Bartonella are vector-borne hemotropic bacteria that infect a wide variety of hosts, including people. While there are PCR assays that can identify individual or groups of Bartonella, there is no reliable molecular method to simultaneously detect all species while maintaining genus specificity and sensitivity. By comparing highly conserved 16S rRNA sequences of the better-recognized Bartonella spp. on GenBank, we selected primers and probes for a genus-specific pan-Bartonella FRET-qPCR. Then, a gltA-based Bartonella PCR was established by selecting primers for a highly variable region of gltA, of which the sequenced amplicons could identify individual Bartonella spp. The pan-Bartonella FRET-qPCR did not detect negative controls  but reliably detected as few as two copies of the positive control (Bartonella henselae) per reaction. There was complete agreement between the pan-Bartonella FRET-qPCR and the gltA-based Bartonella PCR in detecting Bartonella in convenience test samples from China and St. Kitts: cats , Ctenocephalides felis , cattle , and donkeys . Sequencing of the gltA-based Bartonella PCR products revealed B. henselae  and B. clarridgeiae  in cats and C. felis  and B. bovis in cattle  and donkeys . The pan-Bartonella FRET-qPCR and gltA-based Bartonella PCR we developed are highly sensitive and specific in detecting recognized Bartonella spp. in a single reaction. The pan-Bartonella FRET-qPCR is convenient requiring no gel electrophoresis and providing copy numbers, while the gltA-based Bartonella PCR reliably differentiates individual Bartonella species. The use of these PCRs should greatly facilitate large-scale surveillance studies and the diagnosis of infections in clinical samples. Bartonella which are small, intracellular, vector-borne hemotropic Gram-negative bacteria. High prevalences of the organisms have been reported around the world in a wide range of insect vectors and domestic and wild animal hosts including rodents, felines, canines, ruminants, and even bats [Bartonella species have been detected in a wide range of animals [Bartonella species having been implicated in human diseases that range from self-limiting regional lymphadenitis to severe endocarditis [Bartonella infecting people in the Caribbean [There are now over 40 species and subspecies of ven bats –4. In Ch animals –8 with acarditis –11. To taribbean  althougharibbean , mongoosaribbean , bats [1aribbean , 16, dogaribbean , 18, rodaribbean , cat andaribbean , 21, andaribbean .Bartonella species in their vectors and mammalian hosts is often not easy as it is difficult and time-consuming to culture them and serology may be unreliable. Although numerous molecular methods to detect Bartonella have been described [Bartonella [Bartonella, they require a wide range of primers and probes to detect all species. Furthermore, the PCRs have poor sensitivity (with up to 8 × 107 bacteria per g being undetectable in feces) [nuoG of the Bartonella genus and identified B. henselae and B. clarridgeiae in cats in Brazil [Detecting escribed –28, in mrtonella . Althougn feces) , 31 becan Brazil .16S rRNA sequences of 20 Bartonella species in GenBank to develop and validate a highly sensitive and genus-specific pan-Bartonella FRET-qPCR. We also used available sequences for the citrate synthase gene (gltA) and considered a reliable tool for distinguishing genotypes [gltA-based Bartonella PCR to identify and differentiate the species. The development of these PCRs and their use on convenience samples from China and St. Kitts is described below.We used the available enotypes , to deven = 60) were collected from the cats used for blood sampling, and the flea species were identified according to the morphological criteria as described in previous studies [n = 146) sampled during a Trap, Neuter, Release program conducted in 2012. Archived whole blood samples from cattle (n = 98), sheep (n = 30), donkeys (n = 24), and horses (n = 24) collected as described in previous studies [This study was reviewed and approved by the Institutional Animal Care and Use Committee of the Yangzhou University College of Veterinary Medicine, China (YZU-13-58Wang), and of the Ross University School of Veterinary Medicine, St. Kitts (07/02/3MOURA). Convenience whole blood samples in EDTA were collected from cats in China and St. Kitts. Between April 2013 and June 2014, 164 cat whole blood samples were collected from five cities in four provinces in China and stored at −80°C until DNA extraction (see below). The cats sampled in Yangzhou were kept in a shelter, while those from Beijing, Shanghai, and Guangdong were pet cats presenting to veterinary clinics with a variety of conditions. Cat fleas , B. henselae (AJ223778), B. clarridgeiae (NR_03696), B. elizabethae (AB246807), B. bacilliformis (NR_044743), B. bovis (NR_025121), B. capreoli (NR_025120), B. koehlerae (NR_024932), B. alsatica (NR_025272), B. grahamii (NR_029366), B. vinsonii subsp. berkhoffii (DQ2281315), B. vinsonii subsp. vinsonii (NR_037056), B. vinsonii subsp. arupensis (AF214558), B. doshiae (NR_029368), B. queenslandensis (EU111758), B. washoensis (AB519066), B. rattaustraliani (EU111753), B. tamiae (DQ395176), B. rochalimae (DQ683196), B. melophagi (AY724770), Bartonella sp. (U71322), Anaplasma phagocytophilum (AF172167), Anaplasma marginale (AF414873), Wolbachia (KF249715), Brucella melitensis (CP002932), Brucella chilestrain (AY513509), Rickettsia felis (NR_074483), R. rickettsii (L36217), and Coxiella burnetii (D89798)  . These s species . The priBartonella FRET-qPCR was performed in a LightCycler 480® II real-time PCR platform with 20 μl final volumes containing 10 μl extracted DNA. The primers and probes were 1 μM and 0.2 μM in the final reaction system, respectively. The qPCR buffer was established with a final concentration of 20 mM Tris-HCl, 4.5 mM MgCl2, 0.05% Tween 20, and 0.05% Nonidet P-40. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles and 40 low-stringency fluorescence acquisition cycles for 1 sec at 95°C, 12 sec at 58°C, 30 sec at 67°C, and 10 sec at 72°C. Melting curve analysis was performed by monitoring fluorescence between 38°C and 80°C. All the PCRs were run in duplicate in this study.The pan-Bartonella henselae DNA from a cat in St. Kitts [Bartonella samples with the available Bartonella sequences in GenBank. The specificity of the PCR was further verified using the related species Brucella melitensis, Brucella chilestrain, Wolbachia, Coxiella burnetii, Rickettsia felis, Rickettsia rickettsii, Anaplasma phagocytophilum, and Anaplasma marginale .t. Kitts  was usedB. henselae identified in this study. The amplification products were gel purified using the QIAquick Gel Extraction Kit  and quantified using the PicoGreen DNA fluorescence assay . The molarity of the DNA was estimated using the calculated molecular mass of the amplicons and dilutions made to give solutions containing 10,000, 1,000, 100, 10, and 1 gene copies/μl in T10E0.1 buffer. These were used as quantitative standards in the FRET-qPCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples. Twofold dilutions of 10 and 1 gene copies/μl solution were used to determine the minimal detection limit.For quantitative standards, we used amplified DNA of Bartonella FRET-qPCR we established was based on a region of the 16S rRNA that is highly conserved among different Bartonella spp. and is thus ideal for screening samples for the organisms. To differentiate Bartonella spp. that gave positive reactions in screening pan-Bartonella FRET-qPCR, we used a standard PCR that amplifies a relatively highly polymorphic region of the citrate synthase gene (gltA) (439 nucleotides for different Bartonella spp.) and sequenced the products . For the gltA-based Bartonella PCR, we designed, as above for the pan-Bartonella FRET-qPCR, a forward primer (5′-CGTAATGATCTYAGTTAYGCTGCAAA-3′) and a reverse primer (5′-AGAAGTGGATCATTTTGAATRTTBARYTC-3′) that amplify all the 18 better-recognized Bartonella spp. available in GenBank  at the concentration of 1 ng/μl. The calculation of test precision and test variability is based on the CP variation from the CP mean value. Test reproducibility for all investigated quantitative standards was lower in intratest experiments (<3.8%) and was higher in intertest experiments (<6.1%). The reproducibility dropped in lower copies of standards (1 and 10 copies per reaction) in comparison with the high copies of standards .To confirm accuracy and reproducibility of real-time PCR, the intra-assay precision was determined in three repeats within one LightCycler run. Interassay variation was investigated in three different experimental runs, while the quantitative standards  were diluted in 1 × TBartonella FRET-qPCR. Cats from all the sites we studied were positive, mainly from Beijing (11.1%), Shanghai (5.6%), Guangdong (15.0%), Yangzhou (19.4%), and St. Kitts (39.7%)  were positive in the pan-Bartonella FRET-qPCR, while only one donkey (5.0%) from China was positive. Horses, sheep, and donkeys from St. Kitts were negative, but cattle from the island were commonly positive (54.8%) in the pan-Bartonella FRET-qPCR, although cattle from China were negative.Of the 310 cat blood samples we examined, 78 (25.2%) were positive in our pan- (39.7%) . The 60 Bartonella FRET-qPCR were considerably higher in the positive blood samples we collected (106.20/ml whole blood). Fleas positive for B. henselae generally had low copy numbers, but those positive for B. clarridgeiae had considerably higher copy numbers (107.01 vs. 102.88).In general, the copies detected in the pan-gltA-based Bartonella PCR did not amplify the negative controls but did amplify the positive control B. henselae with an amplicon sequence identical to those for this species in GenBank. It also amplified all the test samples that were positive in the pan-Bartonella FRET-qPCR, indicating the tests had similar sensitivity. All of the cats that were positive in the pan-Bartonella FRET-qPCR were also positive in the gltA-based Bartonella PCR. Sequencing of the products of the gltA-based Bartonella PCR revealed B. henselae was most prevalent , followed by B. clarridgeiae   and B. clarridgeiae  were identified with the gltA-based Bartonella PCR. Each of the positive fleas was found on a cat positive for the same Bartonella sp.The ; 19/78) . Both spgltA-based Bartonella PCR that were positive with the cattle from St. Kitts showed only B. bovis was present; this organism was also present in the one positive Chinese donkey  and specific, quantitative, and rapid , because it was designed against a highly conserved region of the 16S rRNA, it did not enable us to determine the Bartonella spp. in the samples. This was possible, however, with the gltA-based Bartonella PCR which was as accurate and specific as the pan-Bartonella FRET-qPCR in identifying Bartonella spp. in our study. Scola et al. first reported that, of the commonly used genetic targets for Bartonella identification, only gltA and rpoB sequences provide sufficient discriminatory power and interspecies diversity to allow discrimination of Bartonella spp. [gltA as the target of our PCR and selected our primers to ensure that, although they only amplified Bartonella spp., they covered a 439 bp hypervariable area of the gene that enabled differentiation of the recognized and poorly characterized Bartonella spp. recorded in GenBank  which comprise the most commonly used samples to date in epidemiological studies. Although the gltA-based Bartonella PCR was more time-consuming, requiring gel electrophoresis to reveal amplicons, it identified all the samples that were positive in the pan-Bartonella FRET-qPCR.Applying our pan-Bartonella henselae and B. clarridgeiae were the species most commonly identified by the gltA-based Bartonella PCR in our study. These are the organisms most commonly associated with cats and their fleas and have already been described to be prevalent in the West Indies [C. felis [B. bovis at a high level in cattle from St. Kitts, we failed to identify the organism in cattle from China. We did, however, find B. bovis in a donkey in China, indicating the organism is present in the country. Bartonella bovis is a recently described species [Bartonella schoenbuchensis and Bartonella chomelii. The latter have mainly been described in Europe or in cattle originating from Europe [B. bovis has been described from widely around the world with prevalences in cattle varying from 0% in Kenya and Japan [B. bovis in equids although horses seropositive against the organism have been described and experimental infections generally cause no clinical signs with no bacteremia and a low-grade and short-duration antibody response [B. bovis has not been determined but is suspected to be a blood-sucking arthropod [t Indies  and Chint Indies  where thC. felis . Althoug species  that is m Europe , while Bnd Japan  to up tond Japan . Althougnd Japan , most innd Japan . There aresponse . The vecrthropod .16S rRNA-based pan-Bartonella FRET-qPCR and the gltA-based Bartonella PCR we developed was based on the alignment of the available recognized Bartonella spp. sequences in GenBank. For completeness, the newly established PCRs should be tested on known positive control samples of DNA of all the recognized and poorly characterized Bartonella spp., but it is no simple matter for one laboratory to obtain all of these samples. Similarly, with the rapid growth in the recognized and suspected Bartonella species, it will be important for ongoing testing of the PCR to ensure it recognizes the newly recognized strains and species. Ideally, the established Bartonella PCRs in this work should be compared with other existing molecular approaches for their sensitivity and specificity and validated fully following the MIQE guidelines for publication of quantitative real-time PCR experiments [The specificity of the eriments .Bartonella species. We also describe a gltA-based PCR that enables the differentiation of the recognized Bartonella spp. The pan-Bartonella FRET-qPCR enabled us to rapidly and quantitatively screen a variety of samples from a number of sources and different geographical areas for the presence of Bartonella spp., while the gltA-based PCR provided a convenient method to differentiate the species involved using only a single reaction.In conclusion, in this report, we describe what we believe to be the first FRET-qPCR that specifically detects all the currently recognized


We investigate if different factors influence whether girls versus boys meet diagnostic criteria for attention-deficit/hyperactivity disorder(ADHD) among children with high ADHD symptoms. Participants were 283 children aged 7–12 from a population-based study. Girls and boys meeting diagnostic criteria for ADHD, based on an objective investigator-based interview, were compared to children who did not meet criteria despite high symptoms on a rating-scale measure of ADHD. We assessed factors that could differentially relate to diagnosis across girls and boys including ADHD symptoms, co-occurring behavioural/emotional problems and impairment, and sex-effects in rater perceptions of ADHD symptoms. While overall similar factors distinguished girls and boys who met diagnostic criteria from high-symptom peers, effect sizes were larger in girls. Emotional problems were particularly salient to distinguishing diagnosed versus high-symptom girls but not boys. Parents rated boys meeting diagnostic criteria as more impaired than high-symptom boys but did not do so for girls, and under-rated diagnosed girls’ hyperactive/impulsive symptoms compared to more objective interview assessment, with the opposite observed in boys. Results suggest girls’ ADHD may need to be made more prominent by additional behavioural/emotional problems for them to meet full diagnostic criteria and that sex differences in parental perceptions of ADHD behaviours and impairment exist. The ratio of boys to girls diagnosed with ADHD in childhood falls in the range of 2:1 to 10:1 , with hiA common explanation for the observed sex differences in referral and diagnosis is that girls with ADHD are more likely to present with predominantly inattentive symptoms, rather than the more potentially disruptive hyperactive/impulsive symptoms, as well as greater levels of internalising symptoms such as anxiety and depression which might lead to alternative diagnoses . In contIf sex-specific stereotypes of ADHD exist, then it is possible parents and teachers may not as readily recognise manifestations of ADHD in girls compared to boys. Furthermore, sex differences in recognition of ADHD may in part reflect bias in the diagnostic criteria, or the way they are applied. For example, if diagnostic criteria are based on a male presentation of the disorder then females may be less likely to meet full diagnostic criteria .The male preponderance could also be due to underlying aetiology of ADHD in relation to a ‘female protective effect’. This model proposes that females require greater genetic and environmental ‘load’ or exposure to factors associated with ADHD to manifest the same degree of impairment and warrant diagnosis as males with ADHD . PartialSeveral methodological limitations exist in the study of sex differences in ADHD. First, many studies of ADHD are comprised of predominantly (or exclusively) male participants, limiting our understanding of ADHD in females; although efforts to investigate ADHD in females are increasing e.g., . FurtherIt is important, especially for clinical practice, to understand more about phenotypic differences in boys and girls with ADHD , includiTo address the methodological issues detailed above, the current study examined data from a population-based twin sample, overcoming issues of referral and clinic bias. Separate trait and diagnostic measures of ADHD were available which extends methodology typically used in population-based samples. We examined what characteristics distinguish girls meeting diagnostic criteria  from girls who do not despite high ADHD symptom levels on a rating-scale measure of ADHD, and if the same distinguishing characteristics operate in boys. We examined core ADHD symptom dimensions, co-occurring behavioural and emotional problems, and impairment. We also assessed sex-dependent biases in parental perceptions of ADHD symptoms by examining whether, despite their offspring meeting diagnostic criteria, parents systematically under- or over-rate relative to the PACS, and if this differs for boys and girls.22.1M = 9.33 years, SD = 0.77) and 130 (38% girls) who showed a high level of ADHD symptoms based on parental report using DSM-5 symptom criteria but did not meet full diagnostic criteria . We refer to the groups as the ‘diagnosed ADHD’ group and the ‘high-symptom’ group. Further details of the PACS is given in the measures section and details on how the diagnosis is made can be found in the supplementary material. We defined a high level of ADHD symptoms as the presence of 5 or more symptoms (out of 18), based on definitions used in previous studies (Participants were 283 children: 153 (21% girls) who met DSM-5 research diagnostic criteria for ADHD based on the Parental Account of Childhood Symptoms (PACS) investigator-rated parental interview  (data was collected for both children in the twin pair regardless of whether one or both had been identified as being at risk for ADHD). Exclusion criteria were: autism spectrum disorder, learning disability, and neurological disability. The current study used a subsample of these children (n = 283) as detailed above.Participants were part of the Developmental Pathways to Hyperactivity and Attention Deficit Study (PHAD), a sub-study of children at risk for ADHD identified from the Twins Early Development Study (TEDS), which is a population-based study of over 15,000 twin pairs born in the United Kingdom between 1994 and 1996, followed up prospectively from the age of 18 months. Details of TEDS is described elsewhere , and det2.22.2.1Parents completed a checklist assessing the 18 DSM-IV items for inattention (9 items) and hyperactivity/impulsivity (9 items) . Each it2.2.2The Parental Account of Childhood Symptoms (PACS) was used to identify children who met diagnostic criteria for ADHD. The PACS is an investigator-rated semi-structured interview developed as a standardised measure for use in assessing and recording accurately the behaviours of children. Parents are asked to describe the behaviour of their child across a range of situations in relation to its frequency and severity, which is then scored according to a standardised operational scale . As such2.2.3The Strengths and Difficulties Questionnaire (SDQ) was used to assess behavioural and emotional problems using mothers ratings . The SDQ2.2.4The SDQ impact supplement comprises items related to overall distress and impairment which can be used to generate an impact score ranging from 0 to 10 . The PACS includes questions on both impairment and school behaviour, as related to the child's inattention and/or hyperactivity/impulsivity problems. The impairment items include: ‘problem is cause for concern’, ‘serious problem perceived/much concern’, ‘no control over behaviour’, ‘serious impairment/social impact of problem’, and ‘interviewer rates problem as markedly or severely abnormal’. Items were scored on likert scales which were used to create dichotomous variables for ‘yes’ or ‘no’ categories . We then calculated a total impairment score for each participant ranging from 0 to 5. School behaviour items were scored as ‘yes’ or ‘no’ and included: ‘child shows distress’, ‘problems getting on with others’, ‘difficulty concentrating’, ‘change of school due to problems’, ‘suspended or excluded’, ‘special educational provision’, ‘complaints about hyperactivity’, and ‘complaints about aggression’ . We did not include the ‘difficulty concentrating’ item in analysis due to it being so closely linked with the definition of ADHD. ‘Change of school due to problems’ and ‘suspended or excluded’ were also omitted from analysis due to so few participants endorsing these items. A total school impairment score was derived for each participant ranging from 0 to 5.2.3cluster(variable) function in Stata 14  and sex . A significant sex-by-diagnostic group interaction would indicate that certain characteristics are greater in diagnosed versus high-symptom individuals and in one sex compared to the other.To elucidate potential sex biases in parent report of ADHD symptoms we examined whether, despite both sexes meeting diagnostic criteria, parents systematically under- or over-rate ADHD symptoms compared to the PACS interview assessment, and if this differs by sex. We tested for an interaction between sex and measure  separately for total scores on the inattention and hyperactivity/impulsivity scale in the diagnosed group of children using linear regression. A significant interaction would suggest ADHD symptom score differs as a function of the type of measure and that this difference is greater in one sex compared to the other, or that the direction of the difference differs in girls and boys.3p = .10, d = 0.20), fulfilling a critical assumption on which the study design lies .Of the 153 children meeting PACS diagnostic criteria for ADHD, 121 were boys (79%) and 32 were girls (21%), giving a male-to-female ratio of 3.8:1. Of the 130 children in the high-symptom group, 81 were boys (62%) and 49 were girls (38%), a male-to-female ratio of 1.7:1. The ratio of diagnosed to high-symptom girls was 0.65:1 compared to 1.5:1 for boys. The diagnosed and high-symptom groups did not differ significantly in the total number of symptoms present based on the ADHD symptom measure  and were more prosocial , compared with equivalent boys , relating to overall distress and impairment, compared to boys meeting diagnostic criteria. Few characteristics distinguished girls and boys in the high-symptom group, except that girls were significantly more prosocial , had lower levels of conduct problems , and were less likely to have complaints about hyperactivity at school  compared to boys , conduct , and peer problems , as well as higher total problem scores   than the high-symptom girls, but the SDQ parent-rated impairment measure did not distinguish the two groups. At school, diagnosed girls received more complaints about hyperactive behaviour  and had higher overall school impairment scores . Girls meeting diagnostic criteria were also 3.89 times  more likely to receive special educational provision (p = .03) than those with high ADHD symptom scores.Compared to high-symptom girls, girls meeting PACS diagnostic criteria had significantly higher reported levels of emotional ( = 0.95) . The PAC3.1.2p = .03, d = 0.32)  and peer problems , and higher total problem scores . Greater impairment was demonstrated in boys meeting diagnostic criteria than high-symptom boys on all the impairment measures  , higher ures see .3.1.3d = 0.65, p = .02), while this characteristic did not distinguish diagnosed and high-symptom boys . Both girls and boys meeting PACS diagnostic criteria were distinguished from their high-symptom peers by greater total problem scores on the SDQ, and specifically conduct and peer problems, but the magnitude of the effect was greater in girls . In addition, magnitude of effect sizes suggested that prosocial behaviour distinguished diagnosed girls versus high-symptom girls but not boys, although this did not reach conventional levels of significance .Overall there were no significant sex by diagnosis interactions, suggesting that many of the same factors distinguished high-symptom children from those who met diagnostic criteria in both boys and girls . There wd = 0.61 vs girls: d = 0.33). Whereas the more objective PACS measure of impairment distinguished children meeting full diagnostic criteria from the children with high-symptoms who did not meet full diagnostic criteria in both girls and boys; however, the magnitude of this difference was greater in girls (d = 1.05 vs 0.78).The SDQ total impact score (parent-rated measure of impairment) only distinguished between the PACS diagnosed and high-symptom boys, with almost double the effect size (boys: p = .07). Problems getting on with others and complaints about aggression similarly distinguished diagnosed boys and girls from their high-symptom peers, but the difference was only significant for diagnosed versus high-symptom boys.The total school impairment score distinguished PACS diagnosed from high-symptom children in both girls and boys, with similar effect sizes. PACS diagnosed boys were 2.38 times  more likely to show distress at school than high-symptom boys, but PACS diagnosed and high-symptom girls were equally as likely to show distress at school . In both girls and boys, the likelihood of children meeting diagnostic criteria having complaints about their hyperactivity at school was greater than in high-symptom children, but the odds were greater for girls  indicating that parents tend to under-rate girls and over-rate boys for the presence of hyperactive/impulsive symptoms compared to the PACS  and ‘exhibits a consistent pattern of restlessness’ (50.1% lower). In diagnosed boys hyperactive/impulsive symptom frequencies were higher in the parent-rated scale than the PACS, except for leaves seat (1.7% lower on the parent-rated measure), runs about (20.6% lower), and interrupts or intrudes (26.5% lower). This pattern was reflected in the total score, and analyses found a significant sex-by-scale interaction for hyperactivity/impulsivity  effect sizes were greater in girls, and were not due to the diagnosed and high-symptoms girls having a greater difference in ADHD symptoms compared to the equivalents groups of boys. The prominence of emotional symptoms in girls meeting diagnostic criteria suggests that this characteristic may be more important to the female phenotype and that girls may express their difficulties differently to boys. Higher rated emotional problems in girls than boys with ADHD has been shown previously . It is pParent-rated impairment using the SDQ did not distinguish between diagnosed versus high-symptom girls; yet it did in the equivalent groups of boys. This is an important finding with regard to sex differences in ADHD as referral based on parent concern requires recognition of impairment, yet parents appear to be less able to spot impairment among girls. One interpretation of these findings is that parents may not be as good at judging impairment in girls, highlighting that objective measures of impairment are especially important in the assessment of girls’ ADHD symptoms . Some diOne characteristic that may influence the perception of impairment is prosocial behaviour. Not only is it clear that social functioning is likely to be linked with perceptions of impairment and coping, socially adaptive behaviour may mask symptoms and impairment to informants . It appeFinally, we also found sex-dependent biases among parental perceptions of ADHD symptoms in children meeting diagnostic criteria. Parents under-rated diagnosed girls hyperactive/impulsive symptoms compared to the more objective accounts from the PACS, with the opposite pattern observed in boys. These findings suggest that sex-specific biases in perceptions of child behaviour may exist. As with the parental under-report of impairment in girls discussed previously, this also has clinical implications for the referral and subsequent diagnosis of girls with ADHD symptoms. Parents may be less likely to take girls for assessment if they perceive them to display less stereotypical ADHD behaviours (as well as being less impaired). It may also contribute to a systematic bias in diagnostic practice, as it may influence the identification and interpretation of symptoms by clinicians if they are relying on parental reports.The present study has several strengths. We extended the methodology typically used in population-based studies by incorporating a comprehensive diagnostic interview and were therefore able to investigate sex differences in the factors that affect whether children with high ADHD symptoms meet diagnostic criteria in a population-based sample. In addition, the use of an objective, investigator-rated interview enabled investigation of potential sex-specific biases in parental report of ADHD symptoms. We have begun to tackle some of the possible contributing factors to the sex differences that could impact the referral and diagnostic process, but replication is needed.However, some limitations should be noted. While sizeable for a study of its kind, there was a mismatch in the number of girls to boys, which is a common issue for studies of sex differences in ADHD. This reduces the power in interaction analyses to detect group differences and if these interactions were small they may have been missed; analyses should be repeated in a larger sample. This is also important to better elucidate findings that approached statistical significance. Also, in the current study we were not looking at who gets diagnosed in clinical settings, but rather investigating sex differences associated with meeting diagnostic criteria in a unique sample with less selection than studies of children who present to clinics. Unfortunately, as with many population-based studies, we do not have information on which individuals were actually referred, and so it is not possible to infer directly what the implications of the findings are on the referral and diagnostic process in clinical practice. Finally, it is important to note that our study was carried out in a twin sample. A concern when using twin samples is the generalisability of results to singletons. For example, relative to singletons, twins are more likely to have lower birth weight  and be bIt is clear that research is needed to identify the best screening methods, most accurate informants, and most appropriate thresholds for the diagnosis of ADHD, which may differ for boys and girls . It is lIn summary, these data suggest that factors which distinguish girls who meet full ADHD diagnostic criteria from high-symptom peers who do not may be somewhat sex specific, with additional behavioural and emotional problems playing a larger role in distinguishing diagnosed from high-symptom girls than the equivalent male comparison. Additionally, we found different parental perceptions of ADHD behaviours as shown by our comparison of parent report to a more objective measure of ADHD symptoms. Such differences may explain why girls are less likely to be referred for their ADHD behaviours. This may also contribute to the relatively low recognition rate of ADHD in girls in clinical practice if girls with ADHD are perceived to display less stereotypical disruptive ADHD behaviours and perceived as less impaired by symptoms than boys, especially in the presence of socially adaptive behaviour and more internalising emotional symptoms. From a clinical perspective, our findings highlight the importance of detailed interview assessments in the diagnostic process, especially for girls who may not be identified with rating-scale measures which are more subject to sex biased perceptions of behaviour, and that emotional problems should not be used to rule out an ADHD diagnoses.


Enterovirus A71 (EVA71) is a human enterovirus belonging to the Picornaviridae family and mostly causes hand-foot-and-mouth disease in infants. Viperin is an important interferon-stimulated gene with a broad antiviral activity against various viruses. However, the effect of viperin on human enteroviruses and the interaction mechanism between EVA71 and viperin remains elusive. Here, we confirmed the EVA71-induced expression of viperin in a mouse model and cell lines and showed that viperin upregulation by EVA71 infection occurred on both the mRNA and protein level. Viperin knockdown and overexpression in EVA71-infected cells indicated that this protein can markedly inhibit EVA71 infection. Interestingly, immunofluorescent confocal microscopy and co-immunoprecipitation assays indicated that viperin interacts and colocalizes with the EVA71 protein 2C in the endoplasmic reticulum. Furthermore, amino acids 50–60 in the N-terminal domain of viperin were the key residues responsible for viperin interaction with 2C. More importantly, the N-terminal domain of viperin was found responsible for inhibiting EVA71 replication. Our findings can potentially aid future research on the prevention and treatment of nervous system damage caused by EVA71 and may provide a potential target for antiviral therapy. The antiviral response of the innate immune system is mainly mediated by the activated expression of interferon stimulated gene (ISG) . HundredEnterovirus A71 (EVA71) is a human enterovirus belonging to the Picornaviridae family and is one of the main pathogens of hand-foot-and-mouth disease, which has had more than 10 outbreaks worldwide since it was first reported in 1969  and affeViperin is an ISG that is highly conserved from lower vertebrates to mammals. It is structurally divided into an N-terminal double alpha helical domain, intermediate SAM region, and the C-terminal domain. The double alpha helical region localizes viperin to the ER and cell membrane lipid raft structure, while viperin antiviral activity is associated with the C-terminal domain. The antiviral area in the C-terminus is highly conserved and is the key domain structure that suppresses replication of hepatitis C virus (HCV) and dengue virus (DENV) . An incrStudies have shown that viperin suppresses the replication of influenza A and HIV-1 by blocking the release of viral particles ,14. MoreIn this study, we showed that viperin mRNA is upregulated in EVA71-infected human astrocytoma cells. Furthermore, we aimed to confirm the effects of EVA71 infection on viperin expression and to investigate the capacity of viperin in functioning as an antiviral protein against EVA71. Moreover, we attempted to verify the interaction between EVA71 and viperin in cells and found that EVA71 upregulated viperin expression in both the mouse and cell infection model. Viperin also interacted with the 2C protein of EVA71 and inhibited viral replication through its N-terminal domain. Therefore, our findings provide insights into the pathogenesis of EVA71 neurological complications and offer a potential target for antiviral therapy2 and 37 °C.Human embryonic kidney 293T cells  and human rhabdomyosarcoma (RD) cells (ATCC-CCL-136) were cultured in Dulbecco’s modified Eagle’s medium . Human neuroblastoma cells SK-N-SH cells (ATCC-HTB-11) were cultured in minimum essential medium . Human astrocytoma CCF-STTG1 cells (ATCC-CRL-1718) were cultured in RPMI 1640 . All cells were maintained in medium containing 10% fetal bovine serum  and incubated at 5% COCells were plated in six-well plates overnight and transfected with 2 µg of plasmid per well or siRNA with the Calcium Phosphate-mediated ProFection Mammalian Transfection System  or Lipofectamine™ 2000 reagent  according to manufacturer’s instructions. CCF-STTG1 cells and 293T cells were plated in six-well plates overnight and infected with EVA71 (BrCr strain) for the indicated time points.The main antibodies used were anti-EVA71 VP1 rabbit poly-antibody (produced in-house), rabbit anti-HA , mouse anti-FLAG , and rabbit anti-viperin . Three siRNA sequences targeting the viperin transcript  and negative control siRNA were designed and synthesized  were ordXhoI and NotI restriction enzyme digestion.FLAG-viperin and GFP-viperin were constructed by inserting the PCR products obtained using the primers described in 5 TCID50/mouse. Subsequently, mice body weight and symptoms were recorded every day. The mice were sacrificed after they displayed oblivious posterior paralysis. Brain tissues of mice infected with EVA71 or injected with control PBS buffer were obtained for immunohistochemistry and RNA isolation.Two-week-old BALB/c mice were purchased from HuBei Center for Disease Control (CDC)  and were infected with 100 µL of high-virulence EVA71 (GZ-CII strain) through an intraperitoneal injection at a dose of 10All studies were conducted in strict accordance with the institutional guidelines for animal research and approved by the Administration of Affairs Concerning Experimental Animals of the People’s Republic of China. All mice were housed under specific-pathogen-free conditions in individually ventilated caging systems. All animal experiments were reviewed and approved in advance by the Ethics Committee of the Animal House Facility at the Wuhan Institute of Virology, Chinese Academy of Sciences .2O2 in methanol for 20 min. The sections were then blocked in TBS containing 10% normal rabbit serum  and 0.2% bovine serum albumin (BSA) for 30 min with shaking at room temperature. Subsequently, brain sections were incubated with a mouse anti-EVA71 VP1 antibody and rabbit anti-viperin antibody (1:200) overnight at 4 °C. The sections were washed in TBS and incubated with Texas Red-labeled and fluorescein isothiocyanate-labeled secondary antibodies  for 1 h at room temperature. The sections were washed in TBS five times, counterstained with DAPI  for 8 min at room temperature, washed in TBS, and then embedded in Mowiol . Slides were cover-slipped with Vectashield Mounting Medium  and imaged under a fluorescent microscope.The brain tissues of mice were embedded in paraffin and cut into 5-μm sections. After deparaffinization and rehydration, the sections were incubated in Tris-buffered saline (TBS) containing 0.1 Triton X-100 for 20 min at room temperature and then washed for 10 min in TBS. To eliminate endogenous peroxidase, the sections were treated with 2% Hg for 15 min at 4 °C and the supernatants collected in 1.5 mL tubes. After adding 2 µL anti-FLAG antibodies to the supernatants, the tubes were gently shaken overnight at 4 °C. Approximately 20 µL of Protein G Agarose beads  washed five times with PBS were then added to the tubes and gently shaken at room temperature for 1 h. The beads were washed with 500 µL 0.02% PBST (1× PBS + 0.02% Triton 100) five times before adding 100 µL 2× sodium dodecyl sulfate (SDS) loading buffer and boiling for 10 min to separate immunoprecipitates from the beads. The immunoprecipitates were then subjected to SDS polyacrylamide gel electrophoresis and western blotting.After 293T cells were transfected with expression plasmids or vectors for 24 h, the cells were harvested and lysed with western and IP lysis buffer containing the protease inhibitor phenylmethylsulfonyl fluoride. The cells were then centrifuged at 12,000× 2) of the standard curve >0.99, and the amplification efficiency ranging between 95% and 110%. GAPDH was used as normalization control for obtaining the relative expression levels of VP1. The primers used are indicated in Total RNA was extracted using TRIzol reagent  according to manufacturer’s instructions. RT-PCR was performed using 1 µg RNA with a random primer and reverse transcriptase . Quantitative PCR was performed using SYBR Green Master Mix  and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Each reaction ran in triplicate following the protocol of our previous study . Calibra293T cells were transfected with GFP-viperin and HA-2C plasmids using a Lipofectamine™ 2000 reagent according to manufacturer’s instructions. After 24 h, cells were washed with 3% normal goat serum (NGS) in PBS , fixed with 4% paraformaldehyde for 15 min, washed, permeabilized with 0.2% Triton X-100 in PBS for 15 min, washed, blocked with 5% NGS and 2% BSA in PBS for 1 h, and then washed three times. For immunostaining of 2C-HA, cells were incubated with rabbit anti-HA antibody overnight at 4 °C. After five washes (10 min each), the cells were incubated with TRITC-conjugated goat anti-rabbit IgG  for 1 h at room temperature. After five washes (10 min each), cell nuclei were stained with Hoechst 33258  and washed three times (5 min each). For ER staining, cells were fixed with 4% paraformaldehyde and then ER-Tracker Blue-White DPX  were applied before fixation. Fluorescent images were obtained using a Perkin Elmer UltraView VOX confocal microscope equipped with 405 nm (for violet fluorescence of the stained cell nuclei), 488 nm (green fluorescence), and 561 nm (red fluorescence) excitation lasers. The cells were imaged with a 60× oil immersion objective.p < 0.05.All experiments were repeated and conducted in triplicate. Data are represented as the mean ± standard deviation (SD) where indicated and used for all statistical analyses with GraphPad Prism 6.0 software . Differences were considered statistically significant when To confirm the effects of EVA71 on viperin expression, we analyzed viperin expression in EVA71-infected cell lines and a mouse model. In EVA71-infected RD cells, the viral RNA of EVA71 was found to be significantly increased at each indicated time point post-infection A, while 4 copies of EVA71 genomic RNA were detected in per mg total RNA extracted for the EV71 infected mouse brain  . After 4se brain B. Additise brain C. This rTo determine the effects of viperin on EVA71 infection, viperin was overexpressed and knocked down during EVA71 infection. 293T cells were transfected with a FLAG-viperin expression plasmid or a control empty plasmid. At 24 h post-transfection, cells were infected with EVA71. As shown in Viperin is localized in and is associated with the ER . In addiTo verify whether EVA71 2C binds to viperin, we utilized a co-IP assay to confirm the interaction between 2C and viperin. FLAG-viperin and HA-2C plasmids were used to co-transfect 293T cells and then the cells were lysed 24 h post-transfection. HA-2C was detected in the precipitate with the anti-FLAG antibody A. FurtheTo identify the viperin domain responsible for interacting with 2C, a series of mutants was constructed based on the functional domains of viperin. As illustrated in To determine the amino acids in the N-terminal domain responsible for binding to 2C, we analyzed interactions between more viperin mutants and 2C using co-IP. The FLAG-tagged viperin, viperin mutants , and HA-2C plasmids were co-transfected into 293T cells. At 24 h post-transfection, the cells were lysed and an anti-HA antibody was used to detect the 2C protein immunoprecipitated with the anti-FLAG antibody. Surprisingly, we found that ΔN-60aa, ΔN-65aa, and ΔN did not interact with 2C, whereas other mutants, including ΔN-50aa, could bind to 2C, similarly to the full-length viperin C. TherefGiven that the N-terminal domain is required for viperin binding to 2C, we examined whether the N-terminal domain is responsible for the inhibition of EVA71 replication. We constructed various mutants of viperin FLAG-N-viperin (only including the N-domain) and FLAG-C-viperin (only including the C-domain). Viperin, N-viperin, C-viperin, ΔN, ΔSAM, and ΔC plasmids were transfected into 293T cells at 24 h post-transfection and then the cells were infected with EVA71 for 12 h. q-PCR and western blotting were used to analyze the effect of viperin mutants on EVA71 infection. Mutants with an N-terminal domain deletion (ΔN and FLAG-C-viperin) exerted minimal effects on the levels of EVA71 viral RNA A and VP1In this study, we showed that viperin was induced by EVA71 infection both in vitro an in vivo and that viperin inhibited EVA71 replication. Furthermore, the 2C protein of EVA71 was found to interact with viperin through its N-terminal domain, which was also the key domain responsible for the antiviral function of viperin. Our findings suggest that viperin suppressed EVA71 infection via interacting with 2C through the viperin N-terminal domain. To the best of our knowledge, our study is the first to report this interaction in human enteroviruses.Activation of the innate immune system following RNA viral infection results in the production of type I IFNs and the production of hundreds of ISGs ,31. IFNsViperin has been demonstrated to inhibit a broad range of viruses from both DNA and RNA viral families, yet the specific antiviral mechanisms of viperin in different viral infections are diverse . The antStructurally, viperin is composed of three distinct domains, an N-terminal domain, a highly conserved central domain containing a radical SAM domain, and a C-terminal domain . The speAt present, the effect of viperin protein on enteroviruses is unknown. As a human enterovirus, the siRNA-mediated knockdown of viperin increases rhinovirus replication in infected epithelial cells . In our Overall, we demonstrated that viperin can be induced during EVA71 infection both in vitro and in vivo and that viperin suppressed EVA71 infection significantly. Furthermore, we found that the 2C protein of EVA71 interacts with viperin. The mechanism behind viperin suppression of EVA71 infection depends on this interaction between 2C and the N-terminal domain of viperin, which interferes with the activity of the EVA71 RC. Our findings may be helpful for the future development of new antiviral strategies that utilize viperin.


The continuity of neurodevelopmental disorders from childhood to adulthood has garnered much attention. However, three studies have recently examined the possibility that adult-onset attention-deficit hyperactivity disorder (ADHD) is a separate neurodevelopmental disorder from childhood-onset ADHD –3. A cohAfter careful consideration of these studies –3, we deSimilar to the studies discussed above, neuroimaging studies have also elucidated the characteristics of each group. Only children with ADHD showed shrinkage in regions of the brain cortex , possiblWe considered the relationship between the expression of ADHD symptoms and social adaptation ability Fig. . IndividPossibly, in cases of adult-onset ADHD, the patients had a good social environment during childhood, with strong family support, a stable school environment to which they could adapt, or sufficient intelligence to compensate for their weaknesses. This may be why they were not prone to adaptation difficulties at that stage . HoweverHence, if individuals with and without neurodevelopmental disorders are both exposed to comparable stress, the former are more likely to experience difficulties in adapting. The difference between childhood-onset and adult-onset ADHD could be whether the difficulties in adaptation that are characteristic of neurodevelopmental disorders occur in childhood or adulthood. It, therefore, seems logical to regard these as forms of the same disease rather than as separate disorders , 8. We a


Updating the worldwide prevalence estimates of attention-deficit hyperactivity disorder (ADHD) has significant applications for the further study of ADHD. However, previous reviews included few samples of Chinese children and adolescents. To conduct a systematic review of ADHD prevalence in Mainland China, Hong Kong, and Taiwan to determine the possible causes of the varied estimates in Chinese samples and to offer a reference for computing the worldwide pooled prevalence. We searched for PubMed, Embase, PsycINFO, Web of Science, China National Knowledge Infrastructure, VIP, WANFANG DATA, and China Science Periodical Database databases with time and language restrictions. A total of 67 studies covering 642,266 Chinese children and adolescents were included. The prevalence estimates of ADHD in Mainland China, Hong Kong, and Taiwan were 6.5%, 6.4%, and 4.2%, respectively, with a pooled estimate of 6.3%. Multivariate meta-regression analyses indicated that the year of data collection, age, and family socioeconomic status of the participants were significantly associated with the prevalence estimates. Our findings suggest that geographic location plays a limited role in the large variability of ADHD prevalence estimates. Instead, the variability may be explained primarily by the years of data collection, and children’s socioeconomic backgrounds, and methodological characteristics of studies. As a major public health problem4, ADHD has been associated with a wide variety of adverse health outcomes for affected individuals6 and severe financial burdens for families and societies7. Concerns have been raised regarding the true prevalence of ADHD among children, the knowledge of which is critical for further service planning, resource allocation, training, and research priorities8. In the last few decades, a host of investigators have made substantial efforts to determine the prevalence of ADHD. Several reviews reported a broad range of prevalence rates, from as low as nearly 1% to as high as nearly 20% among children and adolescents throughout the world14. A comprehensive review including 102 studies worldwide reported a pooled prevalence estimate of 5.3% in children11. Another review covering 86 studies found that the prevalence estimates only employing the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) as the diagnostic criteria varied from 5.9% to 7.1% in children and adolescents14. However, previous systematic reviews seldom selected a sufficient proportion of studies conducted among Asian children and adolescents, and were especially lacking of Chinese samples, despite the fact that China has the largest number of children and adolescents in the world.Attention-deficit/hyperactivity disorder (ADHD) is one of the most common childhood psychiatric disorders, with symptoms including inattention, impulsivity, and hyperactivity15. With the introduction of the Diagnostic and Statistical Manual of Mental Disorders, Third Edition (DSM-III) into China in the 1980s, multifold epidemiological surveys have been carried out on children and adolescents in Mainland China, Hong Kong, and Taiwan, and yielded the prevalence estimates ranging from as low as 0.7% to as high as 14.1%17. Our previous systematic review published in Chinese included 33 studies conducted in Mainland China from 1983 to 2011 and reported a pooled ADHD prevalence of 5.7% in Chinese children and adolescents18.To our knowledge, the first investigation of Minor Brain Dysfunction  prevalence among children was conducted in Mainland China in 198119. Given that the scope of this systematic review falls on the Chinese children and adolescents in Mainland China, Hong Kong and Taiwan, it is noteworthy to mention the features of social-cultural contexts in those three regions. For instance, compared with Hong Kong and Taiwan, the children in Mainland China have experienced more intensely unbalanced development, due to historical reasons and social transformations20. Furthermore the significant gaps in the economic situations among different Urban and rural areas within the Mainland China may fuel the diverse epidemiological aspects of ADHD. More importantly, the long-term one-child policy has notably affected the children’s living surroundings in Mainland China, and thus may increase the chances of suffering the psychological and behavioral problems for the only children due to the lack of playmates, compared to the children with siblings22. It is also worth mentioning that the highly competitive educational system with the increasing academic pressures in China may expose Chinese children to chronic stress, thus increasing susceptibility to mental health problems, including ADHD24. Methodological characteristics, such as screening and diagnostic methods, may be associated with the heterogeneity in prevalence results as well12.Geographical, demographic and cultural factors have been suggested as important variables that contribute to the heterogeneity of ADHD prevalence across studiesIt is clear that an estimated ADHD prevalence from one location fails to represent the overall prevalence among Chinese children, while a systematic understanding of the ADHD prevalence estimates in Chinese children and adolescents may provide a better insight into the overall and subgroup distribution and etiology of ADHD under different social and cultural backgrounds. Furthermore, a meta-analysis that computes the prevalence estimates of ADHD in the three regions will offer the supportive data for the accurate prediction of the worldwide pooled prevalence. Therefore, the purposes of this study are: (1) to estimate the overall and subgroup prevalence estimates of ADHD among children and adolescents in Mainland China, Hong Kong, and Taiwan from 1980 to 2016; (2) to analyze the trends of ADHD prevalence in the three locations in a period spanning the past 3 decades to aid in predicting future trends; and (3) to explore the possible causes of the varied prevalence estimates.15. Only 6 studies (9.0%) were conducted in the first 10 years (1980–1990), and the number of studies remarkably increased from 1991–2000 (10 studies) to 2001–2010 (33 studies). In recent years (2011–2016), 18 studies were carried out. In terms of regions, 49 prevalence rates were determined from samples collected in urban areas, 20 were based on mixed samples from both Urban,& rural areas, and only 1 focused solely on children in rural areas. The time frame of data collection varied across studies, indicating different study designs. The majority of the prevalence rates were from cross-sectional studies, 59 of which collected the data within 1 year. Five cohort studies in Taiwan implemented the data collection for over 2 years. Only 7 studies chose preschoolers as the study population, while over 60% of studies consisted of school-aged children and adolescents. Among the 67 studies, the Conners’ Parent Rating Scale and Conners’ Teacher Rating Scale were the most commonly used screening methods. Over half of the studies employed the diagnostic criteria of the DSM-III/III-R/IV.We screened 4704 abstracts, reviewed 125 full-text articles, and selected 67 studies for the final systematic review. Of these, 13 were published in English and 54 were published in Chinese. Figure Although all estimates from 67 studies were at moderate or low risk of bias, only 1 estimate met all 10 criteria, and 65% were at low risk of bias. The majority of estimates rated poorly for the representativeness of the national population (93%), and the strict measurement of the reliability and validity of the study instrument (85%). Besides, most estimates did not collect ADHD diagnostic information directly from children or adolescents (93%). Summary statistics for risk of bias for estimates are provided in Table The overall and subgroup prevalence estimates of ADHD are shown in Table I2 = 99%; Q = 9121.98, df = 69, P < 0.001), thus, the meta-regression analyses were used to explore the potential causes. In univariate meta-regression analyses , 2001–2010 , and 2011–2016  compared with the period of 1980–1990. The studies with combined samples from both Urban, & rural areas yielded significantly higher ADHD prevalence estimates than those with urban samples . Both school-aged children and adolescents  and preschoolers combined with school-aged children and adolescents  had significantly higher prevalence estimates than preschoolers. The larger sample sizes of 2000–5000  or over 5,000  generated significantly higher prevalence estimates than the sample sizes of less than 2000. There was a significant increase in prevalence estimates when the informants were parents  as well as both teachers and parents  compared with only teachers. The studies that underwent diagnostic procedure  or both screening and diagnostic procedures  displayed significantly higher prevalence estimates than those only with screening procedure. The studies employing Conners-based screening criteria yielded significantly lower estimates than those with other screening criteria, e.g., DSM-III/-III-R , DSM-IV/DISC-IV , Conners combined with DSM criteria . Compared to the studies conducted with the diagnostic criteria of DSM-III/-III-R, the studies using DSM-IV/DISC-IV , CCMD-II/II-R/III , DSM-IV combined with Conners  or ICD-9-CM  as the diagnostic criteria had significantly higher prevalence estimates.Substantial heterogeneity across studies was detected  and preschoolers combined with school-aged children and adolescents  yielded significantly higher prevalence estimates than preschoolers. In addition, the rural areas showed significantly higher prevalence estimates than urban areas .Table 25. This discrepancy can be associated with the fact that a handful of Chinese studies (15 studies) were selected by Thomas et al.25. Meanwhile, our prevalence estimate was pronouncedly higher than the 3.4% reported in another systematic review study that included 48 studies from 1985 to 2012, with only one Chinese study9. Those worldwide ADHD systematic reviews were mainly based on original investigations conducted in Western countries and published in English. Therefore, they neglected a substantial proportion of Chinese investigations and publications, further bringing about both selection bias and publication bias. On the contrary, our pooled ADHD prevalence was highly representative of Chinese children and adolescents, an apparent advantage to generate better population-based benchmarks for Chinese professionals and the public, and to be beneficial for the accurate estimation of the worldwide ADHD prevalence.We identified 67 original studies conducted in Mainland China, Hong Kong, and Taiwan from 1980 to 2016, covering 642,266 children and adolescents. Our prevalence estimate (6.3%) was lower than the 7.2% reported in a worldwide systematic review that included 175 studies from 1977 to 201326, a roster of previous studies that employed the data collected in the USA, UK, and Canada from the 1990s to 2000s exhibited a time trend of mounting ADHD diagnoses and prescriptions of medications for ADHD treatment32. Similarly, the present study showed the investigations implemented from the next 3 decades reported a higher ADHD prevalence rate than those from 1980–1990. The ascending academic pressure emanate from the fierce Chinese educational competition may be associated with the increase in the number of Chinese school-aged children and adolescents with ADHD symptoms.Our study revealed that ADHD prevalence in Chinese children and adolescents arose over time, with slight fluctuations. Even though recent worldwide systematic reviews with meta-analyses showed no evidence of the ascent in the number of children who met the standard diagnostic criteria over the past three decades34, children in rural areas showed a significantly higher ADHD prevalence than their counterparts in urban areas in our study. Nevertheless, caution should be taken in drawing conclusions in that only one selected study consisted of the sample solely from rural areas.We also found that the rates reported by both parents and teachers were higher than those reported by either parents or teachers, corresponding to the stereotype that Chinese children should obey their both parents and teachers, and very active children are generally considered to be either badly behaved or hyperactive, especially in the context of the rising recognition of ADHD in recent years. Additionally, the result from the present study that school-aged children and adolescents had higher prevalence estimates than preschoolers may be explained by the phenomenon that elementary school teachers in China start to demand students follow more behavioral norms, e.g., sitting still in a classroom arrayed with desks and chairs, or standing in line. However, since the mixed-age participants in different grades mostly constituted the selected samples in our review, we were limited to divide the school-aged children and adolescents into elementary school, middle school, and high school children groups to discern the differences in ADHD prevalence among those subgroups. Consistent with the result that children from low-socioeconomic status (SES) backgrounds were more likely to exhibit ADHD symptoms than their peers from high-SES backgrounds11. Nonetheless, it may not be neglected that remarkable differences in the socioeconomic development among the three regions during the last three decades may greatly impact the ADHD prevalence estimates. The previous worldwide systematic review also suggested that the heterogeneity of methodological characteristics may have caused the differences in ADHD prevalence in different locations11. Our review indicated the similar findings that variations of the sample size, study design and screening/diagnostic criteria among the three regions explained the regional differences in prevalence estimates. For instance, although most included studies were conducted in Mainland China, studies in Taiwan had the largest number of participants, and they were more weighted in our meta-analyses. Whereas most studies in Mainland China and Hong Kong were cross-sectional, most studies in Taiwan were longitudinal. In general, compared to studies from Mainland, which wide range of screening/diagnostic criteria were used, most investigators from Hong Kong and Taiwan selected DSM and ICD-based criteria to define the ADHD.While our systematic review included studies specifically conducted in Mainland China, Hong Kong, and Taiwan, no difference was detected in the ADHD prevalence estimates among the three regions after controlling for other factors of the heterogeneity across studies. This finding corroborated the limited function of geographic location in the large variability of ADHD prevalence estimates which was found in the previous review with worldwide samplesFirst, the literature published in the local languages of Hong Kong and Taiwan was not included in our review. Second, the high heterogeneity across studies and publication bias may weaken our ability to precisely estimate the ADHD prevalence among Chinese children and adolescents. Specifically, the pronounced variations in the procedures of screening and/or diagnosis and associated criteria across the studies raised the incomparability across the original ADHD prevalence rates, and thus caused the uncertainty to our pooled prevalence estimates. Third, the ADHD prevalence estimates found in our subgroup meta-analyses cannot adequately discern the differences in economic situations among different Urban and rural areas, and the subgroup estimates cannot be generalized to the only rural areas.This is one of the few comprehensive systematic reviews of ADHD prevalence estimates among Chinese children and adolescents in Mainland China, Hong Kong, and Taiwan over the past three decades. The prevalence estimates of ADHD among children in Mainland China and Hong Kong are similar and consistent with the reported rate in previous reviews. However, Taiwan has significantly lower prevalence than other regions. Even though our results should be interpreted with caution because of the large variability found in the analyses. Moreover, our findings suggest that the geographic location plays a limited role in the heterogeneity of ADHD prevalence estimates in Chinese children. Instead, the variability may be primarily explained by the methodological characteristics of studies, years of data collection, and participants’ socioeconomic backgrounds. Our analyses also indicate that high-quality studies, such as cohort studies or repeated cross-sectional studies, are required to assess the true trend of ADHD prevalence.A search of the literature published in English was performed using PubMed, Embase, PsycINFO, and Web of Science databases. The literature published in Chinese was searched using the China National Knowledge Infrastructure, VIP, WANFANG DATA, and China Science Periodical Database databases. These four Chinese databases include most of the articles published in Chinese among Mainland China, Hong Kong, and Taiwan. The year of publication was confined between 1978 and 2016, because the World Health Organization enacted the International Classification of Diseases, Ninth Edition (ICD-9) in which hyperkinetic disorder was defined  in 1978. The search strategy was composed of search fragments of population (i.e. children and adolescent), disease condition (i.e. attention deficit disorder with hyperactivity), outcome  and geographic location (i.e. China). Four investigators  used the same search strategies for all databases when conducting the literature search (Appendix 1).Three authors  worked on the selection, inclusion, and exclusion criteria. Each author independently conducted a literature search, reviewed abstracts for further full-text reviews, and selected eligible studies according to the preset criteria. Studies with incomplete data or disagreements could not be included in the final analyses unless the three authors reached a consensus.The selection criteria were: (1) original prevalence studies were conducted in the Mainland of China, Hong Kong, or Taiwan; (2) participants aged 18 years old or younger; (3) participants were screened for and/or diagnosed with ADHD; (4) any of the following assessment tools for ADHD was applied: Conners’ Parent Rating Scale (Conners), Conners’ Teacher Rating Scale (Conners), DSM-III, Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition (DSM-III-R), DSM-IV, International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM), International Classification of Diseases, Tenth Edition (ICD-10), Chinese Classification and Diagnosis of Mental Diseases, Second Edition (CCMD-II), Chinese Classification and Diagnosis of Mental Diseases, Revised Second Edition (CCMD-II-R), Chinese Classification and Diagnosis of Mental Diseases, Third Edition (CCMD-III), Diagnostic Interview Schedule for Children-Version 4 (DISC-IV), and others .Inclusion criteria were: (1) the epidemiological survey must have been conducted in the Mainland of China, Hong Kong, or Taiwan; (2) the study must specify the ADHD prevalence rate, rather than that of individual ADHD symptoms, e.g., attention deficit or hyperactivity; (3) participants must have been children or adolescents younger than 18 years old who were native Chinese/Hong Kongese/Taiwanese; (4) the study must have used any of the following standardized assessment tools for ADHD screening and/or diagnosis: Conners, DSM-III, DSM-III-R, DSM-IV, ICD-9-CM, ICD-10, CCMD-II, CCMD-II-R, CCMD-III, DISC-IV, others  or possible combinations; (5) the study must be population based; (6) the sample size was at least 500; (7) the article must be written in Chinese or English.Exclusion criteria were: (1) participants were over 18 years old; (2) participants were migrant children or adolescents; (3) none of the following standardized tools was employed: Conners, DSM-III/III-R/IV, ICD-9-CM/-10, CCMD-II/-II-R/III, DISC-IV or others ; 4) the study was clinic based or patient based; 5) the sample size was less than 500, considering potential lower power due to small sample size.The following key variables were extracted: 1) title of article; 2) years of data collection (the publication year was used as a proxy for studies without this information); 3) geographical locations ; 4) time frame  regions ; 6) age of participants; 7) sample size; 8) procedure of screening and/or diagnosis; 9) screening criteria; 10) source of screening information; 11) diagnostic criteria; 12) overall ADHD prevalence rate; 13) gender-specific ADHD prevalence rates; 14) number of participants with ADHD; 15) gender-specific numbers of participants with ADHD. All the variables were collected and double checked by 2 reviewers , with a third reviewer (T.L.) acting as arbitrator. The description of included studies is shown in the Table et al.35. Each included study was judged by 10 items that assess measurement bias, selection bias, and bias related to the analysis  and an overall assessment of risk of bias rated as low, moderate, or high risk. The more criteria were met, the lower the risk of bias. If the text was unclear, a high risk of bias was then recorded. A study was considered to have a high overall risk of bias if 3 criteria or less were met, moderate risk of bias if 4 to 6 criteria were met, and low risk of bias if 7 to 10 criteria were met.Two reviewers (L.A.N. and T. L.) assessed the risk of bias for each included study using a reliable Risk of Bias Tool for prevalence studies developed by Hoy 36 in both univariate and multivariate models. We applied Begg’s Test and Egger’s test37 to test publication bias. Inferred from the funnel and bias plots  in proportion studies39. The quantity I2 was used to detect the heterogeneity of this meta-analysis40. Next, we fitted a random-effect model to estimate the overall and subgroup pooled prevalence of ADHD using untransformed prevalence rates. To further explore the potential sources of heterogeneity, we conducted the random-effect meta-regression analyses using transformed prevalence rates. Dummy variables were used in our univariate and multivariate meta-regression analyses. All data analyses were performed using Stata 14.0 . A two-tailed P value of less than 0.05 was considered statistically significant.To minimize the effects of extreme prevalence rates on the overall estimates, we stabilized the variance of the study-specific prevalence with the Freeman-Tukey double arcsine transformationBoth univariate and multivariate meta-regression analysis were carried out. In each model, for categorical variable, one group was set as the reference group according to the purpose of analysis. In the final multivariate meta-regression model, the variable time frame was excluded due to the insufficient supportive literature regarding the role of time frame for data collection in the heterogeneity of ADHD prevalence findings. Additionally, the variable procedure of screening and/or diagnosis was included in the final model instead of screening criteria or diagnostic criteria because placing the latter variables in the multivariate regression model would greatly reduce the number of samples and decrease the precision. Additionally, 6 studies did not report their sources of screening information, thus the variable source of information was dropped as well. In summary, the following covariates were finally examined in the multivariate model using the restricted maximum likelihood estimator: years of data collection, geographic location, region, age of participants, sample size, and procedure of screening and/or diagnosis. Stepwise was used to select the significant variables to the model.The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.Appendix 1.Search StrategyAppendix 2. PRISMA checklist


Objective: The aim of this study was to examine the psychometric properties and diagnostic accuracy of the Swedish version of the Wender Utah Rating Scale (WURS) in psychiatric patients with similar symptoms but diagnosed with either attention deficit hyperactivity disorder (ADHD), bipolar disorder (BP), and/or borderline personality disorder (BPD).Methods: A total of 121 patients from an outpatient psychiatric clinic for young adults (18–25 years) were diagnosed using the Structured Clinical Interview for DSM Axis I and Axis II (SCID-I and SCID-II), and ADHD was diagnosed using the Schedule for Affective Disorders and Schizophrenia for School-Age Children (K-SADS). WURS were filled in by the participants and compared with a diagnosis of ADHD according to K-SADS.Results: Internal consistency of the WURS was 0.94. The principal component analysis resulted in a three-factor solution that accounted for 61.3% of the variance. The ADHD group had significantly higher mean scores compared to all other groups. The diagnostic accuracy of the WURS was examined using AUC and ROC analysis, and the optimal cut-off score was 39, with a sensitivity of 0.88 and specificity of 0.70, with AUC 0.87, 95% CI 0.80–0.94, PPV 0.59, and NPV 0.92.Conclusion: The psychometric properties of the Swedish WURS were good. For assessment of adult ADHD, in patients with symptoms of emotional instability, impulsivity, and attention problems but of different origins, a somewhat higher cut-off score than the originally suggested was preferable for identification of ADHD. Attention deficit hyperactivity disorder (ADHD) is mainly characterized by deficiency in sustaining attention, impulsivity, and/or hyperactivity . ADHD caIn clinical practice, the similarity of the behavioural presentation of ADHD, BP, and BPD constitutes a risk for overlooking the symptoms of one of the other disorders. There is therefore a need for diagnostic instruments with satisfactory discriminant validity that can be used for assessment of ADHD in adults, especially in clinical settings where BP and BPD are also common.The Wender Utah Rating Scale (WURS) is a self-report instrument that is designed to retrospectively assess childhood ADHD symptoms, based on the Utah criteria ,11. The The aim of this study was to examine the psychometric properties and diagnostic accuracy of the Swedish version of the WURS-25 in patients diagnosed with ADHD, BP, and/or BPD. We hypothesized that the instrument should have similar internal consistency and factor structure to the original English version, that it should be able to discriminate patients with ADHD from patients with BP and/or BPD, and, finally, that it should show adequate diagnostic accuracy for identifying a diagnosis of ADHD in this phenomenologically homogeneous group.Patients from an outpatient psychiatric clinic for young adults (18–25 years) in Uppsala, Sweden, diagnosed with BPD, ADHD, or BP, were identified from the administrative patient register. Altogether, 759 patients diagnosed with any of the above diagnoses between 1 May 2005 and 31 October 2010 were sent a study invitation to participate in the study by post. Letters were sent to groups of patients on 24 different occasions from 18 August 2008 to 13 May 2011. At the time of investigation, some patients still had ongoing contact with the clinic, whereas others did not. Exclusion criteria were severe psychotic or manic symptoms at the interview appointment. One patient was excluded because of ongoing mania.For a description of the recruitment process, see Among the participants, there were 40 (33.1%) who fulfilled criteria for ADHD. Among those 40, there were 19 diagnosed with ADHD alone, eight had a comorbid BPD, eight a comorbid BP, and five fulfilled criteria for all three diagnoses; however, all were brought together into an ADHD group. The other groups were 17 (14.0%) BPD, 30 (24.8%) BP, 14 (11.6%) comorbid BPD and BP, and finally 20 (16.5%) subclinical cases.P < 0.001). Among our study group, 14.3% were previously diagnosed with BPD, 32.2% with ADHD, and 35.2% with BP.Among all 759 available patients, there were 466 (61.4%) women and 293 (38.6%) men. Among those 230 who participated, there were significantly more women than men  was not a reason for exclusion, but diagnostic criteria were not assessed as part of the study. However, participants filled in the Autism Spectrum Quotient (AQ), and the mean score was 19.3 , supporting a low impact of autistic traits in the study group .n = 66) or more (n = 55) than 30 days between the two tests, including the three without known date placed in the latter group, and diagnostic accuracy was then examined separately in both groups.There were 21 participants who reported on the WURS before the K-SADS interview (Schedule for Affective Disorders and Schizophrenia for School-Age Children) was performed, 44 participants performed both tests on the same day, 53 reported on the WURS after the interview, and the date was missing for three patients. The median time interval between the index test, WURS, and the reference test was zero weeks (SD 51). Patients were grouped in two groups, those with less  also filled in the Montgomery Åsberg Depression Rating Scale–Self assessment (MADRS-S) with a mean score 19.3 (SD 10.2)  .K-SADS is a semi-structured diagnostic interview that examines psychopathology in children and adolescents between the ages of six and 18 years . Since tWURS is a Likert-type, self-report scale that is designed to retrospectively assess childhood ADHD symptoms. The WURS has previously shown good internal consistency and a moderate convergent validity when correlated with the 10-item Parents’ Rating Scale . Both a In the original validation of the WURS-25, Ward et al. reported a diagnostic accuracy for the instrument . With a In this study, participants filled in the 61-item questionnaire, but only the 25 questions known to best discriminate between ADHD and controls were used—the WURS-25.Three medical doctors performed the majority (94%) of all the diagnostic interviews : one specialist in psychiatry (M.R.), one resident in psychiatry (I.K.) and one intern (N.H.). All three had been trained in accordance with the SCID manual by reviewing recorded interviews with a ‘master interviewer’ and by then assessing patients from audio and video recordings, assessing patients in clinical practice, and discussing both performance and appraisal of responses with experienced raters. Four per cent of the interviews were performed by clinicians that previously had shown good inter-rater reliability with the first author.Inter-rater reliability was calculated for the three main interviewers  based on six randomly selected SCID-I interviews (13 protocols), mean prevalence-adjusted bias-adjusted kappa (PABAK) 0.95, range 0.91–0.97; nine SCID-II interviews (23 protocols), mean PABAK 0.85, range 0.79–0.88; and four K-SADS interviews (11 protocols), mean PABAK 0.72, range 0.64–0.81. Not all diagnoses within the K-SADS supplement were assessed, only ADHD. Inter-rater reliability of K-SADS therefore meant agreement upon an ADHD diagnosis or not. Diagnostic reliability was assessed before and during the study as part of regular meetings.post hoc test. The previously suggested cut-off scores (36 and 46) were used for calculations of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for an ADHD diagnosis. Receiver-operating characteristics (ROC) analyses were used for the area under the curve calculation (AUC). We calculated Youden’s J and applied a minimum score of 0.85 for sensitivity and 0.70 for specificity to choose the optimal cut-off  with the mean scores of the individuals with BPD (n = 17), BP (n = 30), BPD and BP (n = 14), and the individuals with subclinical diagnoses (n = 20). In all groups there were more female than male participants; in the ADHD group the percentage of females was 65%, in the BPD group 94%, in the BP group 73%, in the BPD and BP group 86%, and in the group with subclinical diagnoses 55%. The ADHD group had significantly higher mean scores on WURS compared to all other groups. Mean WURS scores for all groups are presented in t [119] = 0.115, P = 0.909).We compared the mean WURS-25 scores for the individuals that met the criteria for ADHD (n = 66), and the group with a time period longer than 30 days (n = 55). Sensitivity, specificity, PPV, and NPV for both the original and the optimal cut-off are presented in The diagnostic accuracy of the WURS-25 was examined using AUC and ROC analysis . The optSince most of the subclinical cases in our study were patients with subclinical ADHD symptoms a complementary analysis considering a minimum of five instead of six criteria for fulfilling an ADHD diagnosis was conducted. Using five criteria as threshold the number of individuals fulfilling the ADHD diagnosis increased from 40 to 53. The sensitivity and specificity for a cut-off score of 36 points was 0.87 and 0.66, respectively, and for a cut-off score of 38, respectively 0.81 and 0.72. The optimal cut-off score was 39 with a sensitivity of 0.81 and specificity of 0.76, with AUC 0.87, and 95% CI 0.81–0.93.The psychometric properties of the Swedish version of the WURS-25 were satisfactory and mirrored the psychometric properties of the questionnaire in other languages. The diagnostic accuracy was good in young adult psychiatric patients with ADHD, BP, and/or BPD, considering that these diagnoses express similar symptomatology.The WURS-25 showed excellent internal consistency in accordance with prior studies ,18. MoreThe items of hyperactivity and inattention loaded on two different factors, which was also the case in previous studies ,18. The Since WURS-25 is a screening tool it is important that its sensitivity is high. The optimal score of 39 in the present study is lower than the cut-off that was suggested in the original validation study . HoweverThis study has several limitations. One is the lack of validated interviews in Swedish for assessing adult ADHD. We therefore had to use the K-SADS, which is an interview for children and adolescents. Furthermore, we did not use the screening questions, only the questions from the K-SADS supplement to evaluate ADHD. This might have affected the estimation of the true ADHD cases. However, all ADHD criteria were explicitly evaluated based on both the interview and medical history. Both WURS and K-SADS are based on the same retrospective data, the patient’s recall from childhood. In many cases a parent had reported childhood symptoms, but this was not mandatory, and for most cases we relied on the patient as the only informant. Another limitation is the use of clinical data, collected during the diagnostic assessments in the clinic. However, the number of diagnostic interviews performed by other clinicians was very limited, and in half of them the diagnosticians were known to be reliable. Inter-rater reliability was calculated on a limited number of interviews, and for K-SADS the agreement was good, but not excellent. A further limitation is that according to the DSM-IV criteria for ADHD the presence of some inattentive and hyperactive/impulsive symptoms before the age of seven is needed, but it is not a requirement for all symptoms to have been present before seven years of age. In accordance with the K-SADS interview structure we asked about the onset at the same time as we evaluated each criterion. This might have caused an underestimation of the true ADHD cases. However, our impressions from the interviews were that most symptoms were present early in life.Strengths include the presentation of separate analyses for those questionnaires filled in more or less than 30 days apart from the reference standard. The absence of a healthy control group might have revealed even greater differences between groups and supported discriminant validity even more. However, our aim was to investigate the psychometric properties of the WURS-25 in a clinical setting considering the difficulties in the assessment of adult ADHD in patients with similar symptoms.


Olefin metathesis is one of the most significant developments of the last 20 years in the fields of organic chemistry, polymers synthesis, and materials science ,4,5,6,7.Molecules entitled “Olefin Metathesis” was to discuss recent developments in all fields of metathesis. This Special Issue consists of five research and two review articles. In the first research article, Pitsikalis and co-workers report the statistical ring-opening metathesis copolymerization of norbornene and cyclopentene by Grubbs’ first-generation catalyst [2(μ-Cl)3Cl4(THF)2]•(THF)3 towards the polymerization of selected cycloolefins [N-heterocyclic carbene complexes by neutral or charged rhodium-based metathesis catalysts [The goal of the Special Issue of catalyst . Moreovecatalyst . These dcatalyst . This isoolefins . In the atalysts . This stN-heterocyclic carbene ligands with stereogenic centers on the backbone [N-heterocyclic carbenes. The second review article, by Dragutan, Simionescu, and co-workers, summarizes the work carried out in the field of controlled “living” ring-opening metathesis polymerization for the preparation of iron-containing organometallic complexes, as well as the advanced applications of these hybrid materials [The next article of the issue reviews the catalytic properties of metathesis ruthenium complexes bearing backbone . Amongstaterials .