, the results of this study cannot be applied to the diagnosis of other clinical or pathological disease presentations in dogs.Another limitation of this study concerns using PCR (qPCR or ddPCR) as a diagnostic method, because demonstration of pathogen DNA in a patient sample does not necessarily mean that live/viable pathogen is present in the patient, nor that that pathogen is the cause of the patient’s clinical disease . For BarBartonella spp.—both those assays currently in commercial use and novel assays that are continuing to be developed–in a variety of clinical settings. These findings also illustrate the limitations and diagnostic complexity associated with determination of Bartonella spp. exposure and infection in clinical patients, particularly as IFA and blood qPCR–the two most common diagnostic assays used by clinicians–had poor diagnostic accuracy. As many epidemiology studies are also based solely on these assays, the lack of sensitivity of these assays illustrated in this and other studies should be considered by researchers attempting to define Bartonella disease manifestations, ecology, transmission, and zoonotic risks.Currently, the “gold standard” of bartonellosis diagnosis (in dogs or humans) continues to rely on a combination of culture, serology, and molecular tests with poor, variable, or undetermined sensitivity, specificity, or both. In the absence of a true gold standard, it is difficult to accurately estimate clinical sensitivity and specificity of diagnostic tests. Without this information, it is impossible for clinicians to critically interpret test results, for epidemiologists to draw appropriate conclusions about population-wide trends, or for vector biologists and ecologists to clarify modes and routes of transmission. When clinical diagnostic tests are assumed to be more sensitive than they are, false negatives are not recognized and serious or fatal cases of bartonellosis may go undiagnosed ,80. FalsBabesia, Bartonella, and hemotropic Mycoplasma spp.) in their blood or tissues [This was a retrospective, observational, descriptive study involving 90 dogs with HSA. Specimens used for this study were previously collected from eight university veterinary hospitals across the United States between May 2008 and November 2011 and were banked by the Canine Comparative Oncology and Genomics Consortium (CCOGC) based on previously published standard operating procedures.  These sa tissues . The CCO tissues .There were 110 eligible dogs from the originally published study ; of thesBartonella spp., including IFA and qPCR on blood and tissue, were performed as part of the previous study [Bartonella antibodies were determined using three Bartonella spp. grown in cell culture  as antigens and following standard immunofluorescent antibody assay (IFA) techniques [Bartonella spp. antigens. For qPCR, each sample was screened for the presence of Bartonella spp. DNA using primers targeting the 16S-23S intragenic transcribed spacer (ITS) region of Bartonella spp., using 5 μL of template DNA as previously described [Bartonella spp. qPCR positive on tissue if one or more of the biopsy samples was qPCR positive . Stringent processing methods were used to avoid DNA carryover during tissue processing [Traditional diagnostic tests for us study . For IFAchniques . Sera weescribed ,82. Valiocessing ,58,83.Bartonella spp. DNA using ddPCR with the same primers and probes employed for qPCR and minor modifications [B. henselae, B. quintana, B. koehlerae or B. vinsonii subsp. berkhoffii genotype II spiked into negative control dog blood samples (at a concentration of 0.5 bacteria/μL) were used to set threshold values and determine the limit of detection. ddPCR amplification of the dog housekeeping gene was also performed using primers and probes employed for qPCR, as a reference target to facilitate quantification. Due to instrument design limitations, digital PCR droplets are not able to be sequenced. WB was performed as previously described using B. henselae SA2 whole-cell protein lysates and goat antidog whole IgG [B. henselae immunodominant proteins with a molecular size of 13, 17, 29, 50, 56, and 150 kDa  [For the current study, each DNA sample was screened for the presence of ications . The proications . DNA exthole IgG . As prevhttps://www.R-project.org/, accessed on 25 June 2020). The percentage of dogs positive for each individual assay, as well as different combinations of assays, were calculated. Diagnostic test results were presented in contingency tables. In the absence of an accepted gold standard for diagnosis of bartonellosis in dogs, two methods were used to estimate diagnostic test parameters: latent class analysis with random effects (RE-LCA) and composite reference standard (CRS) techniques.Data analysis was performed using R 4.0.2 (Bartonella infection is not a binary outcome (infected vs. not infected), models with one, two, and three latent classes were compared. The number of quadrature points was left at default (quadpoints = 21). Model selection was based on BIC [Bartonella infected” or “Not Bartonella infected” was determined with membership probabilities for each class computed from the estimated model parameters. Sensitivity and specificity  were calculated based on marginal outcome probabilities.In LCA models, the true disease status is unobserved  and diagnostic parameters are estimated based on this latent state. Using this method, a single gold standard diagnostic test is not needed, but rather each test is assumed to be imperfect and the probability of a patient being classified into each disease state is determined based on the observed combination of diagnostic test results. Classical LCA is based on the assumption of conditional independence; that is, test results for each subject are assumed to be independent, conditional on the latent state. This assumption is not necessarily met for the patients in this study: conditional independence of IFA and WB cannot be assumed, since both are based on detection of antibodies, and conditional independence of ddPCR and qPCR also cannot be assumed, since both use the same gene targets. There may also be heterogeneity between patients that is not dependent on their classification (infected or noninfected), for example based on infection severity, location, or duration. Therefore LCA with random effects was performed to account for heterogeneity among patients within each classification, with a normally distributed random effect added for each subject. RE-LCAwas done using the “randomLCA” package in R.  The fived on BIC . The minBartonella spp. infection/exposure and decrease the likelihood of false negatives. The inclusive CRS was defined using the combined results of all six assays in parallel: a dog was considered positive on the reference standard if it had a positive result on any one or more of these six assays, and considered negative it was negative on all six assays. Since specificity could not be calculated with this inclusive CRS, a separate CRS was defined for calculation of specificity of each test: for the specific CRS, a dog was considered positive if it had a positive result on any two or more of the six assay, and considered negative it was negative on all six assays or positive on only one assay. For the purpose of this study, a dog was considered to have evidence of Bartonella spp. infection if it had Bartonella spp. DNA PCR amplified on one or more assay, and considered to have Bartonella spp. exposure if it was IFA seroreactive or WB positive. With 90 dogs, we were able to estimate 95% confidence intervals (CIs) for the sensitivity of each test within a margin of ±approximately 11% These CIs for the sensitivity of each test were calculated using the Wilson score interval. Bartonella spp. positive proportions were compared using chi-squared tests with continuity correction. Statistical significance was considered when p < 0.05. To determine agreement between tests, the kappa statistic was calculated [The use of a composite reference standard (CRS) is an alternative method by which to assess test accuracy using a combination of imperfect tests ,63,85,86lculated .


Adult attention-deficit/hyperactivity disorder (ADHD) frequently leads to psychological/social dysfunction if unaddressed. Identifying a reliable biomarker would assist the diagnosis of adult ADHD and ensure that adults with ADHD receive treatment. Pupil diameter can reflect inherent neural activity and deficits of attention or arousal characteristic of ADHD. Furthermore, distinct profiles of the complexity and symmetricity of neural activity are associated with some psychiatric disorders. We hypothesized that analysing the relationship between the size, complexity of temporal patterns, and asymmetricity of pupil diameters will help characterize the nervous systems of adults with ADHD and that an identification method combining these features would ease the diagnosis of adult ADHD. To validate this hypothesis, we evaluated the resting state hippus in adult participants with or without ADHD by examining the pupil diameter and its temporal complexity using sample entropy and the asymmetricity of the left and right pupils using transfer entropy. We found that large pupil diameters and low temporal complexity and symmetry were associated with ADHD. Moreover, the combination of these factors by the classifier enhanced the accuracy of ADHD identification. These findings may contribute to the development of tools to diagnose adult ADHD. As ADHD develops, the deficits of attentional function mostly remain, while impulsivity and hyperactivity become less apparent6. Therefore, it is more difficult to detect the symptoms of ADHD in adults than in children9. Moreover, adults with ADHD frequently experience psychological and social dysfunctions without appropriate treatment12. Thus, the advent of any biomarker to support objective and quantitative diagnosis of adult ADHD is desirable (reviewed in13)14.Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, impulsivity, and hyperactivity18)20. This is caused by overactivity in the locus coeruleus (LC)23, which is a norepinephrine (NE) neural pathway that spans the entire brain and plays a critical role in arousal and attention (reviewed in24). In addition to overactivity of LC, the dysfunction of the right hemispheric LC, which has a crucial role in attention functions25, has been pointed26. However, it is difficult to measure LC activity directly using EEG because the region is located deep inside the brainstem. In contrast, fMRI cannot capture detailed LC activity over time because it has low temporal resolution.Neuro-imaging methods, such as electroencephalogram (EEG) and functional magnetic resonance imaging (fMRI), have revealed abnormal neural activity associated with attentional functions in patients with ADHD (reviewed in29 (review in30) because the LC is the common source for both the sympathetic pathway to the pupil dilator muscle and the parasympathetic pathway to the pupil sphincter muscle32. Therefore, pupil diameter can indicate deficits in attention or arousal, as well as imbalances in the exploration–exploitation trade-off in several psychiatric disorders, including schizophrenia34 and autism spectrum disorder (ASD)38. In particular, since the deficits of attention and arousal functions are the core symptoms of ADHD, the behaviour of pupil diameter may directly reflect the symptoms/pathology of ADHD40. Furthermore, capturing the behavior of pupil diameter using the eye tracker is highly effective in clinical practice because it is cost- and time-effective, widely available, and non-invasive43.Baseline LC activity is strongly reflected in pupil diameter45, healthy aging47, development49, and some psychiatric disorders (reviewed in51), such as ASD52, ADHD53, and schizophrenia55. In addition to EEG/MEG/fMRI studies, those using neural activity estimated by pupil behaviour to detect abnormal LC activity have focused on the complex temporal patterns of pupil diameter58 and asymmetricity of pupil diameters among both eyes59. In particular, Piu et al., Artoni et al., and Nakamura et al. showed that complex temporal patterns reflect attentional functions, arousal level, and the symptoms or pathology of psychiatric disorders58. Poynter demonstrated that pupil asymmetricity reflects attentional load, as well as ADHD symptoms of inattention and hyperactivity26.Accumulating evidence on the neural activity detected by EEG/magnetoencephalography (MEG)/fMRI demonstrates that temporal complexity and asymmetricity reflect cognitive functions64. In particular, as the first mathematical model, Usui and Stark constructed neural systems composed of the sympathetic pathway to the pupil dilator muscle and the parasympathetic pathway to the pupil sphincter muscle; these pathways were driven by common internal neural fluctuation60. Their study succeeded in modeling the autonomous temporal fluctuation of pupil diameter composed of frequency components from 0.04 to 2.0 Hz67, which is called hippus60. Recently Johansson and Balkenius developed a more complex pupil-controlling model comprising the amygdala, LC, cerebellum, and other regions; this model showed that various kinds of responses, including emotional and learning, in complex temporal patterns of pupil diameter appear as a consequence of cognitive tasks64. Furthermore, a recent study of pupil-controlling neural pathways by Liu et al. reported that the parasympathetic pathways from the LC project inhibitorily to both the ipsilateral and contralateral parts of the Edinger–Westphal nucleus (EWN) to control the pupil sphincter muscles, in addition to the ipsilateral parts as previously thought68 . However, the combination of pupil diameter features through machine learning to classify psychiatric disorders has not been developed, even though such an application might improve classification in cases where a single feature is inadequate. Moreover, the kinetics of pupil diameter may more directly reflect LC activity related to a deficit in arousal or attention, whereas the widely used EEG is based on neural activity measured from scalp electrodes. In this context, we hypothesized that analysing the relationship among size, temporal patterns, and asymmetricity of pupil diameters will improve our understanding of deficits in controlling-pupil-diameter neural systems in adult ADHD. Consequently, classification methods that combine these features may contribute to the advent of a biomarker for adult ADHD.Regarding the recent trend of developing diagnosis tools for psychiatric disorders, several identification methods combining several feature values based on neural signals such as EEG/fMRI have been proposed and have exhibited high classification ability in psychiatric disorders74 as a measure of a temporal complexity. Moreover, we analysed transfer entropy (TranEn)75 as a measure of the asymmetricity of left and right pupil diameters. Based on these features, we constructed a classifier using logistic regression and evaluated the ability of classification between healthy adult subjects  and subjects with ADHD.In the present study, to validate our hypothesis, we evaluated the hippus of the resting state in the adult subjects with or without ADHD. Specifically, we analysed the relationship between baseline pupil activity during the hippus and sample entropy (SampEn)40. The sample size for this study was determined based on the sample size used in previous studies regarding the relationship between pupil diameter and psychiatric disorders—including ADHD77. To assess their intelligence levels, all participants were evaluated with the Wechsler Adult Intelligence Scale-Third Edition, revised Japanese edition (WAIS-III), which comprises the intelligence quotient (IQ), verbal IQ (VIQ), and performance IQ (PIQ). To assess the subjects’ ADHD symptoms, they were subjected to the Japanese version of the adult ADHD Self-Report Scale (ASRS)78.This study included 16 subjects diagnosed with adult ADHD and 20 age-, sex-, and intelligence-matched TD subjects2 through a semi-structured interview: the Assessment System for Individuals with ADHD79. The ADHD group consisted of 11 subjects under drug-naïve conditions and five subjects treated with methylphenidate  or atomoxetine . These five subjects stopped taking their ADHD medication on the experimental day. In this study, we defined 1-day medicine-free as being almost equal to drug-naïve, a paradigm which has been used in previous studies80, because the average half-lives of methylphenidate and atomoxetine are 3.5 and 5 h, respectively. The participants with ADHD included ten primarily inattentive (ADHD/I) subjects and six combined inattentive/hyperactive (ADHD/C) subjects. To remove the influence of ADHD medications, a drug-naïve ADHD group was created by excluding the drug-treated subjects from the ADHD group.Subjects with ADHD were recruited from outpatient consultations at Seiwa Hospital, Tokyo, Japan. They were diagnosed based on the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fifth EditionNone of the TD subjects displayed clinically significant levels of ADHD symptomatology, as indexed by the ASRS. In both groups, we set the following exclusion criteria: a current major depressive or manic-depressive episode, a history of psychosis, Wechsler full-scale intelligence quotient mentclass2pt{minim82 for MATLAB  and presented on a 23-in. LCD monitor . SampEn has been widely used to analyse the complexity of temporal patterns with high temporal resolution in the time-series of neural activityr. SampEn h was defined as84. If the time-series becomes complex, h exhibits a large value. The values of h were averaged among epochs. In this study, PhysioToolkit (http://physionet.incor.usp.br/physiotools/sampen/), a toolbox in MATLAB85, was used for the calculation of SampEn.Against 75:t is a time-index, X to Y, X to Y, 67. Therefore, the delay needed to be set sufficiently faster than 2.0 Hz. Additionally, the conditional probability in Eq. . The TranEn 87 with an iteration number of 50. Ten IAAFT surrogate datasets were generated using different random seeds per original pupil diameter. These SampEn and TranEn values were averaged and compared with the value from the original pupil diameters.To investigate whether a non-linear dynamic process was involved in the temporal behaviours of pupil diameters in both TD and ADHD, we used iterative, amplitude-adjusted Fourier transformed (IAAFT) surrogate data analysist-test. To compare the size, SampEn, and TranEn of pupil diameter between the TD and ADHD groups, we used analysis of covariance (ANCOVA) with age as a covariate. To assess the significant main effect of group, post hoc t-tests were used. In t-tests, a two-tailed To compare the SampEn and TranEn values from the original pupil diameter time-series with those from the IAFFT surrogate time-series, we used a paired R among SampEn, TranEn, and size was used for multicollinearity in the logistic regression. For the criteria of multicollinearity, 88. To obtain ROC curves, the probability for ADHD estimated by the logistic regression was utilized. By changing the threshold of this probability to judge ADHD from 0 to 1.0, true-positive and false-positive rates were calculated at each threshold. The performance based on the ROC curve was quantified by the area under the ROC curve (AUC) to determine the overall identification accuracy. AUCs of 1.0 and 0.5 correspond to the case for perfect discriminating ability and the case for random prediction, respectively. For the logistic regression, we used the function for a generalized linear regression model in the Statistics and Machine Learning Toolbox of MATLABTo identify ADHD, a logistic regression based on the SampEn, TranEn, and size of pupil diameter was used. Pearson’s correlation t-test of ANCOVA. The comparison between the TD and ADHD groups indicated that pupil diameter was significantly larger in the ADHD group. The comparison between the TD and drug-naïve ADHD groups showed that pupil diameter was significantly larger in the drug-naïve ADHD group, whereas complexity and symmetricity were significantly smaller. The values of size, SampEn, and TranEn in each subject are represented in Fig. First, an IAAFT surrogate data analysis against SampEn and TranEn was conducted Table . The IAAR as shown in Table R as shown in Table Furthermore, classifiers in the TD and ADHD groups were evaluated by size, complexity, and symmetricity of pupil diameters. Figure P on the size-SampEn plane and size-TranEn plane in cases for classifier of TD/ADHD groups and TD/drug-naïve ADHD groups  were low between the severity of ADHD (ASRS score) and size/SampEn/TranEn  (see Fig. 25. Poynter demonstrated the possibility that subjects with high inattention and impulsivity/hyperactivity exhibited smaller right pupil diameter than the left one, i.e., reduction of symmetricity of the pupil diameters, due to the dysfunction of right hemispheric LC26. Thus, our results of reduction of symmetricity in ADHD are consistent with these findings. Moreover, in the present study, we observed significant reductions in complexity and symmetricity in ADHD when the TD and drug-naïve ADHD groups were compared. However, no such reductions were observed when the TD group and ADHD group including subjects on medication were compared (see Tables Regarding the size, temporal complexity, and symmetricity of pupil diameters in TDs and subjects with ADHD, subjects with ADHD had significantly larger pupil size than TDs, as shown in Table see Fig. . Moreovereview in0 (see Fi60. Piu et al. showed that, in addition to complexity estimated by Shannon entropy, the combination with determinism is effective as a diagnostic tool for identifying the states of neural systems56. Moreover, the importance of evaluation of determinism is supported by the fact that the behaviour of pupil diameters is produced from multiple non-linear neural pathways68. However, the temporal standard deviation, Shannon entropy, and the size of difference of pupil diameters cannot reflect determinism91. In contrast, SampEn and TranEn utilized in this study can precisely detect the determinism of pupil diameters in addition to the evaluations of complexity and symmetricity (see Table In determining ADHD, the advantage of evaluating complexity and symmetricity of pupil diameters by SampEn and TranEn, as used in this study, requires additional discussion. The conventional evaluation method for complexity and symmetricity of the behaviour of pupil diameters utilized temporal standard deviation, Shannon entropy, and size of difference between the right and left pupil diameters92. Applying our proposed method to these responses is important in determining its possible clinical application. Finally, in addition to pupil diameter in ADHD, researchers must evaluate similar profiles in other psychiatric disorders involving attention and arousal deficits or imbalances in the exploration–exploitation trade-off, such as schizophrenia and ASD. We intend to deal with these points in future studies.The limitations of this study must be considered. In this study, as a preliminary study, the characteristics of size, temporal complexity, and symmetricity of pupil diameters in ADHD subjects were revealed in a relatively small sample size. It is necessary for our results to be validated whether these revealed characteristics emerge under different conditions with larger sample sizes, including a large variety of clinical backgrounds among subjects with ADHD. Moreover, the comparisons between the TD and ADHD groups showed different tendencies than the comparisons between the TD and drug-naïve ADHD groups, perhaps because the severity of ADHD differs between subjects on medication and those who are drug-naïve. However, an assessment of the severity before treatment could not be obtained in our study. Regarding the influence of medications, in this study, we defined 1-day medicine-free as being almost equal to drug-naïve. However, it remains possible that these medicines have already yielded some long-term pharmacological effects that endure for more than a few days due to their regular administration. Thus, 1-day off medicine may not be sufficient for an individual to be regarded as being genuinely medicine-free. This influence needs to be evaluated in future studies with a larger sample size consisting of both drug-naïve and drug-treated ADHD subjects. Evaluating the dependencies of pupil behaviours on the specific classification types of ADHD, i.e., ADHD/I, ADHD/hyperactive-impulsive, and ADHD/C types, is important; however, the sample size of this study was too small to allow for such analyses. Additionally, although our study revealed the complexity and symmetricity of autonomous pupil diameter behaviours in both TDs and subjects with ADHD, their variations under cognitive tasks and attention loads remain unclear. However, recent studies indicate that pupil response for cognitive task and light stimulus may reflect the pathology of psychiatric disorders, the performance of attention function, and estimation for sleep qualityBy analysing pupil diameters, we revealed that ADHD is associated with large pupil diameter and low complexity and symmetricity of dynamic pupil diameter behaviours. Moreover, the combination of these factors by machine learning enhances the accuracy of ADHD identification. Applying our proposed evaluation method and our findings may facilitate the development of tools to aid in ADHD diagnosis based on pupil diameter. Since they can indicate deficits in brain function and psychiatric disorders, our methods may be used for other pathologies.


Emotional dysregulation (ED) is currently the most frequently used term to describe children with an impaired regulation of emotional states. Recent research studies speculate whether ED may be a neurodevelopmental disorder itself, a shared risk factor, or a common key feature of several psychiatric disorders, including, among others, attention deficit hyperactivity disorder (ADHD), and bipolar spectrum disorders (BSD). The association between ADHD and ED is one of the main reasons of misconceptions in the definition of boundaries between ADHD and BSD, leading to the frequent misdiagnosis of ADHD as BSD. Since ED is a multidimensional concept, a novel instrument—the Reactivity, Intensity, Polarity and Stability (RIPoSt) scale—was recently developed to assess the different dimensions of ED, which could help in detecting specific ED profiles in clinical youths. Our study included 154 patients, aged 13.8 ± 2.3 years, diagnosed with either ADHD, BSD, or comorbid condition, and a school-based sample of 40 healthy control (HC) adolescents, aged 12.5 ± 1.2 years. The RIPoSt scale and the Child Behavior Checklist were administered to both groups. Our results indicate that affective instability and negative emotionality subscales, as well as negative emotional dysregulation, are higher in BSD, both pure and comorbid with ADHD, while emotional impulsivity is higher in the comorbid condition and similar in the ADHD and BSD alone group; all clinical groups scored higher than HC. Conversely, positive emotionality is similar among clinical groups and within them and HC. Our findings also support the validity of the RIPoSt questionnaire, since the instrument proved to have good-to-excellent internal consistency, and strongly significant positive correlations were found with the CBCL-Dysregulation Profile, which is a commonly used, indirect measure of ED. Hence, the five subscales of the RIPoSt can be reliably used as an effective tool to study the emotional dysregulation in different clinical conditions, to help disentangle the complex relationship between ADHD and juvenile BSD and to provide clinicians with crucial evidence for better diagnostic characterization and therapeutic indications. Children with an impaired regulation of emotional states, including mood lability and instability, severe irritability, low tolerance to frustration, temper outburst, and hyperarousal, have become a diagnostic challenge in the last two decades , 2. The More recent advances tend to interpret ED as a neurodevelopmental, early-onset disorder of the regulation of emotions, often associated to other psychiatric disorders, strongly related with comorbidity between internalizing and externalizing disorders, suggesting that it could be a shared risk factor for both kinds of disorder , or a coMost if not all descriptions in children are focused on the association with ADHD , 9. At lThe association between ADHD and ED is one of the main reasons of misconceptions in the definition of boundaries between ADHD and BSD, leading to the frequent misdiagnosis of ADHD as BSD, or to an overinflated rate of comorbidity between ADHD and BSD. The issue of the boundaries between ADHD and BSD is still difficult to solve, given the partial overlap of symptoms, namely when ED is a prominent feature. Whether ED is an associated feature or a specifier of ADHD—which defines a specific subtype of the disorder—or even a core feature of the disorder—additional to hyperactivity/impulsivity and inattention—or, finally, a comorbidity, is still a matter of discussion . Recent One of the most troublesome issues in the assessment of dysregulated children is the availability of cost-effective and reliable diagnostic measures. To date, the Child Behavior Checklist (CBCL), one of the most used instruments for the assessment of developmental psychopathology , has beeAlthough ED is a multidimensional concept, including emotional reactivity and impulsivity, affective intensity and polarity—both positive or elated and negative or irritable—and behavioral self-control, CBCL does not allow clinicians to disentangle these different components, which may be different in different subjects. Assessing all these components may need different measures, which are currently unavailable in youth , 36. Howr = 0.71–0.84) and internal consistency (Cronbach's α = 0.72–0.95) were high, and concurrent validity was also supported by correlations with the brief TEMPS-M subscales (p <0.001) since cyclothymic and ADHD patients exhibited higher scores than non-clinical controls for each subscale, except for PE.Starting from 60 items concerning reactivity, intensity, polarity of emotional responses, and affective stability, a first validation in both clinical and non-clinical adult subjects led to a 40-item version with four scales . The fouubscales . Discrima priori hypotheses on each single dimension of the construct, since no previous clinical study applied the RIPoSt questionnaire in youths. Nonetheless, according to the theoretical model proposed by Banaschewski et al.  than in ADHD or BSD alone patients and that all clinical groups score higher than a control group of healthy adolescents in all subscales of the questionnaire. We lack specific i et al. , we may Our study included 154 participants  recruited in our third-level Department of Child and Adolescent Psychiatry and Psychopharmacology from 2017 to 2020 . Inclusion criteria were diagnoses of ADHD, BSD, or both, made according to the Diagnostic and Statistical Manual of Mental Disorders—fifth edition (DSM-5) , based oExclusion criteria for the CG were as follows: older than 18 years old or younger than 9 years old; presence of comorbid intellectual disability, as detected through formal psychometric assessment ; and presence of comorbid autism spectrum disorders, schizophrenia spectrum, and other psychotic disorders.Three clinical subgroups were identified in the CG: the ADHD group , consisting of 72 subjects ; the BSD group , consisting of 53 subjects ; and the comorbid ADHD + BSD group, consisting of 29 subjects .A school sample of 40 healthy control adolescents (HC group)  was recruited on a voluntary basis upon engagement of a nearby junior high school in Pisa. Exclusion criteria for the HC group were as follows: older than 18 years old or younger than 9 years old, presence of intellectual disability, and presence of any psychiatric disorder.All participants and parents were informed about assessment instruments, and there was voluntary participation in the study after written informed consent was obtained for assessment procedures from the parents of all children. The institutional review board of our hospital approved the study.t scores of the following subscales, anxious/depression, attention problems, and aggressive behaviors. The reliability coefficients (Cronbach's α) were 0.82, 0.81, and 0.82, respectively.A clinical questionnaire, the Child Behavior Checklist, was used in the both CG and HC samples to support clinical assessment and diagnostic procedures. The Child Behavior Checklist for ages 6–18 years (CBCL-6/18) , 44 is ar = 0.71–0.84) and good-to-excellent internal consistency (Cronbach's α = 0.72–0.95). Concurrent and discriminant validity were also demonstrated to be significant. Thus, the RIPoSt-40 questionnaire proved to be a valid, reliable, and useful tool to assess emotional dysregulation, both in clinical and non-clinical contexts.CG and HC were also assessed by means of the Italian 40-item version of the Reactivity, Intensity, Polarity and Stability (RIPoSt-40) questionnaire , 38, a s* interquartile range) and (third quartile + 2 * interquartile range) and removed. Cronbach's alphas were computed as measures of internal consistency of each subscale of the RIPoSt-40 questionnaire. The χ2 test was used to detect significant differences (p <0.05) between the three clinical groups and the HC group in the distributions of demographic and clinical nominal categorical variables, such as gender and clinical comorbidities. When more than 20% of observations had expected frequencies <5, Fisher's exact test was performed. Analyses of covariance (ANCOVA) were conducted to assess significant differences (p <0.05) between group means in the demographic and clinical variables with continuous distribution, such as subscale scores of the RIPoSt-40 questionnaire while controlling for gender as covariate. A Tukey post hoc test was used whenever ANCOVA led to a statistically significant result in order to identify significant comparisons between couples of groups.Statistical analyses were performed by means of MATLAB® and RStudio® software. For each clinical variable with continuous distribution, outliers were defined as observations lying outside the range between  as covariates.post hoc comparisons are detailed in the table legend. Clinical comorbidities also significantly differed, with the BSD group exhibiting the greatest mean number of comorbid psychiatric conditions, followed by the ADHD + BSD, and then by the ADHD. Specific comorbidities, according to DSM-5, are listed in Our sample included 194 participants, of which 154 were in the CG  and 40 in the HC group. Demographic and clinical characteristics of the four groups are reported in Internal consistency of the RIPoSt-40 questionnaire was initially assessed by computing Cronbach's α coefficients for each subscale. Cronbach's coefficients were generally high for most subscales , except for PE, whose internal consistency was still good (PE: α = 0.814). An excellent reliability value was identified for the NED subscale (NED: α = 0.946).Post hoc comparisons are detailed in the table legend. Notably, the BSD and ADHD + BSD groups scored the highest in the AI, NE, and NED subscales, without significant differences between the groups, and the ADHD group presented significantly lower scores in the three scales, but higher than the HC group. As for the EI subscale, the ADHD + BSD group scored the highest, followed by the BSD and the ADHD groups, which did not differ significantly between them, and finally the HC group, with significantly lower scores. Post hoc comparisons between males and females in the RIPoSt-40 questionnaire subscales revealed highly significant gender-related differences for the AI, NE, and NED subscales, with females scoring higher than males (data not shown).We then compared the RIPoSt-40 subscale scores between the three CG and the HC through ANCOVAs, while correcting for gender distributions as covariate. Age was also initially assessed through a linear multivariate model, though displaying no significant effects on any of the questionnaire subscales and not altering the effect of the other variables of the model; thus, we decided to remove it from the analyses. As shown in r ranging between 0.660 for the correlation between AI and EI and 0.829 for the correlation between AI and NE. The PE subscale was also positively correlated, though less significantly, with the AI, EI, and NE subscales, with coefficients r between 0.184 for the correlation with NE and 0.227 for the correlation with EI. Significantly positive correlations were finally identified between the NED and other subscales of the questionnaire.As shown in We also estimated linear correlation coefficients to detect significant relationships between the RIPoSt-40 and the CBCL-6/18 subscales. The AI, EI, NE, and NED subscales of the RIPoSt-40 questionnaire were highly significantly positively correlated with all syndromes and problems subscales and most DSM-oriented diagnostic category subscales, while the only significant negative relationships of the PE subscale were identified with the anxious/depressed, the withdrawn/depressed, and the affective problems subscales. Notably, the dysregulation profile index of the CBCL-6/18 was highly positively associated with the AI, EI, NE, and NED subscales, while no significant correlation was detected with the PE subscale. Correlation coefficients and statistics are detailed in Five linear multivariate regression models were finally applied to identify statistical associations between the subscales of the RIPoSt-40 questionnaire, as dependent variables, and the presence of psychiatric comorbidities , as independent variables, while controlling for the principal diagnoses (ADHD and BSD). As displayed in This is the first study aimed to explore ED in a clinical sample of children and adolescents using a specific measure, the RIPoSt questionnaire, which includes four dimensions of dysregulation, that is affective instability, emotional impulsivity, negative emotionality, positive emotionality, and the negative emotional dysregulation derived from the sum of the first three dimensions. The first aim of our study was to explore the different dimensions of ED in youth with ADHD, BSD, and the two comorbid conditions. The secondary aims of the study were to preliminarily explore the psychometric characteristics of the RIPoSt questionnaire and to compare this measure with a well-established dimensional measure of psychopathology in youth, the CBCL-6/18, and more specifically with the CBCL-Dysregulation Profile, derived from the three symptom scales of the instrument.The boundaries between ADHD and BSD raised a controversy in the literature, given the partial overlap of symptoms, such as hyperactivity, impulsivity/aggressiveness, and distractibility, particularly when ED is associated. Indeed, when this latter is prominent, inflated rates of comorbidity between the two disorders have been reported in the literature . The greOur findings indicate that AI and NE, as well as the combined NED scale, are mostly related to the BSD, both pure and with ADHD, and can reliably differentiate these conditions from pure ADHD. Similarly, these three dimensions are able to discriminate the dysregulated profile of ADHD youth compared with the heathy controls. A more nuanced difference was shown with emotional impulsivity, which was found to be similar in ADHD and BSD alone and higher in the comorbid condition, and notably, all clinical groups exhibited higher scores than the healthy controls.On the other hand, PE is unable to differentiate clinical and healthy groups and seems a sort of temperamental dimension, which can be found in both patients and healthy individuals, without a significant impairing effect. Moreover, it seems more difficult to be detected, at least compared with NE, and would thus require larger sample size to achieve statistical significance . Also, dA comparison between these results and those reported in a parallel study, conducted on adult patients explored with the same diagnostic tool , is highOur findings also provide a preliminary support to the construct and concurrent validity of the 40-item version of the RIPoSt questionnaire to assess ED. Indeed, the instrument proved to have good-to-excellent internal consistency in both clinical and non-clinical samples. Cronbach's coefficients were high for all subscales and for their combination in the NED subscale, while they were lesser, though still good, for the PE subscale. Furthermore, consistent with clinical findings, the AI, NE, and NED subscales were strongly and positively correlated with each other, while PE was more feebly correlated with the other three dimensions.Construct validity was also assessed in terms of gender-related differences. Indeed, males and females significantly differed in both clinical and non-clinical samples. As expected, girls scored higher in most ED dimensions, namely the AI, NE, and NED subscales, while EI was similar across gender. Moreover, gender differences were also detected in the relative distribution among groups, with ADHD exhibiting strong male prevalence and BSD with an even gender distribution. On the contrary, no age effect was found for any of the dimensions of dysregulation.Correlations between RIPoSt and CBCL subscales further supported the concurrent validity of the new instrument. Indeed, the significant positive correlation between the AI, EI, NE, and NED subscales of the RIPoSt-40 and all syndromes and problems subscales and most DSM-oriented diagnostic category subscales of the CBCL-6/18, but especially their strongest and most significant correlations with the CBCL-DP, indicates that the four subscales and their combination can be used as an effective tool for studying ED in different clinical conditions. On the contrary, PE was limitedly correlated with the anxious/depressed, withdrawal/depressed, and affective problems subscales; thus, it seems to be only related with the affective dimensions of the CBCL, and notably, it did not exhibit a significant correlation with CBCL-DP.Finally, when comorbidities were also taken into account, the AI, NE, and NED subscales presented a positive significant association with multiple anxiety disorder, which has been repeatedly found as a possible precursor of and frequently associated with BSD –49. UnexThis study should be considered preliminary, given some significant limitations: first of all, the lack of a formal standardization of the RIPoSt questionnaire in young people. Indeed, psychometric validation of multiple-item scales is considered to be an integral part, if not a crucial step, of data analysis in most substantive research studies . We largDespite these limitations, our study paved the way for future directions of research in clinical practice. Indeed, a thorough validation of the RIPoSt questionnaire along with an assessment of its psychometric properties is warranted. Our results also need to be further corroborated in larger samples. As pointed out before, the RIPoSt represents a potential clinical tool that may help in disentangling the complex relationship between ADHD and juvenile BSD for better diagnostic characterization and therapeutic indications. Future studies may further explore the longitudinal course of emotional dysregulation in these two partially overlapping disorders and assess the changes in their ED profile after psychopharmacological interventions. Moreover, the questionnaire may be used in the frame of evidence-based psychotherapeutic settings for psychopathological conditions characterized by ED to monitor the clinical course of its different dimensions and provide further evidence of effectiveness. Finally, the assessment of ED dimensions may be also useful in adolescents with conduct disorders, especially comorbid with ADHD, to further characterize the complex relationship between emotional regulation and executive functioning.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by IRCCS Stella Maris Scientific Institute of Child Neurology and Psychiatry, Pisa, Italy. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.GM and AMi: conceptualization. GM, AMi, GS, and SP: methodology. CP, EV, AMo, FP, SB, NL, FD, and ARM: data collection and discussion on the first draft and conclusions. GS and SP: statistical analyses. GM, GS, AMi, and SP: writing first draft. All authors have read and agreed to the published version of the manuscript.GM has received research grants from Lundbeck and Humana, was in an advisory board for Angelini, and has been speaker for Angelini, FB Health, Janssen, Lundbeck, and Otsuka. The remaining 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.