Bartonella, Rickettsia, and apicomplexan parasites  in fleas feeding on small mammals from three different habitat types  in Slovakia. The most common pathogen in the examined fleas was Bartonella spp. , followed by Rickettsia spp.  and apicomplexan parasites . Bartonella strains belonging to B. taylorii, B. grahamii, B. elizabethae, Bartonella sp. wbs11, and B. rochalimae clades were identified in Ctenophthalmus agyrtes, C. congener, C. assimilis, C. sciurorum, C. solutus, C. bisoctodentatus, Palaeopsylla similis, Megabothris turbidus, and Nosopsyllus fasciatus within all habitats. The presence of Rickettsia helvetica, R. monacensis, and rickettsiae, belonging to the R. akari and R. felis clusters, and endosymbionts with a 96–100% identity with the Rickettsia endosymbiont of Nosopsyllus laeviceps laeviceps were also revealed in C. agyrtes, C. solutus, C. assimilis, C. congener, M. turbidus, and N. fasciatus. Babesia and Hepatozoon DNA was detected in the fleas from all habitat types. Hepatozoon sp. was detected in C. agyrtes, C. assimilis, and M. turbidus, while Babesia microti was identified from C. agyrtes, C. congener, and P. similis. The present study demonstrated the presence of zoonotic pathogens in fleas, parasitizing the wild-living small mammals of southwestern and central Slovakia and widens our knowledge of the ecology and genomic diversity of Bartonella, Rickettsia, Babesia, and Hepatozoon.Fleas (Siphonaptera) as obligate, blood-feeding ectoparasites are, together with ticks, hosted by small mammals and can transmit causative agents of serious infections. This study aimed to determine and characterize the presence and genetic diversity of  Fleas (Siphonaptera) are obligate, blood-feeding ectoparasites. They are both vectors and hosts to pathogenic microorganisms and parasites of medical and veterinary importance, playing a role in their dispersal. Pathogens and parasites can be transmitted by fleas by the oral route through the regurgitation of blood meals, by the fecal route through contaminated fecal pellets and in some cases, hosts acquire infection by consuming infected fleas . Fleas cYersinia pestis. Fleas are known vectors of Rickettsia typhi, which causes murine typhus, and Bartonella henselae, which causes cat scratch disease, and also play a role in the transmission of epidemic typhus caused by Rickettsia prowazekii. Flea-borne spotted fever and its agent Rickettsia felis occur throughout the world. Additionally, fleas host endoparasitic helminths [Rickettsia and Babesia and are carriers of Bartonella species. Fleas can potentially ingest tick-borne microorganisms from infected hosts or acquire them through co-feeding with ticks on the same host [The most serious human infection spread by fleas is plague, which is caused by elminths . The majame host ,3. HowevBartonella species are Gram-negative bacteria and are of medical and veterinary importance. They infect the erythrocytes and endothelial cells of mammals. Bartonella henselae, B. quintana, and B. bacilliformis have been found to be responsible for the majority of human bartonellosis cases, whereby B. henselae is the causative agent of the well-known zoonotic infection called cat scratch disease [Bartonella are transmitted through the bite or scratch of infected animals or by different ectoparasitic arthropods, including fleas, lice, and sand flies. The vector role of ticks and mites or of biting and ectoparasitic dipterans have been suggested but not definitely proven [Ctenocephalides felis) is the main vector for B. henselae. Other flea species may also transmit B. henselae or other Bartonella spp.; however, their vector role needs to be confirmed by vector competence studies [Bartonella spp. in Europe [B. henselae and anti-B. quintana antibodies were detected in 23.5% and 24.8% of the examined humans, respectively [Bartonella taylorii, B. rochalimae, B. elizabethae, B. grahamii, B. birtlesii, and Bartonella sp. wbs11 clades was demonstrated in the spleens of small mammals in Slovakia, including those from which the fleas examined in the present study were collected [ disease . Bartoney proven . The cat studies . Cats, dn Europe . In Slovectively . Presencollected .Rickettsia species are obligate intracellular Gram-negative bacteria and are responsible for mild to severe diseases in humans, with a worldwide distribution [Rickettsia helvetica, R. monacensis, Candidatus Rickettsia mendelii, and Candidatus Rickettsia vini, were identified in host-seeking and host-feeding Ixodes ricinus ticks from different habitats [R. helvetica and R. monacensis was also confirmed in the tissue samples of small rodents  [Rickettsia slovaca and Rickettsia raoultii were detected in Dermacentor marginatus and Dermacentor reticulatus ticks [Haemaphysalis inermis ticks, Candidatus Rickettsia hungarica was identified in Slovakia for the first time in 2018 [Rickettsia helvetica, unidentified Rickettsia sp., and rickettsial endosymbionts were detected [Rickettsia felis DNA was reported for the first time in a Ctenophthalmus solutus flea collected from Apodemus agrarius [Rickettsia sp., closely related to R. felis, was detected in A. flavicollis from the same region [R. africae, usually transmitted by ticks, was identified in Ceratophyllus garei fleas collected after a blood meal on reed warblers  that were crossing Slovakia during their spring migration [ribution . They arhabitats ,13,14,15arvalis) , and Ricus ticks ,18. In Hdetected ,19,20, ae region . The preigration .Babesia. These parasites infect a wide range of domestic and wild animals and are transmitted by ixodid ticks of several genera [Babesia microti (strain Jena) and B. venatorum were identified in host-seeking I. ricinus and Babesia canis in D. reticulatus ticks in southwestern (SW) Slovakia [B. microti was also found in rodents and rodent-attached I. ricinus ticks [B. microti is considered a tick-borne pathogen, its presence has been sporadically detected, e.g., in fleas collected from cats and dogs in Poland [Myodes glareolus) in SW Slovakia were found infected with two genetic variants of Hepatozoon sp., Hepatozoon sp. SK1, and Hepatozoon sp. SK2 [Hepatozoon erhardovae, a common blood parasite of bank voles, which is associated with fleas [Babesiosis is a tick-borne zoonotic disease caused by protozoan parasites of the genus l genera . In prevSlovakia ,23. The n Poland  or in fl sp. SK2 . These cth fleas ,27.Bartonella, Rickettsia, Babesia, and Hepatozoon species and determine their potential role in the transmission of etiological agents of zoonotic diseases in three habitat types in Slovakia: Bratislava forest park and a patch of deciduous forest remnants in the campus of the Slovak Academy of Sciences (suburban), a deciduous forest at Fúgelka, located in the Small Carpathian Mountains , and a forest-steppe rural area in the Prievidza district.In the present study, we aimed to examine fleas collected from wild rodents for the presence of In total, 734 flea specimens from 12 species were recovered from the captured rodents: 434 in the suburban habitat, 257 in the natural habitat, and 43 in the rural habitat . Seven sBartonella spp. , followed by Rickettsia spp.  and Babesia and Hepatozoon spp. . The same order of infection rates was found in the rural habitat: Bartonella spp. , and Babesia and Hepatozoon spp. . However, in the natural habitat, a higher proportion of the fleas were infected with Babesia and Hepatozoon spp.  than with Rickettsia . In the suburban habitat, the prevalence of Rickettsia spp.  was higher than that of Bartonella spp. . There was no significant difference between the infection rates in flea males and females. Details are provided below and summarized in A total of 665 fleas represented by 12 species were analyzed for the presence of pathogenic microorganisms . The mosBartonella DNA was detected in 226 fleas . The difference in Bartonella overall prevalence, between flea males and females, was not statistically significant (p = 0.6214). The overall infection rate was 33.98%, ranging from 22.94% in the suburban habitat to 80% (95% CI: 68.31–91.69) in the rural habitat (p < 0.001). The difference in prevalence between males and females was not significant in any of the studied habitats . In the suburban and natural habitats, C. congener was the most infected species with Bartonella, 35.9% (95% CI: 20.84–50.95) and 48.28% (95% CI: 30.09–66.46), respectively, when not considering species with low numbers  and Megabothris turbidus (1 specimen) were positive for Bartonella DNA. Besides, high infection rates were recorded in Ctenophtalmus solutus  and in Nosopsyllus fasciatus . habitat . Infecti numbers . In the Bartonella strains belonged to multiple clusters. Bartonella spp. in the fleas from all three habitats belonged to four clusters: B. taylorii, B. grahamii, Bartonella sp. wbs11, and B. rochalimae. In addition, in fleas from the rural habitat, Bartonella, belonging to the B. elizabethae cluster, were identified. Sequences from this study are identical to those previously identified in rodents from the same study sites [B. taylorii cluster . The number of base substitutions in the sequences, per habitat, are shown in Sequence analysis of the 16S–23S rRNA intergenic spacer region in 145 randomly selected flea DNA lysates showed that the dy sites  and wereC. solutus specimens from the suburban habitat, 3 C. agyrtes from the natural habitat, and 2 C. assimilis, 4 C. solutus, and 1 C. agyrtes from the rural habitat) belonged to the cluster of uncultured Bartonella sp. (DQ155376) and uncultured Bartonella sp. clone PD134, PD135, PD139 (MF039574- MF039576) identified in the rodents from the rural habitat [B. rochalimae  and B. clarridgeiae . The sequence from a C. solutus specimen from the suburban habitat (MK239961) was 85% identical to the uncultured Bartonella sp. clone PD174 (MF039578) [B. rochalimae (KU292577)  identified in the blood of Apodemus sylvaticus from the United Kingdom [Bartonella sp. clone PD130 (MF039573) and clone FU241 (MF039566), originating from rodent samples from the natural and rural habitats of Slovakia [C. agyrtes from the natural habitat and 1 C. sciurorum from the suburban habitat) were 95–98% identical with B. doshiae (AJ269786). In the next cluster, five sequences  were 98–100% identical to B. grahamii (AJ269785), which was previously identified in the blood of woodland rodents in the United Kingdom [Bartonella sp. clone FU80 (MF039560) isolated from the spleen of Apodemus flavicollis and Myodes glareolus found in the natural habitat [C. agyrtes specimen from the natural habitat, the sequence (MK239960) was 91% identical to B. grahamii and uncultured Bartonella sp. FU80.The next cluster was created by eight sequences derived from one  Kingdom , and an  Kingdom  and to tN. fasciatus specimens and one C. assimilis specimen from the rural habitat were 99–100% identical to B. elizabethae (JF766264) isolated from Meriones libycus heart tissue in Georgia [Bartonella sp. clone PD173 (MF039577) isolated from the spleen of A. flavicollis trapped in a rural habitat [Four sequences derived from three  Georgia  and to uRickettsia DNA was found in 127 fleas , with a total prevalence of 19.1% without a significant difference (p = 0.620) between the males and females. The overall infection rate ranged from 0.86% in the natural habitat to 57.78% (95% CI: 43.35–72.21) in the rural habitat and 25.52% in the suburban habitat . The infection rates differed significantly between all habitats (p < 0.001). In the suburban and rural habitats, C. solutus was the most infected species , followed by C. agyrtes  and N. fasciatus , respectively. C. agyrtes was the only species infected with rickettsiae in the natural habitat, with a prevalence of 1.03% (95% CI: 0–2.45).The presence of  habitat . The difgltA gene in Rickettsia–positive fleas showed that the sequences from eight specimens  from the suburban habitat, two C. agyrtes specimens from the natural habitat, and three specimens  from the rural habitat, were identical with Rickettsia helvetica M31 IR SAV (MF673863) and R. helvetica (KY488349)  ; however, amplification of the fragments of the ompA, sca4, and 17-kDa genes was not successful. A fragment of the gltA gene from a C. agyrtes specimen from the suburban habitat was identical with Rickettsia monacensis isolate 31IRM BA (KF258159), previously identified in a host-seeking Ixodes ricinus male collected in Bratislava [gltA and 17-kDA genes from a C. solutus specimen from the suburban habitat belonged to the cluster of Rickettsia slovaca, R. raoultii, R. conori, R. sibirica  , and also with a Rickettsia endosymbiont of Empis bicuspidata (JQ925616), Gymnopternus brevicornis (JQ925600), Gymnopternus metallicus (JQ925549), Argyra vestita (JQ925587), and Tetranychus urticae (KP828066)  , but ompP828066) . UnfortuBabesia and Hepatozoon DNA were detected in 29 fleas , with a total prevalence of 4.36% (p = 1.0). The highest prevalence  was documented in fleas from the natural habitat, followed by the rural habitat  and the suburban habitat . Prevalence differed significantly only between the natural and suburban habitats (p < 0.001). Among the more abundant species, C. agyrtes was the most infected in the natural habitat , followed by C. congener , whereas in the suburban habitat, the infection rates of these two species were similar . In the rural habitat, C. assimilis was the only infected species .of 4.36% . The difBabesia microti and Hepatozoon sp. were identified in 15 (51.72%) and 14 (48.23%) positive fleas, respectively. Babesia microti was detected in five C. agyrtes specimens from the suburban habitat and six C. agyrtes specimens from the natural habitat, with one and two C. congener specimens from the suburban and the natural habitat found, respectively, and also in one P. simillis specimen from the natural habitat. Hepatozoon sp. was found in fleas from all studied habitats . Sequence analysis of the amplified 18S rRNA fragments from the 15 isolates in the fleas revealed a 100% identity with the sequences of B. microti (strain Jena) from host-seeking and rodent-attached I. ricinus ticks  and rodents  originating from the same study sites in the suburban and natural habitats [Hepatozoon sp. SK1, previously identified in M. glareolus from the suburban (KU597243) and natural (KU597245) habitats [nd 14 48.% positivhabitats .Bartonella spp. and rickettsial endosymbionts. The co-infection of three pathogens  was recorded only in a C. agyrtes specimen from the suburban habitat.In all types of habitats, the co-infections of two pathogens were recorded . The totBartonella strains belonging to B. taylorii, B. grahamii, B. elizabethae, and Bartonella sp. wbs11, and B. rochalimae clades, R. helvetica, R. monacensis, and rickettsiae, belonging to R. akari and R. felis clusters and the cluster of R. slovaca, R. raoultii, R. conorii, and R. sibirica, endosymbionts, finding an identity with a Rickettsia endosymbiont of Nosopsyllus laeviceps laeviceps, B. microti, and Hepatozoon sp. in fleas feeding on small rodents.The investigation of communities of fleas that have infested different mammal and bird species has a long tradition in Slovakia, while studies about zoonotic pathogens transmitted and/or carried by fleas are limited. With regards to the flea species spectrum, prevalence, and intensity of infestation in small rodents, we confirmed the previous findings ,31,32 anHaemaphysalis concinna, I. ricinus, and D. reticulatus ticks and Ixodes trianguliceps, I. ricinus, H. concinna, and D. reticulatus ticks, collected from vertebrate hosts as well as blood and tissue samples from small mammals, birds, and free-living ungulates, searching for the presence of rickettsiae, bartonellae, and babesiae [The ecological and bacteriological observations of small mammals and their ectoparasites from different regions and habitat types in Slovakia have shown abundant and diverse communities of microorganisms. In our previous studies, which comprise the same habitats as the present study, we focused mainly on ticks, and analyzed host-seeking babesiae ,13,14,33Bartonella spp. in fleas corresponds with the species spectrum found in rodent spleen samples [Bartonella spp. between small rodents and fleas in the explored habitats, where all analyzed host-seeking I. ricinus ticks were negative [Bartonella spp. in rodents was 64.8%; the highest rate of 73.8% was found in the natural habitat, and the lowest one (56.0%) in the suburban habitat [Bartonella taylorii was the most common species identified in the fleas from this study, similarly to the rodents from Slovakia [Bartonella strains belonging to the B. grahamii, B. elizabethae, and B. rochalimae clades, which were detected in both fleas and rodents from Slovakia, may also represent a potential risk for humans. For example, B. grahamii is a causative agent of human renitis [B. elizabethae causes endocarditis [B. rochalimae causing chronic intra-erythrocytic infections in mammals and infectious endocarditis in dogs [Bartonella species in the fleas from our study is comparable with the diversity in fleas from rodents in Germany [Bartonella spp. In fleas and rodents, which has been demonstrated in Slovakia, as well as in other European countries, supports the assumption that fleas are involved in maintaining the infection cycle in nature. Fleas are capable of serving as reservoirs and vectors for bacteria in different habitat types, and this is also supported by the results of Morick et al. [Bartonella for at least 89 days, and fleas could acquire Bartonella from wild rodents but could not transmit them transovarially.Our findings on the diversity of  samples , which pnegative . The ove habitat . In the Slovakia  and the Slovakia ,35,36. T renitis , and B. carditis , with B. in dogs  and can  in dogs . The divk et al. , who shoRickettsia infections have been analyzed in the tissue and/or blood samples of rodents and in their ectoparasites. Rickettsiae in rodent ectoparasites could be acquired via blood meals from the infected hosts or by co-feeding from infected parasites. The prevalence of Rickettsia spp. was found in a range of 2–43% in different groups of ectoparasites [Rickettsia-positive [Rickettsia helvetica was the most frequently identified species in the fleas, which is in accordance with previous findings [R. monacensis, R. felis, and rickettsiae belonging to cluster of R. slovaca, R. raoultii and rickettsial ensymbionts were also identified in the ectoparasites in the above-mentioned studies, but detection of bacterial DNA in these fleas may be the result of an accidental uptake through feeding on infected hosts [C. solutus, C. agyrtes, and N. fasciatus, which are dominant parasites of the Apodemus species and M. glareolus in lower vegetation zones [Rickettsia species mentioned above is still unclear and needs further investigation.The reservoir role of wild rodents in the rickettsiae life cycle has not been definitively confirmed, although it has been investigated across Europe. arasites ,46,47,48positive ,20,48. Rfindings ,47,48 aned hosts . Ricketton zones ,50. HoweB. microti, and particularly of the bank vole M. glareolus for Hepatozoon sp., has been confirmed in previous studies [I. ricinus ticks with B. microti has been demonstrated using the same study sites as those included in the present study [B. microti was found in fleas in Slovakia for the first time, detections of B. microti DNA in fleas changing hosts and feeding on rodents in natural foci of the parasite is not surprising. Yet this does not confirm either the vector or the reservoir role of fleas. On the other hand, fleas are known as definitive hosts for the sexual development of Hepatozoon spp., while rodents serve as intermediate hosts for the asexual development of the parasite. Hepatozoon erhardovae belongs to the most common apicomplexan blood and tissue parasites in the bank voles of Europe [Hepatozoon sp. was also confirmed in bank voles from the study sites in SW Slovakia [Hepatozoon sp. in Hungary and Slovakia was the same . Overall, our results confirmed previous findings on the role of fleas in the transmission cycle of Hepatozoon spp. infecting rodents.The role of rodents as a reservoir for  studies ,52. The nt study . Althougf Europe . For exaf Europe . The samSlovakia . The speQuercus petraea), with European hornbeam (Carpinus betulus) dominating at lower altitudes and European beech (Fagus sylvatica) dominating at higher altitudes. Bratislava, the recreational forest city park Železná studnička, and the campus of the Slovak Academy of Sciences  represent a suburban habitat with significant anthropogenic impact. The natural habitat at Fúgelka (336–436 m asl) is set in a deciduous forest in the Small Carpathian Mountains. The Prievidza district is located in central Slovakia (289 m asl) and represents a forest-steppe rural area with Carpathian oak-hornbeam woods.Rodents and rodent-attached ectoparasites were collected from three study sites in Slovakia. The locations were described in detail in Kazimírová et al.  and BertApodemus flavicollis , wood mouse Apodemus sylvaticus , bank vole Myodes glareolus , common vole Microtus arvalis , European pine vole Microtus subterraneus , and Eurasian harvest mouse Micromys minutus , were live-trapped during April–June and September–October, 2012–2014, in the suburban and natural habitats, and during May–July, and September–October, 2012–2013, in the rural habitat, using Sherman live traps baited with oat flakes . Rodentails see .Bartonella spp., Rickettsia spp., Babesia spp. and Hepatozoon spp.  were selected for molecular analyses. In total, 665 flea specimens  were analyzed. Fleas were washed with sterile water, dried, transferred to individual tubes, and crushed with a sterile carbon steel surgical scalpel blade . DNA from the fleas was extracted using Dneasy Blood and Tissue Kit (Qiagen) or NucleoSpin Tissue Kit (Macherey Nagel) following the manufacturer’s recommendations. The quantity and quality of DNA was assessed by NanoPhotometer Pearl . DNA samples were stored at −20 °C and later used as templates for the PCR amplifications. Flea samples were screened by PCR-based methods for the presence of Bartonella spp. DNA was examined by a PCR assay targeting the 16S–23S rRNA gene intergenic spacer region (ITS), which generated a product of 420–780 bp [Bartonella spp. DNAs from infected rodents confirmed by sequencing, obtained in a previous study [The presence of 0–780 bp . Positivus study ), and negltA), 17-kDa antigen gene (17-kDa), outer membrane protein A gene (ompA), and PS120-protein encoding gene (gene D) were used to identify the species of Rickettsia [Rickettsia-free ticks and nuclease-free water were used as negative controls in each reaction. DNAs from R. helvetica and R. slovaca, originating from ticks and previously sequenced, were used as positive controls. Additionally, the 23S rRNA gene was used to confirm the presence of R. helvetica in Rickettsia-positive flea samples using TaqMan PCR assay [0–3 × 106 target copies with a sensitivity of 3 copies of the DNA.Four sets of primers for amplifying the citrate synthase gene , and nuBartonella-positive, Rickettsia-positive, and Babesia-positive amplicons were purified and analyzed via sequencing in both directions using Macrogen Inc. . The DNA sequences were compared with those available in GenBank using the Basic Local Alignment Search Tool (BLAST) . New sequences generated in this study were submitted to the GenBank database under accession numbers MK239960-MK239961 and MH784529-MH784535 . A p Value < 0.05 was considered significant. Ninety-five percent confidence intervals (CI) were calculated using an online calculator .The prevalence and mean intensity of infestation (of rodents by fleas) were calculated according to Margolis et al. . DiffereBartonella, Rickettsia, Babesia, and Hepatozoon. Bartonella spp., identified in fleas, are the same as those detected previously in small mammals from the same study sites, suggesting that fleas are involved in the circulation of these bacteria in natural foci. On the other hand, our findings on the presence of tick-borne Rickettsia spp. and Babesia microti in fleas are not sufficient to prove the role of fleas in the transmission cycles. Still, the presence of zoonotic Rickettsia and Babesia species in fleas should be taken with caution. Ectoparasites from free-living and domestic animals should be screened continuously to detect new and emerging infectious agents and discover their role in the circulation of different vector-borne pathogens. Although no flea species is specifically associated with humans, fleas, together with ticks and other ectoparasites, live in close association with domestic animals and humans, who may come into contact with potentially infected fleas and acquire zoonotic infections.The present study provides evidence of the presence of zoonotic pathogens in fleas parasitizing the wild-living small mammals of southwestern and central Slovakia and contributes to our knowledge of the ecology and genomic diversity of


N = 4121). ADHD diagnosis was derived using the Barkley Adult ADHD Rating Scale-IV. Analyses suggested that the self-rated SDQ ADHD subscale had high validity in distinguishing ADHD cases/non-cases in young adulthood  and indicated a lower cut-point for identifying those who may have an ADHD diagnosis in this age group compared to that currently recommended for younger ages. Findings were similar for parent-reports. Our findings suggest that the SDQ is suitable for ADHD research across different developmental periods, which will aid the robust investigation of ADHD from childhood to young adulthood.Attention Deficit Hyperactivity Disorder (ADHD) symptoms typically onset early and persist into adulthood for many. Robust investigation of symptom continuity and discontinuity requires repeated assessments using the same measure, but research is lacking into whether measures used to assess ADHD symptoms in childhood are also valid in adulthood. The Strengths and Difficulties Questionnaire (SDQ) is widely used to assess ADHD symptoms in children, but little is known about its utility in adulthood. The aim of this study was to assess the validity of the SDQ hyperactivity/ADHD subscale to distinguish between cases and non-cases of DSM-5 ADHD at age 25 years in a UK population cohort ( Symptoms tend to decline across childhood and adolescence, but around 65% of individuals with a childhood diagnosis are estimated to show some level of symptom persistence into adulthood  and estiGiven the life-course impacts of ADHD, there is a need to be able to assess symptoms across development to monitor and investigate correlates of symptom persistence and desistence. In particular, there is growing interest in the transition from childhood and adolescence to young adulthood  - a periAdult ADHD has generally been assumed to be a continuation of childhood ADHD and this is supported by findings that adult ADHD symptoms and diagnosis show similar characteristics  when accounting for change in measure and informant . HoweverThe Strengths and Difficulties Questionnaire (SDQ) hyperactivity/ADHD subscale  is a briThe aim of this study was to examine the criterion validity of the SDQ as an assessment of ADHD symptoms in young adulthood, by examining its ability to discriminate DSM-5 ADHD cases from non-cases at age 25 years in a UK population cohort. We also explored whether a different cut-point to that recommended in childhood and adolescence is required to optimize sensitivity and specificity with respect to an adult diagnosis. Follow-up analyses included 1) separate analyses for males and females, and 2) assessing generalizability across subscales by comparing results for the ADHD subscale to the other SDQ  subscales.22.1N = 14,901). Details of this study are provided in the Supplementary Material. Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees. Informed consent for the use of data collected was obtained from participants following the recommendations of the ALSPAC Ethics and Law Committee at the time. Individuals were included in our analyses when ADHD diagnosis data were available at age 25 years  (see below): comparisons between those with and without diagnosis data are given in Supplementary Table 1. Primary analyses were conducted using self-reports as would typically be used in adult mental health services (N = 4121), with secondary analyses conducted using parent-reports (N = 4330) (see below).We analyzed data from the Avon Longitudinal Study of Parents and Children (ALSPAC), a well-established prospective, longitudinal birth cohort study   . The BAA2.3www.sdqinfo.org) total ADHD subscale scores were derived using mean imputation for those with (≤2) of SDQ items missing. Follow-up analyses investigated the other SDQ subscales of emotional problems, conduct problems, peer problems and prosocial behavior . The SDQ can be downloaded from https://www.sdqinfo.org/.Participants also completed the self-rated age 18+ version of the SDQ which includes the five item hyperactivity/ADHD subscale  range 0. In line2.4cvauroc function  curve analyses were used to examine the ability of the SDQ to distinguish between cases and non-cases of ADHD classified using the BAARS-IV. A ROC curve was plotted (sensitivity vs 1-specificity) and the area under the curve (AUC) estimated. Stata's function  was usedfunction .Sensitivity reflects the ability to correctly identify all those with a diagnosis (true positives) whereas specificity reflects the ability to correctly identify all those without a diagnosis (true negatives). Optimum cut-points for ADHD diagnosis were selected as those that best balanced sensitivity and specificity, i.e. assuming that false positives and false negatives are equally undesirable, according to maximal Youden Index (sensitivity + specificity – 1) . Given troccomp function  separately for males and females, using Stata's function  to compafunction , we test33.1p = 2.3×10–07). Mean scores for those who met DSM-5 ADHD diagnostic criteria were 6.70 (SD=1.92) compared to 2.98 (SD=2.02) for those who did not meet diagnostic criteria .The overall mean self-rated SDQ hyperactivity/ADHD subscale score at age 25 years was 3.09 (SD=2.11) with a Cronbach's alpha of 0.67, suggesting acceptable levels of internal consistency. As has been reported elsewhere , the mea3.2p = 0.89).Of those with available data, 2.9% met DSM-5 diagnostic criteria for ADHD at age 25 years (see Supplementary Table 2). At age 25 years, male sex was not associated with an increased likelihood of meeting ADHD criteria , as shown in 3.4The optimum cut-point for distinguishing ADHD cases from non-cases was ≥5, which showed high validity in correctly identifying those meeting diagnostic criteria (i.e. true positives: sensitivity=89.3%) and good validity in correctly identifying those who did not meet diagnostic criteria (i.e. true negatives: specificity=76.7%). This cut-point captured 25.3% of the sample and was associated with a very high probability that those not meeting the cut-point did not meet diagnostic criteria (NPV=99.6%) but a lower probability that those exceeding the cut-point met diagnostic criteria (PPV=10.4%).In line with previous work in younger samples  we also 3.52(1)=0.02, p = 0.89). Sensitivity and specificity values for all possible SDQ subscale cut-points are shown in When examining males and females separately, ROC curve analyses still indicated high validity in distinguishing ADHD cases from non-cases  with similar AUC values across sex : AUC=0.97 (95% CI=0.92–0.98) but with a lower optimum cut-point of ≥4, capturing 13.2% of the sample.4This study aimed to examine the validity of the SDQ as an assessment of ADHD symptoms at age 25 years in a UK population cohort. We found the hyperactivity/ADHD subscale to have high validity in distinguishing those meeting ADHD diagnostic criteria from those who did not. This suggests that the SDQ subscale, which is widely used in child and adolescent populations, is also suitable for use in young adults.Our finding of excellent validity of the self-rated SDQ for measuring ADHD in young adulthood (AUC=0.90) is similar if not somewhat higher than previous work in children and adolescents (using self- and parent-reports) , includiWe also investigated whether different cut-points are required to capture clinically relevant symptoms in young adulthood compared to younger ages. Current recommendations based on identifying the top 10% of the population suggest that in childhood and adolescence, self-rated scores of ≥7 capture those with high symptoms, with scores of 0–5 capturing those close to average . At age The selection of appropriate cut-points depends on the aim and rationale for using the instrument. We selected cut-points based on the premise that false positives and false negatives were equally undesirable , which iWhile our primary analyses focused on self-reports, we conducted secondary analyses using parent-reports (of both the SDQ and to define diagnosis). While adult mental health services may not typically involve parents, research studies often utilize parent ratings at earlier ages, making the validity of the parent-rated SDQ to assess ADHD in young adulthood important for researchers interested in assessing continuity and discontinuity, which ideally requires repeated assessments using the same measure and informant . We founThe validity of parent ratings of their young adult offspring's ADHD symptoms is consistent with recent work in this sample which found parent SDQ ratings of ADHD show similar genetic and neurodevelopmental correlates at age 25 as in childhood, suggesting that these reports capture a similar construct across development  and implOur study has a number of strengths, including the use of a large population sample and the investigation of a measure widely used in childhood and adolescence, which will enable continuity in future investigation of ADHD across different developmental periods. However, findings should also be considered in light of limitations, including non-random attrition which means those included in this sample will not be fully representative of the broader population, as those with elevated risk of psychopathology are more likely to have dropped out of this birth-cohort by the time data were collected at age 25 years . This isIn conclusion, we found the widely-used and freely-available SDQ, used in children and adolescents, to be a valid measure for assessing ADHD symptoms in young adults (at age 25 years). We identified a different, lower cut-point to help more accurately identify those who may have an ADHD diagnosis in this age group. Our findings suggest that the SDQ is suitable for ADHD research across different developmental periods, which will aid the robust investigation of ADHD from childhood to young adulthood.LR, SSA, OE, RBJ and AT conceptualised the study, LR, SSA, OE, RBJ and KL derived the variables, LR wrote the original draft, SSA performed statistical analyses, ES and AT attained the study funding. All authors contributed to the interpretation of data and reviewed/edited the manuscript.None.


Bartonella  encompasses facultative intracellular Gram-negative alphaproteobacteria that parasitize mainly erythrocytes and endothelial cells, as well as macrophages, monocytes and dendritic cells. Although they can infect numerous mammal species and arthropod vectors worldwide, reports of Bartonella infections in marsupials are scarce. In fact, such agents have only been detected in marsupials and/or associated ectoparasites in Australia and the United States of America until the present moment. The present study aimed to isolate and characterize molecularly, morphologically and phenotypically Bartonella infecting free-living marsupials sampled in the Brazilian Pantanal, the largest wetland in South America. Two marsupials were captured in December 2018 and six marsupials in February 2019, totaling eight small mammals sampled: five (62.5%) Thylamys macrurus and three (37.5%) Monodelphis domestica. All blood samples were submitted to qPCR for Bartonella spp. based on the nuoG gene, a pre-enrichment liquid culture and a chocolate agar solid culture. Bartonella sp. was isolated from 3 T. macrurus and one M. domestica. One Bartonella isolate obtained from a T. macrurus blood sample (strain 117A) that showed to be closely related to the Bartonella vinsonii complex and Bartonella machadoae was selected for whole genome sequencing using a hybrid approach based on Illumina NovaSeq and Nanopore sequencing platforms. This strain showed a genome of 2.35 Mbp, with an average C + G content of 38.8%, coding for 2013 genes, and a 29 kb plasmid with an average C + G content of 34.5%. In addition, this strain exhibited an average nucleotide identity (ANI) of 85% with Bartonella species belonging to the B. vinsonii group and 91% with B. machadoae. Phylogenomic analysis based on 291 protein coding genes shared by the genomes of 53 Bartonella species positioned this strain closely to B. machadoae. This new isolated species was named Bartonella harrusi sp. nov., which was characterized as having small capnophilic, microaerophilic and aerobic rods with an absence of pili and flagella. In conclusion, the present work describes the biochemical, phenotypic and genomic characteristics of Bartonella harrusi, a new species isolated from the T. macrurus blood samples of the Brazilian Pantanal. Finally, a review of the taxonomic classification of members of the genus Bartonella is proposed, based on the ANI values accessed by whole genome sequencing analyses.The genus  Bartonella genus comprise Gram-negative, intracellular facultative and fastidious α-2-Proteobacteria ) × 6.022 × 1023 × plasmid/μL copies [DNA from the marsupial blood samples was extracted using the InstaGene™ Matrix kit , according to the manufacturer’s recommendations. A PCR assay based on the mammalian endogenous glyceraldehyde gene 3-phosphate dehydrogenase (gapdh)  was useduoG gene  was usedBartonella sp. were submitted to previously described conventional PCR assays based on six molecular markers: 16S rRNA (400 bp) [gltA (750 bp) [groEL (752pb) [ftsZ (600pb) [rpoB (800bp) [Bartonella henselae previously detected in a cat sampled in southeastern Brazil [For molecular characterization, isolates that were positive in the qPCR for (400 bp) , gltA (7(750 bp) , the 16S(750 bp) , groEL ( (752pb) , ftsZ (6 (600pb)  and rpoB (800bp) . Bartonen Brazil  and sterBartonella sp. The sequences obtained were first submitted to a screening test using the Phred–Phrap software version 23 [Products amplified in conventional PCR assays were purified using ExoSAP-IT  and sequenced using the BigDye™ Terminator v3.1 Cycle Sequencing Kit  and the ABI PRISM 310 DNA Analyzer  . The prirsion 23 ,30 to ashttp://iqtree.cibiv.univie.ac.at/—accessed on 12 December 2021) [The BLAST program (BLASTn)  was useder 2021)  using 10er 2021) . The treer 2021) .In addition, nucleotide sequences were aligned with sequences previously deposited in GenBank and were subjected to a Split-Network-based distance analysis using SplitsTree v4.11.3 software .Bartonella isolated from a blood sample of Thylamys macrurus (strain 117A) was seeded on chocolate agar plates (Novamed) and incubated at 35 °C in duplicates, under the following conditions: (i) aerobic ; (ii) capnophilic  in a CO2 incubator ; (iii) microaerophilic  using Microaerobac® bags (Probac); and (iv) anaerobic  using Anaerobac® bags (Probac).For phenotypic characterization, a Bartonella strain 117A was submitted to light microscopy and scanning electron microscopy (SEM). For light microscopy, the isolate was stained with Gram (Hy-labs) and visualized using 100× objectives. For SEM, the Bartonella isolate (strain 117A) was collected from the chocolate agar plate and fixed in a solution containing 2% paraformaldehyde, 2.5% glutaraldehyde and 0.1 M cacodylate buffer for 1 h and then washed three times in a phosphate buffer. Then, 1% osmium tetroxide was added and the solution was incubated for 30 min. The bacteria were then dehydrated through a graded ethanol series (50–100%) and dried with a critical point dryer (EMS 850). After the critical point, the sample was fixed in metal stubs, and coated with gold (5 nm) in a Danton Vacuum Desk II. Finally, the bacterial cells were visualized by SEM. The samples were analyzed and photographed using a Zeiss Evo 10 scanning electron microscope (10–20 kV).The Bartonella strain 117A isolated from T. macrurus was tested with the identification systems BBL Crystal Enteric/Nonfermenter , API 20E and NF III (Probac), following the manufacturers’ instructions. The enzymatic catalase reaction was carried out with hydrogen peroxide (3%).For biochemical and metabolic characterization, the Bartonella isolated from the marsupial (strain 117A), 10 colony-forming units (CFU) were removed from the chocolate agar plates and deposited into 500 mL of a supplemented BAPGM medium. The inoculum was then maintained at 35 °C with 5% CO2 in a CO2/O2 culture incubator . Every 6 h, 100 µL were collected for 8 days, totaling 32 collections. This volume was subjected to a 9:1 serial dilution in a supplemented BAPGM medium (10−1 to 10−6). One hundred microliters of each of the six dilutions were plated onto chocolate agar plates with a Drigalski loop. Bacterial quantification was performed using the standard plate counting method, determining the number of colony-forming units (CFU/mL). Each plate was stored at 35 °C with 5% CO2 in a CO2/O2 culture incubator . The incubation period of the plates was 72 h and, after this period, colony-forming units per mL (CFUs/mL) were counted.To perform the growth curve of the Bartonella isolate of T. macrurus (strain 117A) previously grown on chocolate agar plates was performed using the commercial kit DNeasy® Blood and Tissue , following the manufacturers’ instructions. For genome assembly, the genomic DNA sample was sequenced by Illumina Novaseq 6000 (PE 2 × 150 bp). A total of 5,871,200 paired-end reads were obtained. These reads were trimmed with Trimmomatic [The genomic DNA extraction from the mmomatic . In addimmomatic . All themmomatic . The assmmomatic .Bartonella isolate (strain 117A) and 53 complete Bartonella genomes retrieved from the NCBI RefSeq database [The assembled genome of the new database  were usedatabase . These gdatabase  and wereBrucella abortus and Brucella ceti  were used as an outgroup.An ML analysis was inferred with the W-IQ-Tree tool  using 10Bartonella harrusi sp. Nov., Bartonella machadoae and the species of the Bartonella vinsonii complex  were calculated with OrthoANIu 1.2 [Bartonella harrusi compared with eight other plasmid sequences from the Bartonella species deposited in GenBank.The average nucleotide identities (ANI) between ANIu 1.2 . In addiBartonella isolate (strain 117A) and the 216 whole genomes of the Bartonella species retrieved from the NCBI RefSeq database [Bartonella species.The whole genome consensus sequence of the new database  were subdatabase . After fdatabase . PrimaryBartonella isolate (strain 117A) and 53 whole genomes of the Bartonella species retrieved from the NCBI RefSeq database [https://www.kbase.us/—accessed on 12 December 2021) [The assembled whole genome of the new database  were alidatabase . After aer 2021) .https://www.kbase.us/—accessed on 12 December 2021) [A search for the occurrence of carbohydrate active enzymes (CAZy) families was performed. For this purpose, the GenomeSet described in the section above was submitted to the dbCAN database for the automated annotation of the active carbohydrate enzymes  using HMer 2021) .Thylamys macrurus and three (37.5%) to the species Monodephis domestica. All eight marsupial blood DNA samples were positive in the PCR for the endogenous mammalian gapdh gene. None of the eight DNA samples extracted from marsupial blood were positive in the qPCR for Bartonella sp. based on the nuoG gene. The efficiency, R2, slope and intercept on the Y axis of the reactions ranged from 91.4% to 103.9%, 0.995 to 0.999, −3.547 to −3252 and 38.571 to 40, respectively  animals belonged to the species ectively .T. macrurus blood samples and two M. domestica blood samples) were positive in the qPCR for Bartonella sp. The number of copies of the nuoG gene fragment per microliter ranged from 5.9 × 102 to 2.93 × 106. The efficiency, R2, slope and intercept on the Y axis of the reactions ranged from 91.4% to 103.9%, 0.995 to 0.999, −3.547 to −3252 and 38.571 to 40, respectively.Out of the eight marsupial blood samples submitted to the pre-enrichment liquid BAPGM culture, four  showed the presence of Bartonella colonies on chocolate agar plates, whose identity was confirmed by qPCR for Bartonella spp. based on the nuoG gene   and one from M. domestica (strain 78A) inferred by the Maximum Likelihood method (ML) and evolutionary model TVM + F + I formed a single and monophyletic cluster. The sequences of Bartonella isolated from marsupials were positioned together with sequences previously detected in Polygenis (P.) bohlsi bohlsi fleas collected from rodents in the Pantanal, state of Mato Grosso do Sul  and in a larger clade with several sequences of Bartonella sp. previously detected in Oecomys mamorae rodents in the Brazilian Pantanal, state of Mato Grosso do Sul , rodents from the Brazilian states of Maranhão, Ceará and Rio de Janeiro , rodent-associated genotypes from Peru (KF021604) and B. machadoae (CP08714). Furthermore, this clade was closely related to the clade of Bartonella genotypes detected in bats and Streblidae bat flies (Strebla guajiro)  and Bartonella sp. previously detected in Cricetidae rodents from Peru and the USA  (The phylogenetic tree based on the sequences of the 16S rRNA C1-phy) .gltA, groEL, ftsZ, rpoB and ITS) obtained from four Bartonella isolates from two marsupials, one from the T. macrurus species  and one from the M. domestica species (strain 78A), were identical to each other. The genealogical analysis of the nucleotide sequences by Network, using the program Splitstree v4.11.3 [Bartonella isolates of T. macrurus and M. domestica, was related to Bartonella genotypes previously detected in rodent-associated fleas in the Pantanal . Cluster #2 consisted of Bartonella genotypes previously detected in rodents from the Brazilian states of Mato Grosso do Sul, Maranhão, Ceará and Rio de Janeiro , rodents from Peru (KF021604) and B. machadoae. Cluster #3 contained Bartonella genotypes previously detected in rodents from Peru and the USA . It was inert for all the tested carbohydrates  and negative for arginine, lysine and tryptophan arylamidase activities. Furthermore, it was negative for esculin, DNAse activity, β-D-galactosidase nitrate and McConkey growth. It showed sensitivity to Polymyxin B. 3.6.The B. harrusi growth curve showed moderately slow growth in the supplemented BAPGM medium. The bacteria entered the logarithmic phase 30 h after the inoculum preparation and maintained an average quantification of 107 CFU/mL until 90 h of the experiment. After this period, the bacteria entered the decline phase  and B. machadoae, respectively  presented a low quality both for completeness (below 70%) and percentages of contamination (above 15%)  A,B, and analyzed . Furthert groups . Among tt groups .Bartonella using the genome available in Genbank showed similar profiles: all species analyzed (n = 53) had the capacity to oxidize hydrogen and oxygen, as well as to fix carbon. Some Bartonella species  were not able to oxidize sulfur because they did not have related genes. The most diverse species found were the Bartonella sp. Strain HY038 and Bartonella apis, which presented in their genomes genes related to nitrogen fixation, the oxidation of urea, selenium, arsenic and halogenated compounds, a profile that was not observed in any other Bartonella species. Interestingly, the Bartonella sp. strain HY038, Bartonella apis and Bartonella tamiae showed genes related to the use of C1 compounds , 2  and 3 . Family 4 of the group of glycoside transferases was found only in a few species, including B. machadoae recently isolated from rodents in the same region where the present study was performed, but not in the Bartonella sp. strain 117A described in the present study , enzymes classified into families 24, 94 and 103 of the group of glycoside hydrolases enzymes, families 19, 29, 30, 51 and 84 of the group of glycoside transferases, and family 11 of the group of carbohydrate transferases were found in all the analyzed genomes . Howevernt study .Bartonella species analyzed, there was a difference in the number of occurrences depending on the analyzed genome, with variations from 1 to 12 genic regions  in Australia. Bartonella DNA was also detected in Ixodes tasmani ticks collected from koalas (Phascolarctos cinereus) [Candidatus Bartonella bandicootii’ and ‘Candidatus Bartonella woyliei’ were molecularly detected in fleas (Pygiopsylla tunneyi) from bandicoots (Perameles bougainville) and in fleas (Pygiopsylla hilli) and ticks  collected from woylies (BIettongia penicillata), respectively [Bartonella DNA was not detected in marsupials  in the Pantanal of Mato Grosso do Sul [Bartonella bacteremia, which might have hampered the detection of this agent by qPCR directly from the blood. The Bartonella bacteremia in the marsupials sampled by [nuoG gene. Similar results were found herein when marsupial blood DNA samples were submitted to qPCR assays. In the present study, Bartonella DNA was found in marsupial blood samples only after passage in a BAPGM liquid medium (a pre-enriched liquid culture) and the isolation of Bartonella spp. on a chocolate agar. The present work brings the first detection and isolation of Bartonella in marsupials in the Americas.There are few works in the literature that have investigated the role of marsupials as reservoirs of r et al.  isolatedinereus)  in Austrectively . When ito do Sul . This fimpled by  was probBartonella spp., as previously reported in [Bartonella spp. in dog blood samples, they found a positivity of 8.1%; on the other hand, when they performed a molecular diagnosis directly from the blood DNA samples, a positivity of 4.7% was found. Our research group has been using this diagnostic platform and showing the advantages of using the “liquid medium” for the successful diagnosis and isolation of Bartonella henselae in blood samples from cats [Bartonella machadoae from rodent blood samples sampled in the Pantanal of Mato Grosso do Sul [The use of a BAPGM pre-enrichment medium and isolation on a chocolate agar brings greater sensitivity in the diagnosis of orted in  where, wrom cats . More reo do Sul .Bartonella sp. isolated from a blood sample of T. macrurus showed microbiological and morphological characteristics identical to the B. machadoae recently described by our research group [2. Additionally, this strain showed aerobic, capnophilic and microaerophilic characteristics. It was characterized by rounded and smooth colonies (1–2 mm) after 4–5 days of plating on a chocolate agar. Similarly to B. machadoae, the strain 117A was not able to grow under anaerobic conditions. Such growth characteristics are similar to those reported in other species of the genus Bartonella, such as in B. elizabethae, B. henselae (the Houston-3 strain), B. clarridgeiae, B. quintana and B. vinsonii subsp. Vinsonii [B. kosoyi and B. krasnovii isolated from Rattus rattus and Synosternus cleopatrae fleas collected from Gerbillus andersoni, respectively, did not grow under strictly aerobic and anaerobic conditions [Regarding the phenotypic characteristics, the strain 117A of ch group , showingVinsonii . In contnditions .Bartonella strain 117A was negative for oxidase, catalase, urease, indole production, acetoin production and nitrate reduction. These characteristics were similar to those found in the biochemical profiles of the Bartonella species of the B. vinsonii complex [B. machadoae [Bartonella species: while B. krasnovii was able to ferment sucrose, citrate and arabinose, Bartonella kosoyi was able to ferment sorbitol, melibiose, rhamnose and glucose, in addition to showing a positive reaction to arginine [B. machadoae, Bartonella harrusi sp. nov. was not able to ferment the abovementioned carbohydrates [The  complex  and B. machadoae . Howeverarginine . On the hydrates .B. machadoae (unpublished data) required a shorter time to enter into the logarithmic phase and presented a higher peak of CFU/mL, remaining longer in the stationary phase, the Bartonella strain 117A needed more time to reach the highest CFU/mL count, in addition to remaining in the stationary phase for a shorter time. When compared with other species of the genus, B. henselae (using a supplemented RPMI 1640 medium) and B. quintana (using a DS2 medium formulated to favor the cell growth of insect cells) showed curves similar to that presented by the Bartonella strain 117A, both requiring a longer time to enter into the logarithmic phase and, similarly to the present work, they did not remain in a stationary phase for a long time [Regarding the bacterial growth curve, the results of the present study showed discrepancies between the growth curves of the two isolated species: while ong time .BadA) and flagella (FlaA and FlaB). The absence of pili or flagellum is shared with other rodent-associated Bartonella species, such as B. vinsonii [B. japonica, B. silvatica [B. heixianziensis [B. machadoae [B. bacilliformis, B. schoenbuchensis, B. capreoli, B. chomelii, B. clarridgeiae and B. rochalimae have flagella. The presence of pili has been described in rodent-associated Bartonella spp. such as B. tribocorum [B. alsatica [B. acomydis, B. jaculi, B callosciuri, B. pachyuromydis [B. fuyuanensis [B. kosoyi and B. krasnovii [The Bartonellae isolated in the present study did not show pili or flagella at SEM, which was confirmed by the absence of genes encoding adhesin , do Amaral et al.  demonstra et al. , have leerformed  , since they have an ANI value below 95% among themselves. On the other hand, B. schoenbuchensis, Bartonella chomelii, B. capreoli and B. melophagi should be grouped as a subspecies of B. schoenbuchensis (the species described first among the four cited), since they show ANI values above 95% among themselves. Finally, based on the Bartonella species  with the highest number of genomes deposited in the database, the variation of the ANI values within the same species ranged from 97 to 100%. Therefore, we propose a cut-off point of 95 < ANI < 97% for the classification of new Bartonella subspecies  bohlsi bolshi, Polygenis occidentalis occidentalis, Polygenis platensis and Craneopsylla minerva minerva) in the biomes of Pantanal (the state of Mato Grosso do Sul), Pampa and the Atlantic Forest, in the state of Rio Grande do Sul [Bartonella genotypes detected in fleas from central-western and southern Brazil were found in regions where the circulation of B. machadoae, a species recently described in rodents, was also reported [B. machadoae in Amblyomma sculptum ticks collected from wild boars (Sus scrofa) in southeastern Brazil. Thus, it is necessary to evaluate the diversity of hosts, vectors and the zoonotic potential of B. machadoae and B. harrusi, considering that both are closely related to Bartonella species belonging to the B. vinsonii complex, which have already been shown to have zoonotic potential in several regions in the world [Despite the vectors involved in the transmission of this new e do Sul ,68. Intereported  detectedhe world ,70,71,722) at 35 °C. Colonies grown on chocolate agar are smooth and circular. The time required for growth to reach colonies of 1.0–2.0 mm is 4 days at 35 °C. They are negative for oxidase, catalase, urease, indole production, acetoin production and nitrate reduction. They are unable to ferment the carbohydrates sorbitol, melibiose, rhamnose and glucose, albeit they showed a positive reaction to arginine. The type strain (117A) carried a single circular chromosome of 2.23 MB, with an average G + C DNA content of 38.8% by mol. and a 29,892 bp circular plasmid with an average G + C content of 34.5%. The closest species with a valid published name is B. machadoae (91.98% ANI). Bartonella harrusi sp. nov. (strain 117A) is stored at the Laboratory of Immunoparasitology, Department of Pathology, Reproduction and Single Health, Faculty of Agrarian and Veterinary Sciences/Universidade Estadual Paulista, UNESP, Jaboticabal, SP, Brazil.The bacterial cells of this new proposed species are rods (1–2.0 µm long and 0.3–0.6 µm wide) without flagella and pili, showing excellent growth under capnophilic conditions (5% COBartonella in marsupials from the Americas. Bartonella harrusi sp. nov. was isolated from blood samples of T. macrurus in the Pantanal of Mato Grosso do Sul. The diagnostic platform combining qPCR results based on the nuoG gene from blood samples in a pre-enrichment liquid culture and chocolate agar isolation proved to be efficient in detecting Bartonella in marsupials. A phylogenomic analysis based on 291 genes and an ANI value of 91.98% obtained by the WGS of Bartonella 117A allowed the separation of Bartonella harrusi sp. nov. from Bartonella machadoae. A bioinformatics analysis applied to complete genomes should be used for the classification and taxonomic review of members of the genus Bartonella based on ANI values.The present work presents the first description of


Attention Deficit Hyperactivity Disorder is a neurodevelopmental disorder with three presentations: inattentive, hyperactive/impulsive and combined. These may represent an independent disease entity. Therefore, the therapeutic approach must be focused on their neurobiological, psychological and social characteristics. To date, there is no comprehensive analysis of the efficacy of different treatments for each presentation of ADHD and each stage of development. This is as narrative overview of scientific papers that summarize the most recent findings and identify the most effective pharmacological and psychosocial treatments by ADHD presentation and age range. Evidence suggests that methylphenidate is the safest and most effective drug for the clinical management of children, adolescents and adults. Atomoxetine is effective in preschoolers and maintains similar efficacy to methylphenidate in adults, whereas guanfacine has proven to be an effective monotherapy for adults and is a worthy adjuvant for the management of cognitive symptoms. The psychosocial treatments with the best results in preschoolers are behavioral interventions that include training of primary caregivers. In adolescents, the combination of cognitive and cognitive-behavioral therapies has shown the best results, whereas cognitive-behavioral interventions are the most effective in adults. Pharmacological and psychosocial treatments must be adjusted to the ADHD presentation and its neurocognitive characteristics through the patient’s development. Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by persistent inattention, hyperactivity and impulsivity behaviors . ADHD isThe Diagnostic and Statistical Manual of Mental Disorders (DSM-5) distinguishes three clinical presentations of ADHD: predominantly inattentive, predominantly hyperactive/impulsive and combined .The inattentive ADHD presentation is the most commonly diagnosed (53.7%), followed by the combined type (26.8%), while the least diagnosed is the hyperactive/impulsive (19.5%)  see Tab.Sex and age of patients are associated with ADHD presentation. In women, inattentive presentation is more frequent, while in men the combined type predominates. In contrast, the hyperactive/impulsive presentation shows similar incidences between men and women . AccordiThe diagnosis of ADHD presentations may vary throughout the life of the patient . PreschoThe first clinical manifestations of ADHD can appear from the third year of life . HoweverThe present systematic review examines the efficacy reported in pharmacological, neuropsychological, or psychosocial treatments in ADHD according to age or presentation. We searched for articles from the National Library of Medicine by using the PubMed search engine and the terms: efficacy of neuropsychological treatments, OR pharmacological treatments, OR novel psychological treatments OR psychosocial treatments combined with the terms ADHD, ADHD-presentations, OR inattention, OR impulsivity, OR hyperactivity. The initial search in the MEDLINE database comprised 141 papers. Of these, 56 references were eliminated because: a) they compared alternative or unregulated therapies  with pharmacological interventions and b) the efficacy of treatments  was evaluated when AHDH was comorbid with diverse neurodevelopmental disorders. Only 85 references were selected because they met inclusion criteria: a) Type of paper  and b) Efficacy of pharmacological treatments  combined or not with neuropsychological treatments (cognitive training) or psychosocial treatments  in preschoolers, children, adolescents and adults with a diagnosis of ADHD. For description of the mechanism of action for drug medication in ADHD, we also included animal models. The current review included English- and Spanish-language reports published between the years 1991 and 2021. Then, the pre-selected papers (85 documents) were subjected to methodological quality assessment by two independent reviewers. After this process, 7 reports were eliminated because they contained confirmative information, or their evidence did not approach the aim of the review. Finally, only 78 references were utilized for this review and all the selections followed PRISMA guidelines .Of the 79 selected references, 34 were used for analysis of the current pharmacological treatments. The conventional treatment for ADHD is based on the use of nervous system stimulants (amphetamines and methylphenidate)  and non-After MPH oral administration, MPH reaches sufficient plasma levels in one hour  and blocThe long-term therapeutic effect of MPH depends on the dose, route of administration and treatment duration . Seven- In most cases, MPH recovers cognitive performance in patients with ADHD, regardless of whether they have comorbid psychiatric disorders or not . In healMPH is effective for the management of oppositional defiant disorder (ODD) and reduces the incidence and severity of assaults . ODD is ATM is a norepinephrine reuptake inhibitor that increases neurotransmitter levels in the synaptic space . In presATM is highly recommended for controlling the hyperactive/impulsive presentation in pre-escolar patients. Pharmacological presentation seems to be a significant factor that modifies the efficacy of every drug, for instance, ATM shows a similar size effect when compared to immediate-release vehicles of MPH (SMD −0.05), but a poor size effect when compared to the osmotic-release vehicle of MPH (SMD 0.031) . However2a adrenergic receptors in the prefrontal cortex, which facilitates neural signaling in this region [Guanfacine was initially used against hypertension, but recent studies demonstrated its efficacy as an adjuvant drug for ADHD management in children and adolescents. The ideal dose of guanfacine to control ADHD symptoms is between 4 and 7 mg/kg . In aduls region  and redus region . Guanfacs region . For theRecent studies show that the efficacy of MPH, ATM and guanfacine varies according to the stage of development of the patient. MPH has better results in the management of central symptoms in late childhood, adolescence and adulthood. ATM is less effective for the management of symptoms in late childhood and adolescence but has similar efficacy to MPH in adults . AlthougTo summarize the findings of neuropsychological treatments, we analyzed 10 of the 79 selected references. Recently, a systematic review that compared 34 meta-analyses reported that children and adolescent patients with ADHD present a worsening in neurocognitive performance when compared with typically developing subjects . SeveralDysfunction in executive functions varies for each ADHD presentation. Children and adolescents with combined or inattentive subtypes show severe deficits in the control of inhibitory responses, whereas those with the hyperactive-impulsive subtype show less executive impairment . PatientOther cognitive variables also present differences among ADHD presentations. Inattentive and hyperactive-impulsive types are inversely correlated to intelligence, alertness, response variability, processing speed and delay in the aversive stimulus so it is important to incorporate the evaluation and treatment of these cognitive tasks in both presentations. Notably, the predominance of inattention symptoms seems to be strongly associated with cognitive deterioration . ConsequBehavioral techniques, based on rewards or punishments, that seek to generate more adaptive behaviors.Cognitive techniques that identify maladaptive beliefs and replace them with others that generate better adaptation, i.e., cognitive restructuring or rehabilitation.Combination of cognitive and behavioral techniques at a group or individual level.Training in organizational and/or social skills.To evaluate the efficacy of psychosocial treatment in ADHD, we analyzed 21 of the 79 selected references. In this regard, the efficacy of psychosocial treatment in ADHD has been a discussion subject in the scientific literature . MethodoThe psychosocial intervention in patients with inattentive- or combined-type ADHD that is based on training in improving organization skills regarding school materials showed a moderate improvement in inattention . In earlCognitive interventions in children and adolescents have a moderate size of effect on improving inattention and a small effect size on controlling impulsivity and hyperactivity . SimilarUp to 70% of ADHD patients have comorbid psychiatric disorders. Depression and anxiety are the internalizing comorbid disorders more prevalent in patients with inattentive and combined presentations ,68,69,70Externalizing disorders, such as oppositional-defiant disorder, are psychopathologies that occur with a higher incidence in children with the combined presentation ,76 and aIn the preschool stages (3–6 years), the most prevalent presentation of ADHD is the hyperactive/impulsive subtype. In childhood (6–12 years), the combined type predominates in boys, whereas the inattentive type is the most common ADHD presentation in girls. The hyperactive-impulsive ADHD commonly progresses to a combined presentation that, in turn, evolves to the inattentive type in adults see .Due to cognitive and behavioral comorbidities, the most complex presentations for clinical management are inattentive and combined types. ATM is the drug with better results in preschool children because it has fewer side effects as compared to MPH. ATM is also recommended for the therapeutic management of adolescents and adults with abuse of drugs. MPH is very efficient for clinical management of late childhood and adolescents with inattentive and combined presentations because it improves working memory, intelligence, academic skills and ODD comorbidity. Guanfacine is also recommended as an adjuvant treatment in childhood and adolescents to improve academic symptoms. Guanfacine can also be useful for handling adults. In parallel, neuropsychological treatments act as adjuvant therapy, whereas the most effective psychosocial treatment in early childhood is behavioral intervention that includes training for parents.Late childhood is benefited by cognitive strategies that improve inattention, whereas cognitive-behavioral approaches reduce hyperactivity and impulsivity. In adolescents and adults, the most effective approach is the combination of cognitive and cognitive-behavioral therapies see .


In over half of pediatric cases, ADHD presents with comorbidities, and often, it is unclear whether the symptoms causing impairment are due to the comorbidity or the underlying ADHD. Comorbid conditions increase the likelihood for a more severe and persistent course and complicate treatment decisions. Therefore, it is highly important to establish an algorithm that identifies ADHD and comorbidities in order to improve research on ADHD using biorepository and other electronic record data.It is feasible to accurately distinguish between ADHD in isolation from ADHD with comorbidities using an electronic algorithm designed to include other psychiatric disorders. We sought to develop an EHR phenotype algorithm to discriminate cases with ADHD in isolation from cases with ADHD with comorbidities more effectively for efficient future searches in large biorepositories. We developed a multi-source algorithm allowing for a more complete view of the patient’s EHR, leveraging the biobank of the Center for Applied Genomics (CAG) at Children’s Hospital of Philadelphia (CHOP). We mined EHRs from 2009 to 2016 using International Statistical Classification of Diseases and Related Health Problems (ICD) codes, medication history and keywords specific to ADHD, and comorbid psychiatric disorders to facilitate genotype-phenotype correlation efforts. Chart abstractions and behavioral surveys added evidence in support of the psychiatric diagnoses. Most notably, the algorithm did not exclude other psychiatric disorders, as is the case in many previous algorithms. Controls lacked psychiatric and other neurological disorders. Participants enrolled in various CAG studies at CHOP and completed a broad informed consent, including consent for prospective analyses of EHRs. We created and validated an EHR-based algorithm to classify ADHD and comorbid psychiatric status in a pediatric healthcare network to be used in future genetic analyses and discovery-based studies.In this retrospective case-control study that included data from 51,293 subjects, 5840 ADHD cases were discovered of which 46.1% had ADHD alone and 53.9% had ADHD with psychiatric comorbidities. Our primary study outcome was to examine whether the algorithm could identify and distinguish ADHD exclusive cases from ADHD comorbid cases. The results indicate ICD codes coupled with medication searches revealed the most cases. We discovered ADHD-related keywords did not increase yield. However, we found including ADHD-specific medications increased our number of cases by 21%. Positive predictive values (PPVs) were 95% for ADHD cases and 93% for controls.We established a new algorithm and demonstrated the feasibility of the electronic algorithm approach to accurately diagnose ADHD and comorbid conditions, verifying the efficiency of our large biorepository for further genetic discovery-based analyses.NCT02286817. First posted on 10 November 2014. ClinicalTrials.gov, NCT02777931. First posted on 19 May 2016. ClinicalTrials.gov, NCT03006367. First posted on 30 December 2016. ClinicalTrials.gov, NCT02895906. First posted on 12 September 2016.ClinicalTrials.gov, The online version contains supplementary material available at 10.1186/s11689-022-09447-9. ADHD is the most prevalent neurodevelopmental disorder in children , 2. It iBecause ADHD exists on a spectrum and presents in various forms , 5, patiRobust phenotype algorithms typically use empirical and/or machine learning algorithms, combining multiple data sources to achieve high positive predictive values (PPVs) in identifying cases and controls . A thoroGiven the prevalence of comorbidities with ADHD, we believe an algorithm including comorbidities would be more representative of the patient population and permit a greater yield of ADHD cases within a large database. To address this, we developed a multi-source/multi-approach EHR rule-based algorithm with NLP text mining allowing for a more complete extraction of the patient’s medical record. Chart abstractions were completed to add evidence and assess confidence in the ADHD and comorbid diagnoses. Our objective was to create and validate an EHR-based algorithm to classify ADHD and comorbid psychiatric status in a pediatric healthcare network to be used in future genetic analyses and discovery-based studies. The underlying genetic etiology may be different for children with comorbid psychiatric conditions versus ADHD in isolation. We believe these cases represent a group of subjects that may be less responsive or amenable to traditional ADHD medications and may represent a suitable cohort for testing new non-stimulant compounds for ADHD.The rule-based phenotyping algorithm was developed using data from the CAG pediatric biorepository database at CHOP Fig. . We extrThe algorithm for detecting psychiatric phenotypes draws information from multiple sources within the EHR  to provide a thorough picture of the patient’s medical record. The EHR includes emergency department, inpatient and outpatient visits, date of visits, reason for visits, admission details, diagnosis codes (ICD-9/ICD-10-CM format), growth measurements, medications prescribed, imaging, laboratory test results, patient problem list, and referrals made. This information is captured and moved into the repository in a structured format. The algorithm was constructed using information from CAG internal and external published or publicly available psychiatric disorder algorithms , 20–23.or ICD10 codes beginning with “F90.,” or prescriptions for ADHD-specific medications in the subject’s EHR. To account for the various possible issues in diagnosing ADHD, we required more than one ADHD “hit” at different visits for inclusion. At minimum, the patient must have the following: (1) two separate diagnosis days; (2) two separate ADHD medication prescription days; (3) one diagnosis day and one ADHD medication day on separate calendar days. Later, we added to the algorithm; (4) one prescription and one result from an abstracted note; or (5) one diagnosis code and one result from an abstracted note. All cases had a diagnosis of ADHD at 4 years or greater, keeping with the American Academy of Pediatrics Clinical Practice Guidelines for ADHD [ADHD cases were defined by the presence of ICD9 codes beginning with “314.,” Keywords were selected to search for ADHD phenotypes  and analyzed. Abstractions were searched using the ADHD or comorbidity keywords and medications used in the structured search. These results were added to the ADHD and each comorbidity algorithm as further criteria adjustmentsThe CAG database was searched for subjects with one or more of nine psychiatric diagnoses: anxiety, autism, major depression, oppositional defiant disorder (ODD), conduct disorder (CD), tic disorders, Tourette syndrome, schizophrenia, and/or bipolar disorder. Subjects with mild/moderate intellectual disability (ID) and learning disabilities (LD) were also included. Algorithms for each condition were created and modified from previously published or publicly available algorithms  number of unique diagnosis days, (2) number of unique ADHD medication days, (3) whether an ADHD phenotype was located in psych abstraction , (4) whether an ADHD medication was located in psych abstraction , and (5) whether a subject was noted to have psychotherapy or neurocognitive therapy . The number of sources can be from 1 to 5 based on the categories above. High confidence is defined as (1) total score of ≥ 20, (2) total score > 9 and number of sources 4–5, or (3) number of diagnosis days > 9 or number of ADHD medications > 9 and number of sources 2–3. Moderate confidence is defined as (1) subjects not defined in high or low confidence. Low confidence is defined as (1) number of diagnosis days + number of ADHD medications = 2 and total score < 3 or (2) number of diagnosis days = 0 and total score < 10 and number of ADHD medications > 2 and no abstraction sources.A total of 51,293 subjects were extracted from the CAG database based on the study inclusion criteria. Of these, 16,902 subjects were classified as controls, and 10,368 were classified as positive cases for one or more of the 10 psychiatric conditions . In addNeuroimaging data such as voxels from functional magnetic resonance imaging (fMRI) will be an exciting new data type to begin quantifying the ADHD and/or other psychiatric disorders as we characterize correlating brain regions.In terms of treatment improvement of ADHD or other related psychiatric and neurodevelopmental disorders, medication and psychosocial treatment need to be objectively measured for efficacy such as in the Preschool ADHD Treatment Study (PATS) and Multimodal Treatment Study of Children with Attention-Deficit/Hyperactivity Disorder (MTA).The advantages of an accurate and high-performing automated algorithm approach to cohort building versus chart review are substantial, particularly with extremely large databases. Chart abstraction required approximately 20–30 min per participant, representing a significant obstacle to scalability. This means the 1079 abstractions would have taken at a minimum about 360 h, or 9 weeks, worth of analyst time. Although the algorithm took several weeks to create, most of this effort was devoted to designing inclusionary/exclusionary criteria, a necessary step for manual processes as well. Establishing an ADHD algorithm that considers comorbidities reduces the amount of time of building and running all comparisons. Critically, subsequent iterations can be run in a matter of minutes, meaning the prospect of keeping pace with an ongoing biorepository is relatively straightforward. These considerations, coupled with high PPVs from validation efforts, urge confidence that the algorithm is a robust and valuable tool for identifying case/control datasets for genetic and discovery-based analyses.Additional file 1: Table S1. ADHD Inclusion/ Exclusion Table. ICD codes and direct terms of ADHD and ADD were used, as well as terms that were specific towards ADHD phenotypes and medications. Table S2. Anxiety Inclusion/ Exclusion Table. ICD codes and terms that were specific towards anxiety phenotypes and medications were used. Table S3. Autism Inclusion/ Exclusion Table. ICD Codes as well as terms that were specific towards autism phenotypes were used. Table S4. Conduct Disorder Inclusion/ Exclusion Table. ICD codes and terms that were specific towards conduct disorder phenotypes were used. Table S5. Oppositional Defiant Disorder Inclusion/ Exclusion Table. ICD codes and terms that were specific towards oppositional defiant disorder phenotypes were used. Table S6. Major Depressive Disorder Inclusion/ Exclusion Table. ICD codes and terms that were specific towards major depression phenotypes and medications were used. Diagnosis of major depressive disorder was present on at least two (2) distinct calendar days that are at least thirty (30) days apart and not more than one hundred and eighty (180) days apart. Table S7. Bipolar Disorder Inclusion/ Exclusion Table. ICD codes and terms that were specific towards bipolar disorder phenotypes and medications were used. Diagnosis of bipolar disorder was present on at least two (2) distinct calendar days that are at least thirty (30) days apart and not more than one hundred and eighty (180) days apart. Table S8. Schizophrenia and Psychoses Inclusion/ Exclusion Table. ICD codes and terms that were specific towards schizophrenia and psychoses phenotypes and medications were used. Table S9. Tic Disorders Inclusion/ Exclusion Table. ICD codes and terms that were specific towards tic disorder phenotypes were used. Table S10. Tourette Syndrome Inclusion/ Exclusion Table. ICD codes and terms that were specific towards Tourette syndrome phenotypes were used. Table S11. Intellectual Disability Inclusion/ Exclusion Table. ICD codes and terms that were specific towards intellectual disability phenotypes were used. Table S12. Learning Disability Inclusion/ Exclusion Table. ICD codes and terms that were specific towards learning disability phenotypes were used. Table S13. Case Exclusion Table. ICD codes and terms used for case exclusions. Table S14. Control Exclusion Table. ICD codes and terms used for control exclusions. Table S15. Control Syndromes Exclusion Table. ICD codes and terms used for control syndrome exclusions. Table S16. Psychiatric Conditions Prevalence in Extracted Subjects. Numbers and percent of psychiatric disorders and comorbidities in all cases and Psych Positive Cases. *has at least of the 10 psychiatric conditions  of algorithms for each psychiatric disorders and comorbidity of ADHD measured.


Previous studies have looked at the effects of physical activity interventions on the symptoms of ADHD, although few have compared the motor behavior of children with ADHD versus those without. This exploratory study provides detailed information on the patterns and intensity of physical activity and sedentary behavior in children with ADHD as measured by Actigraph GT3X accelerometry, as well as the differences in physical activity in the different presentations of ADHD; (2) Methods: A cross-sectional design was used with a sample of 75 children, aged 6 to 12 years, with and without ADHD. The ADHD group had a previous diagnosis, determined by clinical assessment based on DSM-5 criteria; (3) Results: Physical activity levels were higher in children with ADHD compared to children without ADHD, but there was no difference in sedentary time between groups during weekdays or weekends. Physical activity decreased with age, with significant differences in the ADHD group, who exhibited more minutes of moderate Physical activity in 6–7 year-olds than 10–11 year-olds during weekdays and weekends; (4) Conclusions: Sedentary time increased by age in children without ADHD, and there was a decrease in moderate-intensity physical activity time in children with ADHD by age. Attention Deficit Hyperactivity Disorder (ADHD) is one of the most common neurodevelopmental disorders, with an incidence of 5–7% of the school-age population, and is more common in boys than in girls . In SpaiAccording to Sempere-Tortosa et al. , there aAccordingly, the inexactness of the diagnosis of ADHD—relying on non-objective criteria —coupled According to the recommendations of the World Health Organization (WHO), children and adolescents aged between 5 and 17 should do an average of at least 60 min of moderate or intense physical activity (MVPA) per day and may expect additional benefits with more activity . DespiteIt is also important to mention sedentary lifestyles. Sedentary time is defined as periods involving energy expenditure ≤1.5 metabolic equivalents (METs) during waking time . DecreasLin, Yang, and Su  showed tMeasurement of physical activity is as important as it is complex, and there are many ways it can be quantified. As Aparicio-Ugarriza et al.  noted, rThe sample comprised 75 children aged 6 to 12 years old, with an average age of 9.47 (SD = 1.73), from public and private elementary schools in northern Spain. A control group of 28 children without ADHD and a group of 47 ADHD children were included.The study complied with the ethical regulations of the Declaration of Helsinki  and was The sample was split into two groups: the “ADHD” group of students diagnosed with ADHD, attending state-funded and private schools in the Principality of Asturias, and the “Non-ADHD” group of students in a state school. The sample of children with ADHD was provided by two different hospitals dealing with ADHD in a single health authority region. The evaluation was carried out by a clinician, with the families’ approval, in order to verify the inclusion of the participants in the study. The participants came from different schools selected for ease of accessibility, all from the same area of the Principality of Asturias, and all following the same teaching system. There were no differences between the schools since Spanish schools follow a state curriculum, with a school schedule of 25 h per week and the same number of hours of physical education for all participants—2 h per week. Schools were not analyzed separately to determine whether there were differences in intensity between them.To be included in the study, subjects in the ADHD group had to meet the following requirements (based on the DSM-5 criteria ): (1) muIn order to define the groups and to determine the relationship between the variables in relation to the different presentations of ADHD, clinicians confirmed the DSM-5 criteria based on the results from the Farré and Narbona test , EvaluatA variety of data collection tools were used in the study based on the variables to be included.This information was provided by the school, the parents, or the students.The physical activity level of the students was objectively measured with ActiGraph-GT3X accelerometers  . ParticiThe categories of intensity are given in The Assessment of Attention Deficit with Hyperactivity Scale (EDAH) by Farré and Narbona  measures2 = 0.01 (d = 0.20), medium when ηp2 = 0.059 (d = 0.50), and high when ηp2 = 0.138 (d = 0.80).All of the collected data was stored in an SPSS 25.0 database. To analyze whether there were statistically significant differences between the groups, univariate and multivariate analyses of covariance (ANCOVA and MANCOVA (with covariates age and sex)) were performed. The independent variable was first the group (ADHD and control) and then the ADHD presentation groups . The dep = 0.231) or BMI (p = 0.161). The groups were not offset by sex since ADHD is more frequent in boys than in girls (χ2(1) = 4.587); (p = 0.032). The descriptive body composition and socio-educational variables of the 75 children participating in the study are shown in p = 0.703), where nonparametric tests were used in vigorous PA . The ADHD group also spent more minutes during weekdays than the non-ADHD group in light PA (F(1.75) = 35.994; p < 0.000; d = 1.43), moderate PA (F(1.75) = 43.986; p < 0.000; d = 1.58;) and vigorous PA (F(1.75) = 16.983; p < 0.000; d = 0.98;). There were statistically significant differences in sedentary minutes between the groups during weekdays (F(1.75) = 7.398; p < 0.01; d = 0.65), with higher values of sedentary behavior in children with ADHD than in the group without ADHD. Medication had no statistically significant effects on the weekday variables p = 0.952.The variables sex and age in the MANCOVA analysis were not statistically significant as covariates . The ADHD group also spent more weekend minutes than the Non-ADHD group in light PA (F(1.75) = 21.129; p < 0.000; d = 1.10), moderate PA (F(1.75) = 32.599; p < 0.000; d = 1.36), and vigorous PA (F(1.75) = 25.026; p < 0.000; d = 1.19). There were also statistically significant differences between the groups during weekends in sedentary time (F(1.75) = 6.939; p < 0.05; d = 0.63) with more minutes of sedentary time in the ADHD group than the Non-ADHD group. As with weekdays, medication had no statistically significant effect on weekends p = 0.667.Similarly, during the weekends, the ADHD group had higher counts than the Non-ADHD group, with the differences being statistically significant (F(1.75) = 20.085; p < 0.05; d = 3.25). Likewise, differences between the sexes were found in the ADHD group weekday PA variables, with significantly higher values for boys in weekday counts (F(1.47) = 10.9; p < 0.01; d = 5.13) and moderate weekday PA (F(1.47) = 4.969; p < 0.05; d = 3.46). In the Non-ADHD group, there were differences between the sexes in weekday and weekend PA variables, with significantly higher values for girls in weekday counts (F(1.28) = 4.476; p < 0.05; d = 0.80), moderate PA (F(1.28) = 8.051; p < 0.01; d = 1.08), light PA (F(1.28) = 9.818; p < 0.01; d = 1.19) and sedentary time (F(1.28) = 4.388; p < 0.05; d = 0.79).Differences between the sexes were found for weekend PA variables, with significantly higher values for boys in weekend vigorous PA (F(1.75) = 4.851; p < 0.05; d = 0.82), moderate PA (F(1.28) = 6.39; p < 0.05; d = 0.96), light PA (F(1.28) = 12.145; p < 0.01; d = 1.32) and sedentary time (F(1.28) = 7.248; p < 0.05; d = 0.37) with higher values for girls. In the Non-ADHD group, no differences were found for either weekday or weekend vigorous PA.Similarly, during the weekends, there were significant differences in counts (F(1.28) = 4.644; p = 0.002, p = 0.000), as well as in the weekend count means of the control group compared to the Hyperactive/Impulsive and Combined groups , the control group exhibited lower values in both cases.Both during the week and at weekends, the hyperactive/impulsive and combined groups of students had statistically significantly higher counts than the control group. They also spent statistically significantly more time than the control group in light, moderate, and vigorous physical activity, both during the week and at weekends. The only statistically significant difference between the control group and the inattentive group was in the amounts of moderate physical activity, both during the week and at weekends; the inattentive group had higher values than the control group. There were statistically significant differences in the weekday count means of the control group compared to the Hyperactive/Impulsive and Combined groups  and in the mean amounts of weekend light physical activity between the control, Hyperactive/Impulsive and Combined groups . There were also statistically significant differences in mean weekday minutes of moderate activity between the control group and all three ADHD presentations, with lower values for the control group compared to the Inattention, Hyperactive/Impulsive and Combined groups , with a similar pattern for weekend minutes of moderate activity , which was repeated with the mean amounts of weekend vigorous activity between the control group and the Hyperactive/Impulsive and Combined presentations .A similar pattern was evident in the mean amounts of weekday vigorous activity between the control, Hyperactive/Impulsive, and Combined groups , respectively, with higher values for the Hyperactive/Impulsive and Combined groups compared to the non-ADHD group.Similarly, statistically significant differences were found in mean weekend minutes of vigorous physical activity between the control group and the Hyperactive/Impulsive and Combined presentations , with significantly more minutes of moderate PA in 6–7 year-olds than 10–11 year-olds  on weekdays.Looking more deeply at the age range using Scheffé’s post hoc test for the ADHD group in p = 0.045), and there were higher levels for 8–9 year-olds than 10–11 year-olds . There were no differences between age in the other physical activity and sedentary behavior variables for the ADHD group (p > 0.05).Similarly, at weekends, 6–7 year-olds exhibited more minutes of moderate PA compared to 10–11 year-olds  with an increase in minutes in these variables as a function of age. Children aged 8–9 had more sedentary minutes on weekdays than those aged 6–7 , and children aged 10–11 had more sedentary minutes than those aged 6–7 . On weekends, sedentary minutes also increased as a function of age, with 10–11 year-olds having more than 6–7 year-olds . There were no differences between ages in the other physical activity variables for the Non-ADHD group (p > 0.05).We did find age-related differences in the non-ADHD group for minutes of light physical activity on weekdays (d = 4.53, MD = −328.18, This study produced valuable findings about the amounts of moderate physical activity in children with and without ADHD, additionally demonstrating whether the participants did the amount of physical activity that the WHO recommends for children. Children with ADHD did more PA than non-ADHD children, but about 92% of the sample in both groups complied with the WHO guidelines for PA in children , with a In this sample of children with and without ADHD, there was no difference in the objective measurements of physical activity between boys and girls, although differences were found within the non-ADHD group by sex. Our results are consistent with previous studies, which found sex differences in the amount and intensity of PA in non-ADHD children . We did,Sedentary time has been shown to be more important than moderate or high activity in predicting ADHD symptoms as a diagnostic measure of activity . HoweverOther studies using objective measurements to analyze movement, via infrared ,52,53 orIn line with Volkmar’s  results,Previous research has reported similar mean effect sizes in children and in adolescents , althougThis study has some limitations. It did not consider other comorbid disorders, nor were the use of stimulant treatments or their effects on ADHD subjects’ locomotor activity examined. The main limitation of our study was the sample size of the subgroups for the different presentations of ADHD. A lack of previous studies providing information on amounts of different intensities of activity or activity related to ADHD presentations on both weekdays and weekends makes it difficult to compare the patterns and motor behavior based on the intensity of physical activity of children with ADHD versus those without ADHD. It would be interesting to record the intensity of physical education classes, as well as to record whether children engage in active transportation. Other limitations of the study are the size and accessibility of the sample—our sample size was small, and it was a pilot study—as well as not examining the influence of variables such as ethnicity/race or socioeconomic status. ADHD is a disorder with a higher prevalence in boys than in girls, which may imply a limitation in the homogeneity of the sample. One of the strengths of this study is the use of GT3X accelerometry to measure physical activity and sedentary time patterns in children with and without ADHD. Future studies might find it interesting to determine the diagnostic utility of accelerometry in patients with this disorder, looking to see whether there is variability when analyzing other ethnicities and lifestyles and the interaction of age and sex on motor symptoms based on a larger sample.In conclusion, greater physical activity was associated with ADHD and ADHD presentations in children with ADHD compared to Non-ADHD children. We found that 92% of our 6–12-year-old population did sufficient levels of physical activity, although children with ADHD had higher levels of MVPA. Children with ADHD did not present differences in the amount or intensity of physical activity by sex, while in children without ADHD, girls demonstrated more activity and a greater intensity of physical activity than boys. We reported an increase in sedentary time by age in Non-ADHD children and a decrease in moderate-intensity physical activity time in ADHD children by age. There is a need for additional work to investigate these topics more fully. A metanalysis might be helpful in terms of conceptualizing how/why increased gross motor movements may manifest in children with ADHD .