Neisseria species two, Neisseria meningitidis, and Neisseria gonorrhoeae, cause invasive disease: the other eight are carried asymptomatically in the pharynx, possibly modulating meningococcal and gonococcal infections. Consequently, characterizing their diversity is important for understanding the microbiome in health and disease. Whole genome sequences from 181 Neisseria isolates were examined, including those of three well-defined species  and genomes of isolates unassigned to any species (Nspp). Sequence analysis of ribosomal genes, and a set of core (cgMLST) genes were used to infer phylogenetic relationships. Average Nucleotide Identity (ANI) and phenotypic data were used to define species clusters, and morphological and metabolic differences among them. Phylogenetic analyses identified two polyphyletic clusters (N. polysaccharea and Nspp.), while, cgMLST data grouped Nspp isolates into nine clusters and identified at least three N. polysaccharea clusters. ANI results classified Nspp into seven putative species, and also indicated at least three putative N. polysaccharea species. Electron microscopy identified morphological differences among these species. This genomic approach provided a consistent methodology for species characterization using distinct phylogenetic clusters. Seven putative novel Neisseria species were identified, confirming the importance of genomic studies in the characterization of the genus Neisseria.Of the ten human-restricted  Neisseria includes a number of species that colonize the epithelium of animals1. Human-restricted species are Gram-negative, oxidase and catalase positive bacteria with two different morphologies: coccoid ; and rods (Neisseria elongata and Neisseria bacilliformis). All are asymptomatic commensal inhabitants of the mucosa, but two are also potentially pathogenic species: (i) N. meningitidis, which colonizes the oropharynx, sometimes causing meningitis and/or septicaemia; and (ii) N. gonorrhoeae, which typically inhabits the genitourinary tract, causing local and occasionally systemic gonococcal disease2. The remaining eight species are generally harmless inhabitants of the oro- and/or nasopharynx.The genus Neisseria phenotypic taxonomy reliably identifies the three species most studied at the time of writing: N. meningitidis; N. gonorrhoeae; and N. lactamica. This approach has, however, presented difficulties in defining and identifying other species3, which has led to the application of molecular approaches such as DNA-DNA hybridization (DDH)3 and phylogenetic analyses of nucleotide sequence data of the 16S ribosomal gene4. Analysis with single loci, such as 16S rRNA genes, poorly differentiate Neisseria species, due to distortions resulting from low resolution and horizontal genetic transfer (HGT)5. Phylogenies based on multi-locus sequence analysis (MLSA)4, such as (i) the seven loci used in Multi Locus Sequence Typing (MLST)6, (ii) the 53 ribosomal genes of ribosomal MLST (rMLST)7, and (iii) the 246 loci of the Neisseria genus core genome MLST (cgMLST)3, have greatly improved Neisseria species classification. These approaches led to the reclassification of some Neisseria species, with the consolidation of N. subflava biovar sublfava, perflava and flava, and Neisseria flavescens into a single species named Neisseria subflava. Isolates previously classified as Neisseria sicca were identified as variants of N. mucosa3 and “Neisseria mucosa var. heidelbergensis”, was shown to be genetically identical to Neisseria oralis8. A single-locus sequence species identification assay, which uses a fragment of the ribosomal gene rplF, can identify different Neisseria species reliably, but as this is a single locus test, it is not on its own an appropriate tool for the definition of novel species9. Measuring the Average Nucleotide Identity (ANI) between genomes pairs has also facilitated bacterial taxonomic research and been proposed as an alternative to DDH11, which is highly specialized, labor intensive and difficult to standardize and compare among laboratories12.Conventional Neisseria (NPN) are rarely isolated and characterized, mostly because they seldom cause invasive disease, except in immunocompromised individuals1; however, their possible role in modulating nasopharyngeal carriage of N. meningitidis has increased interest in them. Several studies have indicated that nasopharyngeal colonization with N. lactamica can be protective against N. meningitidis infection15 and that N. lactamica has potential to be used in the design an outer-membrane vesicle (OMV) meningococcal vaccines16. Neisseria are known to be abundant in the human microbiome17, reinforcing the importance of understanding the diversity of this genus. This has been exemplified by the findings that: (i) in both high-income countries and in the African meningitis belt, the incidence of carriage of N. meningitidis increases with age as that of N. lactamica declines19; (ii) the risk factors for carrying N. meningitidis in the African Meningitis Belt (AMB) are inversely related to those of carrying NPNs20; and (iii) the microbiota has a major influence on other diseases, including pneumonia21 and cystic fibrosis23.Non-pathogenic N. polysaccharea has been described as polyphyletic, potentially containing more than one species. In particular, isolate 15883, identified by Berger in 1985 in Germany24, has been proposed as a new species25. The increase in the number of non-N. meningitidis carriage isolates collected in previously under-sampled and geographically distinct regions20, combined with the concomitant decrease in the cost of whole genome sequencing (WGS), have allowed an increasing number of NPN isolates to be sequenced, although numbers remain low compared to N. meningitidis and N. gonorrhoeae.rplF nucleotide sequences, isolates genetically close to isolate 15883, were identified in MenAfriCar carriage studies, suggesting that this putative novel species, which had not been found since Berger, could be present in the African continent20. Similar non-defined isolates were identified in other studies conducted in and outside of the African meningitis belt28 and in the UKMenCar4 carriage study29. Here, phylogenetic relationships of members of the human-restricted Neisseria species were examined using WGS data to characterize their genetic and phenotypic diversity and clarify the identity of isolates belonging to the Neisseria genus but currently of unknown species.Based on the phylogeny of partial f_rplF sequence data identified four clusters including monophyletic N. meningitidis and N. gonorrhoeae clusters, and more diverse N. polysaccharea and Nspp isolates ; five among N. meningitidis ; six among N. polysaccharea ; and six among Nspp isolates .WGS data of the 181 isolates included in this study  all the isolates from Nspp 1 (N = 36) and Nspp 8 (N = 1) were collected in Mali; (ii) Nspp 2 (N = 20), Nspp 4 (N = 14) and Nspp 5 (N = 21) were collected in Malawi; (iii) Nspp 3 (N = 9) came from Chad; (iv) Nspp 7 (N = 2) from The Gambia; and (v) Nspp 9 (N = 1) from Germany. The only cluster with isolates from different locations was Nspp 6 collected in Malawi (N = 2) and in the UK (N = 1). Similarly, all N. polysaccharea 2 isolates were collected in The Gambia (N = 6) and Mali (N = 7), two countries of the African meningitis belt, whereas N. polysaccharea 1 included isolates both from Europe and Africa. Even within the N. polysaccharea 1 cluster, these remained polyphyletic with clusters separated by deep branches. The ten isolates from Malawi (N = 10) formed a divergent branch and the remaining three African isolates, from Mali (N = 1) and The Gambia (N = 2), clustered more closely together compared to European isolates  to 96.6% (between Nspp 1 and Nspp 2). Only three pairwise comparisons yielded values >95%, these were between: (i) Nspp 1 and Nspp 2; (ii) Nspp 1 and Nspp 9; and (iii) Nspp 2 and Nspp 9. Using a threshold of 95% ANI, these findings indicate that these should be considered as distinct bacterial species. All the remaining ANI values were lower than 95%  in Germany in 198424.57 isolates including clusters Nspp 8 cluster comprised a single isolate collected in Mali in 2010; the name “Neisseria maigaei” is suggested, after the late Aziz Maiga who played an important role in the isolate characterization of the MenAfriCar study in Mali.The Neisseria uirgultaei sp. nov. is proposed for the nine isolates from cluster Nspp 3 , after Sir Brian Greenwood, the director of the MenAfriCar project (greenwood is uirgulta in Latin).The name Nspp 7, the name “Neisseria basseii” is suggested, after the town of Basse in The Gambia, where the isolates were collected.As only two isolates have been identified in Neisseria blantyrii is proposed for the 14 Nspp 4 isolates , after the city of Blantyre in Malawi where the isolates were collected.The name Neiseria viridiae sp. nov. is proposed for the 21 Nspp 5 isolates , after Jenny MacLennan (nee Green) who collected and characterized all the isolates from Malawi and The Gambia included in this study (green is viridi in Latin).Neisseria benedictiae” is suggested for the three Nspp 6 isolates, after Julia Bennett (bennet is benedictus in Latin) who pioneered the genomic work of N. bergeri isolates and the speciation of Neisseria with cgMLST approaches , sharing at least one allele, and similarly for Nspp 1 and Nspp 9 (N = 145) and Nspp 2 and Nspp 9 (N = 134)  Fig. , consistN. polysaccharea clusters, designated N. polysaccharea 1 and N. polysaccharea 2, had an ANI value of 94.6%, confirming that they can be considered as two distinct species , suggesting that the cluster was not homogeneous and probably included more than one species. Three additional genomes of the N. polysaccharea 1 cluster were selected to attempt to characterize this further  visually appeared to be moister. The size range of the colonies varied from 0.1 mm to 1 mm; N. meningitidis colony size range extended to 2 mm . The putative novel species exhibited edges with a regular shape and smooth texture; however, N. bergeri  exhibited filaments covering their extracellular membranes, “pili-like” structures could also be seen connecting cells of “N. maigaei” (Nspp 8), “N. blantyrii“(Nspp 4) cells were covered in an intense filamentous network, “N. viridiae” (Nspp 5) and “N. benedictiae” (Nspp 6) exhibited visible “bulbs” on the surface of their extracellular membranes. Conversely, “Neisseria uirgultaei” (Nspp 3) possessed a smooth surface with no visible extracellular elements and “Neisseria basseii” (Nspp 7) showed few visible filaments and bulbs  of representative isolates from each proposed species revealed that they were all coccoid bacteria, with a mix of completely formed diplococci and incompletely formed diplococci, which were characterized by a “heart-shaped” morphology Fig. . Variatirii“Nspp  cells weTM system identified the putative novel species as GGA, ONPG, and Tributyrin negative, Leucine and Proline Aminopeptidase positive. They were also positive for the maltose degradation and negative for lactose degradation. Results were inconsistent for glucose and sucrose when repeated: N. bergeri subcluster 1  and 2 , “N. uirgultaei” (Nspp 3), “N. basseii” (Nspp 7) and “N. viridiae” (Nspp 5) being mostly positive for glucose after at least 3 repeats, and N. bergeri subcluster 3 , “N. maigaei” (Nspp 8), “N. blantyrii” (Nspp 4) and “N. benedictiae” (Nspp 6) mostly negative. Similarly, “N. viridiae” (Nspp 5) and “N. benedictiae” (Nspp 6) were mostly positive for sucrose while all the other species were negative. GGA, proline aminopeptidase and lactose/ONPG tests were useful for distinguishing new species from N. meningitidis and N. lactamica; however, none of these allowed a distinction from N. polysaccharea  reactions were useful to distinguish putative novel species from N. lactamica and N. meningitidis but no biochemical test could distinguish them from N. polysaccharea  became an approach of choice33. The analysis of the seven MLST loci has proven to be sufficient to distinguish N. meningitidis, N. gonorrhoeae, and N. lactamica34, for example, which single-locus 16S RNA sequence analysis has failed to achieve3. Although housekeeping genes have predominantly been used, there have been no specific guidelines on the loci or the number of loci to be chosen for MLSA. The increased availability of whole or nearly complete genome sequence data allows species characterization at a higher resolution, as demonstrated through the use of the Neisseria genus cgMLST to identify multiple Neisseria species3.The concept and definition of species in prokaryotes continues to be subject to debate, as do the best methods for microbial taxonomy35 adopted here combines rMLST, MLSA, and ANI scores, using nucleotide sequence data of core genes, supplemented with a quantitative measure of genetic relatedness . Three putative novel Neisseria species were assigned names, as clusters contained more than three representative isolates: N. uirgultaei sp. nov., N. blantyrii sp. nov., and N. viridiae sp. nov. The remaining clusters had fewer than three isolates and therefore will not be formally named; however, the species names  are proposed for future reference. Identical ANI results were obtained using the full draft WGS  remained polyphyletic.The phylogenomic approachess Fig. . This aless Fig. . The 95%son Fig. , suggestANI Fig. . However37, countries of the African Meningitis Belt38, and in Malawi (outside the belt)28 have led to the collection of a large number of Neisseria isolates; however, the NPNs were not always characterized at the genomic level. Our results indicate a phylogeographic clustering of genotypes, with isolates from the same country clustering together  as opposed to 15–19 years old (UK), may have played a role in the differences observed.A major limitation when undertaking taxonomic studies is ensuring the natural diversity of the bacterial population has been appropriately sampled. Studies are often geographically limited, leading to an underestimation of diversity. The different nasopharyngeal carriage studies undertaken in the UKher Fig. . Even thNeisseria isolates from various geographical regions will improve the taxonomy of this genus. Isolates previously characterized as “N. bergeri”, based on nucleotide sequences of the rplF locus20, included additional putative novel Neisseria species, such as those identified in this study. Genome sequences obtained from the Malian collection of the MenAfriCar project found that most of Nspp genomes were classified as N. bergeri sp. nov. using the 95% cgMLST ANI analysis presented here; however, one was found to form a distinct putative species, “N. maigaei”. Although the sequencing of the f_rplF locus provides species identity for previously defined species, our study confirms that it does not provide sufficient resolution for the characterization of new species. Each of the clusters identified had distinct f_rplF alleles except N. benedictiae (Nspp 6) and N. basseii (Nspp 7) which shared allele 69, consistent with recent HGT or shared ancestry.WGS analysis of additional carried N. polysaccharea isolates were indistinguishable using standard biochemical tests, despite being clearly distinct by 95% cgMLST ANI. However, additional tests including biochemical analyses, pH, or chemotaxonomy  combined with a larger numbers of isolates, including additional type strains, may identify further differences39. Phenotypic tests are labor intensive, and the interpretation of the results can be subjective. The DiatabsTM tests were, overall, more straightforward than API®-NH tests, which have a strict McFarland Standard concentration. Sugar degradation by the DiatabsTM were accurate for the N. meningitidis and N. lactamica controls but were elsewhere unstable with repeats giving different results. On the other hand, the degradation of sugars assessed by the API®-NH were highly reproducible, but failed to give the expected results for N. meningitidis24. Coccoid cells, some of which exhibiting distinct extracellular membrane structures, were visible following SEM analyses  were identified in the pubMLST Neisseria database; some of these had been recorded as “Neisseria bergeri”, following preliminary analysis28. The majority of the Nspp were collected in carriage studies performed in Africa and included: (i) 57 isolates collected from Ndirande, Blantyre, Malawi, from41; (ii) 37 isolates collected in Mali and 9 from Chad38; and (iii) 2 isolates obtained in Basse, Gambia26. Two additional isolates were collected in Europe: the first in Germany in24; and the second in the United Kingdom29.A total of 107 Nspp isolates were sequenced de novo using the Illumina MiSeq platform. Isolates were cultured on blood agar plates (BAP) and incubated overnight for 16–24 hours at 37 °C, in 5% CO2. For 96 of the isolates, genomic DNA was extracted using the Wizard genomic DNA Purification kit (Promega) following the manufacturer’s instructions; for the remaining nine isolates from Chad, DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen), as previously described42. DNA samples were sequenced by pair-end sequencing on an Illumina HiSeq, with read lengths of 100 (N = 55), 125 (N = 46) and 150 (N = 4) base pairs at the Wellcome Trust Sanger Institute, using previously described methods43.All the African 44: all odd-numbered kmer lengths between 21–99, 21–124 and 21–149 were sampled for read lengths of 100, 125, and 150 base pairs respectively, using the VelvetOptimiser software (v2.2.4)45 to automatically establish the optimal assembly parameters for Velvet (default optimization functions used). The assembled contigs were deposited in the Neisseria PubMLST database46 which uses the Bacterial Isolate Genome Sequence Database (BIGSdb) software47 and to the European Nucleotide Archive .2.0844: Nspp sequenced de novo, and the two European strains previously sequenced and deposited into the http://pubMLST.org/neisseria database, 74 other genomes deposited in the database were chosen as described below, to complete the genome collection analyzed in this study. These represented three known human-associated Neisseria species and included all the genomes available at the time of analysis for N. polysaccharea (N = 45), all but one finished genomes of N. gonorrhoeae (N = 9) and representative genomes of the most common clonal complexes of N. meningitidis (N = 20)43, with a preference for finished genomes when available. Additional information and meta-data for each of the 181 genomes are available in Supplemental Table In addition to the 105 African f_rplf, a fragment of the ribosomal gene rplF9, (ii) 51 of the 53 rMLST loci (BACT0060 (rpmE) and BACT0065 (rpmI) were excluded as they are known to be paralogous in Neisseria species); and (iii) a newly defined cgMLST scheme (Human-restricted Neisseria cgMLST v1.0), including 1441 loci present in at least 95% of isolates in this collection. All the sequences were aligned using MAFFT in pubMLST49.A hierarchical gene-by-gene approach to Whole Genome Sequence (WGS) analysis was applied to all 181 genomes, including the analysis of: (i) N. meningitidis finished genome FAM18 (Accession number: AM421808) as a reference with parameters set at a minimum identity of 70%, a minimum alignment length of 50%, a BLASTN word size of 20 base pair and a core threshold of 95% ; (ii) Proteinortho, assembled draft genomes were downloaded from PubMLST for each isolate and used as input files for the annotation program Prokka, using default settings with the option “–addgenes” and “–compliant”50. The protein sequence outputs of Prokka were used as input files for Proteinortho, with default settings, to detect orthologous group of genes51.Two methods where used in parallel to generate the list of core loci using: (i) the Genome Comparator tool of PubMLST was used to compare all genomes, using the Both methods generated a list of core loci present in 95% of the genomes included in the analysis; 1421 loci were identified by Genome comparator and 1366 by Proteinortho. An inclusive list of 1441 loci including any that were considered core to 95% of isolates by both methods was generated and used in the phylogenetic analysis. Within these 1441 loci, 1114 had complete sequences in all the genomes, whereas the remaining 327 fell at the beginning or the end of a contig in at least one genome and were classified as incomplete genes and excluded from the phylogenetic analysis  as an out-group. The f_rplF and rMLST phylogenies were generated in MEGA6 using the Maximum likelihood method with the following options: General Time Reversible model52, 100 bootstrap replications, Gamma distributed rate with Invariant sites and 5 discrete Gamma Categories. The cgMLST phylogeny was generated in FastTree53 using the General Time Reversible model (“-gtr”) and all other defaults options. Tree annotations were performed in MEGA654.Phylogenetic analyses were performed for Neisseria species included in the paper published by Bennett et al., which included the available type strains genomes3.Another rMLST phylogeny was reconstructed with the Neighbor Joining model in MEGA6 using the 181 genomes previously described and all 40 additional human-restricted 55.Pairwise allelic variations at the core genes, between each identified clusters, were assessed using the allele-overlap script comparing alleles of each gene between two defined sets of genomesN. polysaccharea cluster. An online ANI tool, “ the ANI calculator”57 was used to calculate a two-way ANI between each pair of selected isolates using the method described previously11. Pairs of isolates with ANI values higher than 95% were considered as indistinguishable at the species level as suggested by the program. The analysis was also computed using the whole genomes sequences of the isolates.The Average Nucleotide Identity (ANI) was calculated by comparing concatenated sequences of cgMLST loci from representative isolates of each phylogenetic cluster of interest: 13 isolates for the analysis of the whole phylogeny and 5 for the further characterization of the Nspp1-9, Np1-2, and three positive controls: (i) N. meningitidis (pubMLST ID: 35956/isolate ID: Z1318)58; (ii) N. polysaccharea (pubMLST ID: 19095/isolate ID: CCUG 18031)59; and (iii) N. lactamica (pubMLST ID: 8851/isolate ID: 020-06)59 , 90% for 10 min, 95% for 10 min, 100% for 20 min and in pure ethanol three times for 20 min each. Ethanol was removed, and the samples were dried by incubating specimens in a 50% Hexamethyldisilazane (HMDS) and 50% ethanol solution for 15 min, followed by incubation in pure HMDS for an additional 15 min. Once HMDS was removed, samples were left in a fume hood overnight to allow HMDS fumes to evaporate. Samples were mounted on SEM stubs with a conductive carbon adhesive, then coated with 10–15 nm of gold using a Quorum Technologies Q150R ES sputter coater and were imaged with a Zeiss Sigma 300 field emission gun SEM at an accelerating voltage of 2 kV.Scanning Electron microscopy (SEM). Sub-cultured isolates were prepared for SEM after 20 hours of incubation, following adaptation of a protocol previously describedOxidase tests were conducted with the oxidase test disks (Sigma-Aldrich) following the manufacturer’s instructions. Catalase tests were undertaken by putting the bacteria in contact with a drop of hydrogen peroxide; the formation of bubbles was considered a positive reaction. Haemolysis was assessed for each sample by visual inspection of the cells on BAP.TM (Rosco Diagnostica) were used according to the manufacturer’s instructions to test for sugar degradation of glucose, maltose, sucrose, and lactose, and other selected microbial enzymatic properties including: the ability to enzymatically hydrolyse aminopeptidase substrates like gamma glutamyl aminopeptidase (GGA), leucine aminopeptidase and proline aminopeptidase; the hydrolysis activity of the enzyme β-galactosidase on the orthonitrophényl-β-galactoside (ONPG) substrate; and the enzymatic hydrolysis of tributyrin into butyric acid and glycerol often used for the differentiation of Moraxella catarrhalis from Neisseria species. The results were assessed following the instructions of the test  for de novo sequenced isolates are provided in Table The Accession numbers for the Supplemental figures and tables


Retinopathy of prematurity (ROP) is a vascular proliferative disorder of the developing retina and a significant cause of childhood blindness around the world. The incidence of ROP is affected by many factors, and the incidence rate varies from country to country. The purpose of this study is to report the incidence and risk factors of ROP in neonatal intensive care unit (NICU) of Guangzhou First People’s Hospital in China.A retrospective review was performed on 436 premature infants who were consecutive ROP screened in the NICU of Guangzhou First People’s Hospital from March 2013 to October 2017. The single-factor analysis and the logistic multivariate regression analysis were used to detect risk factors of ROP.Total 436 premature infants were consecutive ROP screened, 138 (31.65%) were found ROP, and 61(13.99%) were treated. The single-factor analysis revealed that the incidence of ROP was associated with multiple births, gestational age, birth weight, mechanical ventilation, intravascular hemolysis, the number of operations and blood culture results. The logistic multivariate regression analysis revealed that gestational age; birth weight, mechanical ventilation, minimum SaO2 and daily weight gain were independent risk factors for ROP onset. Forty-nine patients underwent retinal laser photocoagulation with recurrence 20 patients. Twelve patients underwent anti-VEGF drug (Ranibizumab) via intraocular injection with 5 patients of recurrence.The incidence of ROP in NICU of Guangzhou China will match those in middle-income countries, but higher than high-income countries. Anti-VEGF drugs could be preferred as a good treatment method for zone 1 ROP and aggressive posterior ROP. Retinopathy of prematurity (ROP) is one of the major causes of blindness in children and is the most common cause of retinal vasculopathy in premature or low-birth-weight infants . The phyIn recent years, with the development of neonatal medical technology, an increasing number of severe premature infants have survived, including cases of prematurity associated with in vitro fertilization (IVF), multiple births and infants with very low birth weight, very low gestational age, congenital dysplasia, immaturity, septicemia, severe infections and multiple surgeries after birth. The survival rates of extremely immature premature infants < 26 weeks and < 1000 g worldwide have continuously increased, and the incidence of ROP has been increasing in parallel –8.This study retrospectively analyzed the screening and treatment of ROP in severe premature infants who were admitted to the Neonatal Intensive Care Unit (NICU) of Guangzhou First People’s Hospital from March 2013 to October 2017. The results revealed that ROP was common in severe premature infants with risk factors and that anti-VEGF treatment of ROP produced a curative effect. Guangzhou First People’s Hospital is a comprehensive Grade 3A hospital in economically developed regions of China with advanced neonatal care. The purpose of this study was to analyze the prevalence and risk factors of ROP in advanced neonatal care hospitals in a single centre of China.The medical ethics committee of Guangzhou First People’s Hospital approved this study. The patient-related data collected in this study include the following: name, age, and clinical diagnosis. The names were replaced by numbers, and the relevant data of the patient was encrypted and stored in the main study. We had obtained the written informed consent of the parents of the children.A retrospective analysis was performed on premature infants who underwent ROP screening in the NICU of Guangzhou First People’s Hospital from March 2013 to October 2017.According to the 2004 and 2014 China ROP screening guidelines , ROP scrThe first fundus examination was performed at 4 to 6 weeks after birth or at 32 weeks of postmenstrual age (PMA). The following methods and procedures were used: (1) the primary screening was performed by binocular indirect ophthalmoscopy with appropriate mydriasis. Infants with suspected ROP were examined with Retcam (retina camera). (2) The surgeon determined the use and method of treatment based on the results and images got from the Retcam examination. (3) Examination results and treatment recommendations were recorded in detailed electronic medical records. They are location of the disease into zones , the circumferential extent of the disease based on the clock hours (1–12), the severity of the disease (stage 1–5) and the presence or absence of “Plus Disease”. And screen or treatment information, which included follow-up, treatment methods. (4) Follow-up treatments and condition progression were recorded in detailed electronic medical records according to the international staging and classification standard for ROP . They arLaser photocoagulation and anti-VEGF drug (Ranibizumab) were used in 61 premature infants with ROP who reached the therapeutic threshold. Forty-nine with ROP in zone 2 or zone 3 underwent retinal laser photocoagulation, and 12 with ROP in zone 1 or aggressive posterior ROP (AP-ROP) were given anti-VEGF drug (Ranibizumab 0.25 mg) via intraocular injections. All patients with ROP recurrence were retreated with laser photocoagulation. Infants with ROP were monitored until 54 weeks of PMA as the study endpoint.Data related to the objectives were collected from the electronic medical record. Data included the following: (1) birth data , (2) relevant data obtained within 30 days after birth  and congenital dysplasia, etc.), (3) long-term follow-up data for infants with ROP .2 test. A multivariate logistic regression analysis was used to determine independent risk factors for ROP. P < 0.05 was defined as the threshold for a statistically significant difference.SPSS 17.0  was used for all statistical analyses. A single-factor analysis was used to analyze correlations with ROP and identify a normal fundus (ROP (−) or ROP (+). All measurement data are presented as percentages (%) and were measured with the χThe total number of newborns was 11,275 in Guangzhou First People’s Hospital from March 2013 to October 2017. Four hundred thirty-six severely premature infants met the ROP screening criteria and completed a first ROP ophthalmological examination at which their general condition was recorded. Among these patients, 256 (58.72%) were boys, and 180 (41.28%) were girls. The incidence of all stages ROP was 31.25% (80) girls and 32.22% (58) boys. The treatment of ROP was 12.89% (33) girls and 15.56% (28) boys. There are 286 (65.60%) were single births, 116 (26.60%) were twin births, and 34 (7.80%) were gestations births. The incidence of ROP was 33.57% (96) in single births, 23.28%(27) in twin births, and 44.12%(15) in gestations births. The treatment of ROP was 12.24%(35) in single births, 13.79%(16) in twin births, and 44.12%(29.41) in gestations births. The average gestational age was 33.84 weeks (min 26 max 39 weeks). Overall, 342 of the patients (78.44%) had a gestational age ≤ 32 weeks, and 94 patients (21.56%) had a gestation age > 32 weeks. The incidence of ROP were 31.56% (117) in gestational age < 32 weeks and 22.34% (21) in gestational age > 32 weeks. The treatment of ROP were 16.4%(56) in gestational age < 32 weeks and 5.3%(5) in gestational age > 32 weeks. The mean birth weight was 2160.23 g (min 1015 max 3690 g). Birth weights ≤1500 g were recorded in 251 cases (57.57%), weights 1501–2000 g were recorded in 109 cases (25%), and weights > 2000 g were recorded in 76 cases (17.43%). The incidence of ROP were 46.61% (117) in birth weights ≤1500 g, 16.51% (18) in birth weights 1501-2000 g, and 3.95% (3) in birth weights > 2000 g. The treatment of ROP was 21.51%(54) in birth weights ≤1500 g, 6.42% (7) in birth weights 1501-2000 g, and 0 in birth weights > 2000 g. One hundred forty-two of the patients (32.57%) were mechanical ventilation > 96 h group, and 294 patients (67.43%) were mechanical ventilation < 96 h group. The incidence of ROP were 54.22% (77) in mechanical ventilation > 96 h group, and 20.75%(61) in mechanical ventilation < 96 h group. The treatment of ROP were 28.17% (40) in mechanical ventilation > 96 h group, and 7.87%(21) in mechanical ventilation < 96 h group.)According to the screening results and treatment, all children were divided into two groups: ROP (−) and ROP(+). Four hundred thirty-six severely premature infants, 298 patients (68.35%) were ROP (−), and 138 patients (31.65%) were ROP (+). In ROP(+) group, 61 patients (44.20%) received treatment (12 patients revieved anti-VEGF drug treatment and 49 patients received laser treatment). The ROP (+) and ROP (−) groups were included in a univariate analysis. The results showed that the incidence of ROP was correlated with many factors including multiple birth, gestational age, birth weight, mechanical ventilation, IVH, number of operations, and positive blood culture (Table 1P < 0.05), which were introduced into a logistic regression equation for a multi-factor unconditional logistic regression analysis. The independent risk factors that were introduced into the logistic regression model included gestational age, birth weight, mechanical ventilation, oxygen saturation, and average daily weight gain  via intraocular injections. Ranibizumab was injected into the vitreous with 0.25 mg per dose. In the laser photocoagulation treatment group, 21 patients (42.86%) exhibited recurrence of ROP. In the Ranibizumab treatment group, 5 patients (41.67%) had recurrence of ROP. All infants with ROP recurrence were retreated with laser photocoagulation. The conditions of all infants were controlled after retreatment until 54 weeks of PMA. No serious complications such as retinal detachment occurred in any patient in our study. The results of the analysis showed that both the number of gestational weeks and birth weight were significantly lower in the Ranibizumab treatment group than in the laser photocoagulation treatment group and the untreated ROP (+) group , and 13.99% (61 cases) of premature infants with ROP required treatment. These values are similar to those in many Chinese reports (from 10.8 to 17.6%) –28; HoweROP is a disease involving multiple factors, and its exact etiology is not known. According to many reports, gestational age and birth weight are currently recognized as the primary risk factors for ROP . Our resWe also analyzed the correlation between ROP and individual factors in this study; the results showed that the factors significantly associated with the incidence of ROP include IVH, invasive examinations, and positive blood culture. However, the mechanisms by which these factors cause ROP are not clear. We speculate that these factors may lead to vital signs instability, and long-term, high-flow oxygen absorption is usually given as a treatment method.In a logistic regression analysis, gestational age, birth weight and unmonitored supplemental oxygen were indicated as the primary risk factors for ROP . Our stuWith the rapid development of perinatal medicine and continuous improvements in treatment facilities, the survival rate for premature and low-birth weight infants has substantially improved, and the incidence of ROP has also significantly increased in China. Many studies have suggested that the up-regulated expression of VEGF plays an important role in the development of ROP , and antIn report of the developed countries, the ROP that need treatment includes zone 1, any stage with plus disease; zone 1, stage 3, with or without plus disease; and zone 2, stage 2 or 3, with plus disease. The ROP that need watch and wait includes zone 1, stage 1 or 2 without plus disease and zone 2, stage 3 without plus disease. However, there is no uniform treatment threshold for ROP in China , 43. DueRetinal laser photocoagulation is a standard treatment for ROP. However, it can result in many problems, such as a wide range of retinal damage and the loss of vision in the photocoagulation area, and is not suitable for some patients, such as those with ROP in zone 1 or AP-ROP –46. AntiIn our study, children with ROP in zone 2 or 3 were treated with traditional retinal laser photocoagulation, whereas children with ROP in zone 1 and posterior polar invasion ROP were given Ranibizumab via intraocular injections. The recurrence of ROP in the laser treatment and anti-VEGF treatment groups were 42.86 and 41.67%, respectively. Because anti-VEGF drugs may induce systemic risks in premature infants, all infants who had ROP recurrence were retreated with laser photocoagulation.The conditions of all children were controlled after retreatment was given until 54 weeks of PMA. No serious complications such as retinal detachment occurred in any patients in our study. We speculate that may be related to the fact that children with a very low birth weight (< 1000 g) and gestational age (< 26 weeks) were not included in this study. An analysis of the two groups showed that the gestational age and birth weight were significantly lower in the anti-VEGF treatment group than that in the laser treatment group. This result is similar to other reports , 53–57.In conclusion, in this study, we systemically analyzed the incidence of and risk factors for ROP in severely premature infants treated in the NICU at Guangzhou First People’s Hospital. The results of our study suggest that the incidence of ROP is 31.65% which will match those in middle-income countries, but higher than high-income countries. Anti-VEGF drugs could be preferred as a good treatment method for zone 1 ROP and aggressive posterior ROP.


Echinococcus granulosus have been a focus of particular interest in biology and medicine. The determinants and underlying molecular mechanisms of Echinococcus development in different host settings is largely unknown. The phenomenal bi-directional development of E. granulosus protoscoleces into multi-proglottid and/or microcysts, is a fascinating feature of the parasite cultivation. Calmodulin (CaM) is the major intracellular Ca2+ binding protein in plant and animal organisms. Many Ca2+-related processes in the physiology of eukaryotic organisms are CaM-dependent, however little is known on the role of CaM in platyhelminths growth and development. Small interfering (si) RNA-induced manipulations of the genes involving in the parasite development is an opportunity to explore novel approaches for cystic echinococcosis (CE) prevention and management. Regarding the fundamental role of CaM in cellular function of the parasites, in this study, we investigated the molecular and morphological changes induced by siRNA on CaM in different in vitro stages of E. granulosus. Three developmental stages of the tapeworm, protoscoleces, microcysts and strobilated worms, were cultivated in vitro in mono- and di-phasic media and three delivery methods, i.e. electroporation, soaking and electro-soaking, were used for RNA interference. The level of mRNA suppression as well as the phenotypic changes of the parasites were measured. Following RNA interference, EgCaM mRNA suppressions of 65–99% were recorded in different stages of the tapeworm as compared to untreated/unrelated siRNA controls. Lower viability, growth retardation, morphological abnormalities as well as EgCaM expression suppression were documented in the parasite implying potential of siRNA technology for the prevention and management of CE.Among parasitic helminths, biological features of  E. granulosus is a zoonotic platyhelminth causing cystic echinococcosis (CE) in human and livestock. The life cycle of E. granulosus is complex and required two mammalian hosts, leading to many morphological, biochemical, and physiological alterations in the parasite. Echinococcus metacestode called hydatid cyst, develops in the viscera of herbivorous intermediate hosts (livestock and humans) and can infect carnivorous final host (most commonly dogs) via feeding with infected organs. This parasite has a worldwide distribution and it has been estimated to infect 2–3 million people across the globe1.Echinococcus, and an external host-derived adventitial layer formed in response to the parasite2. As a model organism Echinococcus granulosus has been demonstrated as a valuable biological entity influencing advancements in developmental biology of flatworms3.The gravid worm shed eggs in dog small intestine which are disseminated in the environment via defecation. Infection of the intermediate host occurred through ingestion of infective eggs, followed by the release of oncospheres in the small intestine. The oncosphere subsequently migrates via the portal system to various organs and tissues, mainly liver and lungs, where it develops into a unilocular, fluid-filled cyst comprised of an inner germinal layer which responsible for production of the infective stage, a structure called protoscolex (PSC), an extracellular matrix in the middle called laminated layer that is unique to the genus Echinococcus cultivation in appropriate diphasic and/or monophasic media the phenomenon of bi-directional development of E. granulosus protoscoleces into multi-proglottid and/or microcysts, is a fascinating feature of Echinococcus biology. This provides us an opportunity to study genes involving in the parasite development and to manipulate the parasite for disease management and prevention6. Understanding the determinants and molecular mechanisms in the parasite development in different host settings offers suitable tools for prevention and control of CE2.Following 8. Wang et al. have used Immunohistochemical localizations on different stages of the parasite, suggesting the expression of E. granulosus CaM (EgCaM) in the tegument tissues, protoscoleces parenchymal region, germinal layer as well as in the adult stage. The study showed physiologically active regions for EgCaM expression and suggested the crucial role of Ca2+ signaling pathway in the growth and development of the helminth and its function between E. granulosus and its host9.Calmodulin (CaM), is a multifunctional intermediate calcium sensor protein expressed in all eukaryotic organisms. The functions of CaM include Ca2+ binding and alteration of calcium signal transduction pathway to regulate a multitude of biological processes, such as cytoskeletal assembly/reorganization, activation of phosphorylase kinase, abiotic stress responses, neurotransmission, smooth muscle contraction, metabolism and cell motility10. Molecular methods based on RNAi or Post-Transcriptional Gene Silencing (PTGS) are the new millennium tools that provide information on gene function and characterization of the genes involving in regeneration and development. RNA interference mediated by siRNA with perfect homology to their target can cause silencing of specific genes for experimental and therapeutic purposes12.In recent decade, there has been increasing interest in RNA interference (RNAi) technology in the field of biology and development. Our understanding of the genetic basis of development in invertebrates is limited. Small interfering RNAs (siRNAs) provides us a valuable tool to improve our knowledge on the developmental biology of PlatyhelminthesFasciola hepatica leucine aminopeptidase (LAP) with siRNAs as well as double-stranded RNAs (dsRNA), suppressed mRNA and protein expression for up to 3 weeks in the newly excysted juveniles13. RNAi has been used to suppress a number of E. multilocularis endogenous genes expression in protoscoleces14. Protoscoleces electroporation of genes encoding the 14–3–3 and elp with siRNA resulted in reduced expression of up to 21.8 and 35.5% of the genes respectively, compared to untreated control. In addition, the target proteins have been significantly reduced on day 1514.RNAi has been successfully applied to numerous helminth organisms to date. Silencing of the gene encoding E. granulosus. There are reports for schistosomes16, the liver flukes Fasciola gigantica and F. hepatica18 and Caenorhabditis elegans19. Suppression of calmodulin mRNA by feeding Schistosoma mansoni with dsRNA resulted in a phenotype characterized by waves of contraction in adult worms but not in schistosomula16. RNAi has also been used to determine the biological function of F. hepatica calmodulin in vitro. Stunted growth and lower viability were documented after treating the in vitro cultured flukes with dsRNAs and siRNA20. The purpose of the present study was to determine the effect of RNA interference on EgCaM expression in different developmental stages of E. granulosus in vitro.CaM-specific RNAi technology has been utilized to determine the functional importance of calmodulin in several helminth parasites, however there are very few data on the role of CaM function in E. granulosus (Accession Number MG832791). E. granulosus protoscoleces were successfully cultivated in both monophasic and diphasic media. The protoscoleces started to develop proglottization in diphasic culture and the first proglottids were observed on day 28. However, the full grown three-proglottid strobilated forms were developed after 55 days of cultivation. Egg production in the terminal proglottid was not observed under the culture conditions , however the fluorescence pattern was distinct and readily distinguishable in the parasites tegument treated with siRNA.  was observed following electro-soaking in all developmental stages of us Table . It has et al. successfully suppressed 14-3-3 and elp genes expression by 21.8% and 35.5% respectively. Also, in the analysis of viability, 14-3-3, and elp siRNA-treated samples displayed 58.0 ± 23.0, and 55.1 ± 14.6% viability on day 15, respectively, compared to the untreated control14. This is in line with our data on the viability of protoscoleces after siRNA-mediated CaM suppression  in juvenile S. japonicum, inducing death or changes in the parasite morphology37.After 8 days of siRNA treatment on the strobilated forms, 65–89% EgCaM expression suppression was observed Table . In a reE. granulosus leads to decreased motility and strobilar contractions  are proved to play a key role in the motility-related functions in invertebrates40. However, using both RNAi and the CaM inhibitor, Trifluoperazine, McCammick et al. showed a significant increase in motility, migration and movement of the juvenile F. hepatica20.In the strobilated worms we noted significant phenotype changes including severe morphological abnormalities and reduced body contractions from day 2 onwards Fig. . It seemons Fig. . While pin vitro data to in vivo experiments is difficult. Therefore, cautions have to be made in the interpretation of culture data at the host level41. As in vitro-reared E. granulosus do not develop as much as they do in the hosts, we could not precisely predict the behavior of the helminth in its natural habitat in the host42. As the dsRNAs are generally induced more persistent effects on target genes, this study would be supplemented by using an EgCaM-specific dsRNA. However, using dsRNAs has its own limitations and challenges44. Another prospect for RNAi-induced gene silencing in E. granulosus is to investigate the interference impacts at protein level using western blotting and other protein-based tools as well as further in-depth in vivo studies followed by deep sequencing analyses.Generalizations of E. granulosus. The results indicate the significant effect of siRNA treatments on the phenotype as well as the gene expressions in all development stages of E. granulosus cultured in mono- and di-phasic media using different delivery methods. The study demonstrated that EgCaM is essential for viability, growth and development of the protoscoleces. Different phenotypic changes and transcript knockdowns observed in the present study encourage further investigations towards the development of novel therapeutic agents against cystic echinococcosis.The present study used siRNA for suppressing EgCaM in different developmental stages of in vitro cultivation, were obtained from a single sheep liver cyst. Therefore, the microcysts and strobilated worms were derived from the cultivation of protoscoleces obtained from the same cyst.Parasite specimens were obtained by dissection of livers from naturally infected sheep at the municipal abattoir of Kerman, southeastern Iran with consent from animal handlers and the abattoir veterinary officer. Animals were slaughtering as part of the normal daily practice in the abattoir. The study was approved by the University Ethics Review Committee (code 95000288). All procedures were performed according to the University guidelines and regulations. The infected organs were immediately transferred to the Helminthology Lab, Dept of Medical Parasitology, Kerman University of Medical Sciences. After careful examinations of fertility and viability, one single viable cyst was finally selected for further experiments. Genotyping has been carried out using mitochondrial cox1 PCR-sequencing and the sequence was submitted to GenBank. Protoscoleces used for 4 protoscoleces in 0.9% NaCl solution with a viability rate of at least 95%. Before cultivation the viability of the protoscoleces was checked by 0.1% aqueous eosin under a light microscope45. Two layers of sterile gauze were used to release protoscoleces from the brood capsules.Under sterile conditions the hydatid fluid containing protoscoleces was aspirated with a 50 ml syringe and then the laminated/germinal layer were removed. The aspirate as well as the cyst layers were carefully washed five times with PBS containing 100 U/ml penicillin and 100 μg/ml streptomycin (PBS-PS). The number of protoscoleces per ml was adjusted to 1 × 10in vitro culture in diphasic and monophasic media to reach the strobilated as well as the microcyst forms, respectively, according to the method described by Smyth et al.45. The diphasic medium was S.10E.H, consisted of two phases: (i) the liquid phase, containing 260 mL of CMRL 1066 medium , 100 mL of heat-inactivated fetal calf serum , 36 mL of 5% yeast extract  in CMRL 1066, 5.6 mL of 30% glucose (Sigma-Aldrich) in distilled water, 1.4 mL of 5% dog bile in PBS, 20 mM HEPES (Sigma-Aldrich), 10 mM NaHCO3 supplemented with penicillin (100 IU/mL), streptomycin (100 mg/mL), and (ii) the solid phase that is bovine serum coagulated at 76 °C for 20–30 min46. The medium was changed every 7 days and viability and morphological development of the parasite was observed under an invert microscope .The protoscoleces were used for 2 flasks incubated at 37 °C with 5% CO2. The medium was changed every 8 days. The flasks containing the protoscoleces were monitored weekly under an optical microscope to check the growth status of the microcysts48.For monophasic culture the protoscoleces were cultivated in Dulbecco’s minimal essential medium (DMEM)  containing 10% heat-inactivated FBS (Gibco), 2 mM glutamine (Sigma-Aldrich), penicillin (100 IU/ml), and streptomycin (100 mg/ml) at the bottom of 25 cmhttps://www.invivogen.com/sirnawizard). After prediction, all siRNAs were manually re-checked relative to their position, target site, length of siRNA, nucleotide content and specificity of siRNA (off targets), and finally the best was chosen. The siRNA was synthesized commercially by TAG Copenhagen A/S . The sequences of the EgCaM siRNA were as follows: sense 5′UCGUUAAAGUCAAUAACACCC3′-fluorescein and antisense 5′GUGUUAUUGACUUUAACGAAU3′ –fluorescein. Another negative (non-silencing) control siRNA was purchased as an unrelated siRNA .We used several online softwares for siRNA design i.e. siDirect (sidirect2.rnai.jp/), siRNA Design , BLOCK-iT RNAi Designer (www. invitrogen.com/rnai), and RNA wizard (E. granulosus: (1) no-treatment group without any RNAi intervention, (2) unrelated/irrelevant Negative Control siRNA (siR-Ctrl) group that was transfected into the parasites using three transfection techniques i.e. soaking, electroporation and electro-soaking, (3) EgCaM-specific siRNA group delivered by soaking, (4) EgCaM-specific siRNA group delivered by electroporation and (5) EgCaM-specific siRNA group delivered by electro-soaking49. EgCaM-specific and Negative Control siRNAs were delivered into the parasites of corresponding treatment groups using three transfection techniques. No interventions were made on the parasites in no-treatment groups. For siRNA delivery, in 6-well plates different stages of the parasite with three separate batches of 5000 protoscoleces, 15 microcysts and 15 strobilated worms were treated by soaking, electroporation and electro-soaking in the presence of 50 nM/ml of fluorescent siRNA50. For soaking, protoscoleces were cultured in 2.5 ml DMEM supplemented with siRNAs and a lab-made transfection reagent was then added to each well and maintained for 6 hours51. For electroporation, protoscoleces were removed from the culture, resuspended in 200 μl of electroporation buffer containing the fluorescently labeled siRNA to give a final concentration of 50 nM, transferred to a 2 mm gap cuvette, and were subjected to time constant protocol  using Gene Pulser II 31. For electro-soaking following electroporation, the parasites were maintained in a culture media containing siRNAs for 6 hours. After treatment, the parasites were rinsed thoroughly in PBS to remove unincorporated siRNA molecules. A fluorescent microscope  was used to examine siRNA delivery. The parasites viability, as revealed by flame cell activity, were recorded daily for eight days and any phenotypic changes e.g. motility, transparency and tegumental alterations were examined. In addition, the size of the protoscoleces was measured using a calibrated microscope. The motility of the strobilated worms from each group of target siRNA and controls were measured as the number of body contractions per minute  using a commercial kit . Furthermore, RNA was quantified by measuring the absorbance ratios at 260 and 280 nm by spectrophotometry . cDNA was then synthesized from 100 ng RNA in a total volume of 20 μl using miscript®II Reverse Transcriptase Kit  according to the manufacturer’s instructions.Real-time qPCR (RT-qPCR) was carried out in a Rotor-Gene Q System . The primers specifically designed for EgCaM RT-qPCR were CalF . The primers of the ACTB (encoding β-actin) were ACTBF (5′-ATG GTT GGT ATG GGA CAA AAG G-3′ and ACTBR 5′- TTC GTC ACA ATA CCG TGC TC-3′). Relative quantification of gene expression levels was carried out by using SYBR green PCR Master Mix with 5 µl 2X QuantiNova SYBR Green PCR, 0.4 µM primer, 2 µl 0.5X diluted Template cDNA and 2 µl RNase-free water with a final volume of 10 µl. All samples were run in duplicate and underwent 40 cycles of 95 °C, 2 min for initiation, 94 °C, 5 sec for denaturation, and 60 °C, 10 sec for annealing.−ΔΔCT method was employed, using β-actin as the reference gene for each sample. Results obtained from the parasites treated with the unrelated Negative Control siRNA were used as calibrators. Negative Control siRNA target gene was unknown, as it is the proprietary information of Qiagen®. According to the manufacturer, the unrelated siRNA has been proven to have no significant effect on cell proliferation, viability, or morphology.For relative quantification, 2www.graphpad.com). P values of less than 0.05 were considered significant.Differences between and within groups were assessed for statistical significance using one-way or two-way ANOVA test using GraphPad Prism 7.0 Software (Supplementary Figure S1


We have shown that exposure of rats to lipopolysaccharide (LPS) during gestation induces autistic-like behaviors in juvenile offspring and pioglitazone post treatment corrects social and communication deficits. The first objective of the present study was to evaluate the cognition of the rats, because this is also a behavioral sphere committed in autism. Second, biomarkers related to pioglitazone pathways and autism were studied to try to understand their mechanisms. We used our rat model of autism and pioglitazone was administered daily to these young offspring. T-maze spontaneous alternations tests, plasma levels of brain-derived neurotrophic factor (BDNF), beta-endorphin, neurotensin, oxytocin, and substance P were all studied. Exposure of rats to LPS during gestation induced cognitive deficits in the young offspring, elevated BDNF levels and decreased neurotensin levels. Daily postnatal pioglitazone treatment abolished cognition impairments as well as BDNF and neurotensin disturbances. Together with our previous studies, we suggest pioglitazone as a candidate for the treatment of autism, because it improved the responses of the three most typical autistic-like behaviors. BDNF and neurotensin also appeared to be related to the autistic-like behaviors and should be considered for therapeutic purposes. Summary: Exposure of rats to lipopolysaccharide during gestation induced autistic-like behaviors in the juvenile offspring. Daily postnatal pioglitazone treatment abolished cognition impairments as well as brain-derived neurotrophic factor and neurotensin disturbances. Autism spectrum disorder (ASD) is a developmental disorder that is characterized by social, communicative and cognitive deficits and has a higher prevalence in males . The incWe have shown that exposure of rats to lipopolysaccharide  during gestation [gestational day (GD) 9.5] induces socialization and communication deficits as well as repetitive/restricted behavior in juvenile offspring. These behavioral impairments are found just in males and not in the female offspring . TherefoThere is no effective treatment for ASD, and a small number of drugs are authorized by the US Food and Drug Administration (FDA) . These dThere are pilot studies showing that pioglitazone treatment associated with other pharmacological and behavioral therapies induce some clinical improvements in autistic patients. For example, daily pioglitazone treatment attenuated irritability, lethargy, stereotypy and hyperactivity in some autistic children, without significant side effects . SimilarUsing our rat model of autism, we have demonstrated that pioglitazone treatment corrects social and communication deficits as well as elevated plasma IL-6 levels . The firP=0.0037). Prenatal LPS exposure decreased T-maze spontaneous alternation in the offspring compared with the control group (P<0.01). Although postnatal pioglitazone treatment with 0.25 mg/kg/day did not affect the performance in the T-maze task, postnatal pioglitazone treatment with 1.0 mg/kg/day increased spontaneous alternation in the rats prenatally exposed to LPS  to the same levels as those in the control group. Thus, prenatal LPS induced cognitive inflexibility, and postnatal pioglitazone treatment abolished these impairments.F(3/28)=5.43, P=0.0045]. Prenatal LPS exposure (LPS+DMSO group) increased the total BDNF levels compared with the control group . Both doses (0.25 and 1.0 mg/kg/day) of the post-treatment with pioglitazone decreased BDNF levels in the rats that were prenatally exposed to LPS  to the same levels as those in the control group.F(3/28)=3.15, P=0.0407 and F(3/28)=8.41, P=0.0004, respectively], whereas oxytocin and substance P levels were not affected by the treatments . Prenatal LPS exposure (LPS+DMSO group) did not affect beta-endorphin levels compared with the control group (SAL+DMSO). Both pioglitazone doses (LPS+PI0.25 and LPS+PI1.0 groups) increased the beta-endorphin levels compared with the LPS group . Prenatal LPS exposure increased neurotensin levels compared with the control group . Although postnatal pioglitazone treatment with 0.25 mg/kg/day did not affect the neurotensin levels compared with the LPS group it recovered the levels to the same levels as those in the control group. Postnatal pioglitazone treatment with 1.0 mg/kg/day increased the neurotensin levels in the rats prenatally exposed to LPS  to the same levels as those in the control group. Thus, prenatal LPS decreased neurotensin levels, and postnatal pioglitazone treatment recovered this disturbance.The most typical symptoms found in ASD are social, communicative and cognitive deficits, such as cognitive inﬂexibility and repetitive/restricted behavior . We haveConsidering no effective treatment yet exists for autism, we started to search for new treatments for autistic symptoms using our rat model. We selected pioglitazone because although it is originally an anti-diabetic drug, it also exerts anti-inflammatory effects in several cell types . MoreoveWe have demonstrated that pioglitazone treatment corrects social and communication deficits . Our preBDNF is a member of the neurotrophins family and is found in the central nervous system and peripheral blood. It is involved with morphological, developmental and neuronal processes, such as synaptogenesis and synaptic plasticity . SeveralBoth doses of postnatal pioglitazone treatment (0.25 and 1.0 mg/kg/day) abolished the BDNF disturbance induced by prenatal LPS. Although there are few studies about the relation between pioglitazone and BDNF, it is known that memory impairment, BDNF disturbance and oxidative damage induced by β-amyloid in an animal model of Alzheimer's disease are reversed by chronic administration of pioglitazone . MoreoveTachykinin 1 gene (TAC1), which is located in a candidate region for ASD  induced cognitive inflexibility and elevated BDNF levels in juvenile male rat offspring, which are described as autistic-like symptoms. We also found reduced plasma neurotensin levels after prenatal LPS exposure. Daily postnatal pioglitazone treatment corrected the cognition impairments, as well as BDNF and neurotensin disturbances. Together with our previous studies , we suggin vivo techniques when available. The experiments were performed in accordance with good laboratory practice protocols and quality assurance methods.This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the School of Veterinary Medicine, University of São Paulo, Brazil (permit no. 2824/2012). All efforts were made to minimize the suffering, reduce the number of animals used and utilize alternatives to Rattus norvegicus) of 15–17 weeks of age and weighing 220–275 g were used. The rats’ housing, nutritional conditions, determination of GD 0, pregnancy monitoring, weaning and litter sexing during the experiments were the same as previously described by our group  was dissolved in sterile saline (50 µg/ml LPS in a 0.9% NaCl solution) and administered intraperitoneally (i.p.) to pregnant dams at a dose of 100 µg/kg on GD 9.5 (n=13). This GD of LPS exposure as well as the dose was chosen based on our previous studies (n=6). Each dam (LPS and control) received 0.2 ml/100 g of each solution (LPS and vehicle solutions).LPS  also received pioglitazone (Sigma-Aldrich) or its vehicle dimethyl sulfoxide  daily from PND 21 until 29. Rats received one of two pioglitazone doses: 0.25 and 1.0 mg/kg/day (i.p.) based on the pharmacokinetics of the drug , the autistic children's prescriptions  and rat n=8 rats per group): the SAL+DMSO group (control group), the LPS+DMSO group (LPS group), the LPS+PI0.25 group and the LPS+PI1.0 group. The experimental groups are described in Four groups were investigated  followed by Fisher's LSD multiple comparison tests were used to compare the parametric data between the four groups. The results are expressed as the median (minimum and maximum) or the mean±s.e.m. In all cases, the results were considered as statistically signiﬁcant at P<0.05.Homogeneity and normality were verified using a Bartlett's test or


AbstractAves between August 2013 and October 2018 as part of a LIFE research project aiming to preserve and restore three coastal wetlands from Praia da Vitória . Systematic monthly observations were carried out for five years in order to provide a checklist and monitoring of bird species and subspecies observed in three sites: Paul da Praia da Vitória (PPV), Paul do Belo Jardim (PBJ) and Paul da Pedreira do Cabo da Praia (PPCP). Main objectives were to determine their ornithological richness while also adding data to the overall knowledge of Azorean Avifauna and to monitor seasonal and between-year variation on species abundance.The data presented here come from field observations of Aythyaamericana ; Chlidoniasleucopterus  and Tringabrevipes . One species is a new record for Terceira Island: Lophodytescucullatus .During a five-year observation period (2013-2018), a total of 82,985 birds belonging to 108 species/subspecies were observed. From this, 16,663 were in PPV, 11,793 from PBJ and 54,529 from PPCP. The total richness was 55, 40 and 85, respectively. Three species are first records for the Azores:  The Azorean Avifauna has been described in several publications, the most recent being Paul da Praia da Vitória (PPV), Paul do Belo Jardim (PBJ) and Paul da Pedreira do Cabo da Praia (PPCP) – were studied during the LIFE – Coastal Wetlands Restoration Project and are known as a high avifauna site attracting birdwatchers and which include an important number of species  -  species . PereiraThis manuscript is the third contribution in a series of papers that characterise the biota of the three coastal wetlands from this area .The aim of this work is to inventory the avifauna present in the three coastal areas of Praia da Vitória , focused on the LIFE-CWR Project, Paul da Praia da Vitória (PPV) Fig. , Paul doInventory of bird species in three coastal wetlands from Terceira Island (Azores)The inventory was conducted during five years between August 2013 and October 2018 by experienced birdwatchers: Sofia Goulart, Mariana R. Brito and Sónia Santos.Terceira Island  is one of the nine islands of the Azores archipelago, located in the North Atlantic, roughly at 38°43'49''N 27°19'10''W . The cliThis study covers a small coastal area with 3.58 km extension between PPV and PPCP.http://lifecwr.com/index.php/pt/observacao/registos-de-observacao/registos-de-observacao-2). Whenever needed, several field-guides were used  using a Swarovski 20-60 telescope and Opticron Verano HD 10-42 binocular. Photographs were made with a Canon 60D camera , Macaronesia, Portugal.38º42'09''N and 38°44'12''N Latitude; 27º03'46''W and 27°02'39''W Longitude.AvesData range: August 2013 – October 2018.Creative Commons Public Domain Waiver (CC-Zero)LIFE_CWR_TER_Aveshttp://ipt.gbif.pt/ipt/resource?r=azores_birdshttp://islandlab.uac.pt/software/ver.php?id=341Birds from Praia da Vitória marshes Darwin Core Archive50http://ipt.gbif.pt/ipt/resource?r=azores_birdsversion 1In this data table, we include all the records for which a taxonomic identification of the species was possible. The dataset submitted to GBIF is structured as a sample event dataset, with two tables: event (as core) and occurrences. The data in this sampling event resource have been published as a Darwin Core Archive (DwCA), which is a standardised format for sharing biodiversity data as a set of one or more data tables. The core data table contains 2003 records. One extension data table also exists. An extension record supplies extra information about a core record. The number of records in each extension data table is illustrated in the IPT link. This IPT archives the data and thus serves as the data repository. The data and resource metadata are available for downloading in the downloads section. The versions table lists other versions of the resource that have been made publicly available and allows tracking changes made to the resource over time. In Suppl. material Scolopacidae (32 species) and Anatidae (24 species) were the most diverse while three species corresponded to 47.8% of all observed/identified birds , Charadriusalexandrinusalexandrinus  and Arenariainterpres . The Order Charadriformes, with 63,671 individuals observed, corresponds to 75.7% of all birds from this work , Chlidoniasleucopterus (Temminck 1815) and Tringabrevipes (Vieillot 1816). One species is a new record for Terceira Island: Lophodytescucullatus (Linnaeus 1758). Aythyaamericana is a Nearctic occasional migrant duck, Chlidoniasleucopterus is a Palearctic tern and Tringabrevipes is a shorebird breeding in northeast Siberia. All other species have previously been recorded at several levels of relative abundance, both as breeding native (ten taxa), breeding Azorean endemic (seven taxa), breeding Macaronesian endemic (one taxon), breeding introduced (four taxa) and vagrant   wetlands: Paul da Praia da Vitória (PPV), Paul do Belo Jardim (PBJ) and Paul da Pedreira do Cabo da Praia (PPCP).File: oo_268688.xlsxGoulart S et al.


We conducted a study in which 99mTc-Rituximab was compared with filtered 99mTc-sulfur colloid (fTcSC) for sentinel lymph node (SLN) detection in patients with breast cancer.99mTc-Rituximab and fTcSC, the interval time is separated by ≥2 days. 2. Then, 91 patients were selected for a randomized controlled trial . All patients were administered either agent at the 6- and 12-o’ clock positions by subareolar injection technique. SLN mapping was then performed 2 h after injection. 3. Serial dynamic images were further acquired for 2 h in 31 patients .The study is divided into three parts. 1. Initially, 25 patients were selected for an internal controlled trial to received both 99mTc-Rituximab and 99mTc-SC were 98.5% vs 98.7, 100% vs 98.4%, respectively. The mean number of SLNs identified by 99mTc-Rituximab and fTcSC was respectively 2.72 and 3.28, with a significant difference of P = 0.013 (paired sample t-test). The difference exists in the internal mammary and clavicular area, not in the axillary. The mean number of axillary sentinel lymph node biopsy (SLNB) for 99mTc-Rituximab and fTcSC was 2.95 vs 3.14, respectively, and no significant difference existed. 99mTc-Rituximab also exhibited a significantly faster injection site clearance rate when compared with fTcSC .The identification rate of lymphoscintigraphy and SLNB in all and axilla regions for 99mTc-Rituximab based imaging demonstrated a fewer number of secondary lymph nodes and had faster injection site clearance rate.No significant difference was observed in identification rate and number of axillary SLN imaging and SLNB, between the two tracers. Compared to fTcSC, ChiCTR1900024990 .www.chictr.org.cn,  Axillary nodal involvement in breast cancer has a significant impact on tumor staging, treatment, and prognosis. The concept of SLN, first described in 1977 by Cabanas and colleagues, and is defined as the first lymph node that receives afferent lymphatic drainage from a primary tumor .99mTc-sulfur colloid (99mTc-SC), whereas, in Europe, the most used tracer is 99mTc-HSA nanocolloid [Despite the widespread use of SLNB for breast cancer, there is still uncertainty in regards the optimal agent for SLN identification. Radiopharmaceuticals and vital blue dye (VBD) are now commonly used. The most common agent used in the United States is ocolloid . Still, An ideal radiopharmaceutical for SLN detection should exhibit rapid clearance from the injection site, rapid uptake, high retention within the first draining lymph node, and low uptake by distal lymph nodes. These characteristics can be fulfilled based on the properties of novel ligand-receptor or antigen-antibody specific binding agents for SLN mapping . Lymphos99mTc-Rituximab for SLN detection in animal models and confirmed that it exhibits the desired properties of rapid clearance from the injection site as well as low accumulation in distal nodes. In this work, we carried out a clinical trial to confirm these characteristics in human subjects and to further compare the detection efficiency between 99mTc-Rituximab and fTcSC.CD206 and CD20 are current research focuses of tracer targets for tracer development in regards SLN agents. The CD20 antigen is specifically highly expressed at the surface of almost all normal and malignant B cells, and is neither shed from the cell surface nor internalized after binding to specific antibodies  has repoPatients diagnosed with breast cancer who underwent SLNB, in the period of Oct 2016 to Oct 2017, participated in this study. Procedures were followed according to the EANM and SNMMI practice guidelines . The inc99mTc-Rituximab at first, and fTcSC 2 days later. Twelve patients first received fTcSC, and followed by 99mTc-Rituximab 2 days later. Secondly, 91 patients participated in a randomized controlled trial, and there were 41 patients in the 99mTc-Rituximab group and 50 patients in the fTcSC group. Thirdly, 31 subjects participated in the injection site clearance rate trial. And 22 versus 9 patients were administered with 99mTc-Rituximab versus fTcSC, respectively. Serial dynamic images were acquired for 2 h. The age range of the subjects was between 32 to 76 years old  method, described by Li Nan [99mTc-Rituximab was standardized as 74 MBq in 1 ml on the day before operation, and 37 MBq in 1 ml on the operative day. The fTcSC was radiolabeled, boiled for 3 min, and passed by a 200 nm membrane for filtration, and the radiochemical purity was always > 91%. The injection dose of fTcSC was 74 MBq in 1.5 ml on the day before operation, and 37 MBq in 1.5 ml on the operative day.Each patient was administered with an imaging agent at the 6- and 12-o’clock positions, by applying the subareolar technique with both subcutaneous and intra-parenchymal injection, regardless of the site of breast cancer. Administration site was then massaged for several minutes. Two hours later, a lymphoscintigram was acquired, including planar imaging and fusion tomography, in order to localize the SLNs, which is divided into three areas: ALN, internal mammary lymph nodes (IMLN) and clavicular lymph nodes. The SPECT (GE Discovery NMCT 670) acquisition conditions were set as matrix, 128 × 128; zoom, 1; peak energy, 140 keV; window width, 20%.n = 31), dynamic imaging of the injection site was immediately performed after the injection, at 30-min intervals for 2 h. All images (256 × 256) were acquired for 3 mins. The standard source was placed on the level of the contralateral clavicle of the breast lesions. The clearance rate constant (kc) and half-life (Tc) at the injection site were calculated using decay corrected counts, obtained from the nuclear images of the injection site.For the third group of patients , injected by subareolar and subcutaneous technique, was used to trace blue-stained nodes. During operation, γ detector identified all nodes with counts rate greater than 10% of the nodes with the highest count rate, that is, hot nodes [All excised lymph nodes were examined by intraoperative frozen section and printing slice cytology analysis. Classification of SLN metastases followed according to the 8th edition of AJCC, and in our study a positive SLN was identified, when it contained metastatic tumors with sizes > 0.2 mm, and then the lymph node basin was completely dissected.99mTc-Rituximab and the fTcSC. A difference with a P value < 0.05 was considered statistically significant.Chi square analysis or Fisher’s exact test was used to compare clinical data, tumor features, and SLN identification rates between groups. The Wilcoxon rank-sum statistical test was also used to compare the number of SLNs detected by the two agents. T-test was applied to calculate significant differences in the kc and Tc values between the 99mTc-Rituximab and fTcSC. As for clavicular lymph nodes, the identification rate detected by 99mTc-Rituximab was 8%, which was less than fTcSC, 28%. Still, this difference was not statistically significant, due to the small sample size.Twenty-five patients were enrolled in the internal-controlled trail in which paired injections of each agent were administered, at separate times and on different days. And lymphoscintigraphies were compared. Both imaging agents achieved a high SLN imaging identification rate of 96%. Both tracers failed to detect SLNs in one patient who performed SLNB after neoadjuvant chemotherapy. No statistical difference was observed in the identification rate of ALN and IMLN, detected between 99mTc-Rituximab and fTcSC were respectively 68 vs 82, (mean 2.72 vs 3.28), and the difference had statistical significance (P = 0.013). 99mTc-Rituximab identified fewer or the same number of SLNs, while all of them were detected by fTcSC, with a 82.9% concordance rate (68/82). There was no statistical difference in the average number of ALN detected between 99mTc-Rituximab and fTcSC . A significant difference between the two agents was observed in the number of both IMLN and clavicular lymph nodes, and the total number of SLNs identified by 99mTc-Rituximab or fTcSC was 8 vs 12, 2 vs 9, respectively. Figure 99mTc-Rituximab detected 2 SLNs while fTcSC detected 4 SLNs.The total number of SLNs identified by e P = 0.03. 99mTc-99mTc-Rituximab and fTcSC tracers was 2.61 and 2.92, respectively, whereas the number detected by SLNB was 2.95 and 3.13, respectively. The metastatic rate for 99mTc-Rituximab and fTcSC was 17.07 and 20%, and no statistically significant difference existed between the two agents. Also, no significant difference between 99mTc-Rituximab and fTcSC was observed in the directive function of operation. We found that fusion tomography for SLN localization, combined with 99mTc-Rituximab, had lower identification rate and detected fewer numbers of clavicular lymph nodes than fTcSC, and this difference was statistically significant (P < 0.05).There was no significant difference in age, breast type, tumor size, pathological type, tumor location and molecular typing between the two groups (P > 0.05). The identification rate of SLNs imaging and SLNB was 100% for both groups. The mean number of SLNs detected by imaging with 99mTc-Rituximab was less than fTcSC, but only in randomized controlled trial, the difference was significant. In addition, we have found that the both agents had similar numbers of ALN and SLNB. 99mTc-Rituximab had fewer number of total SLN, IMLN in internal-controlled trial and fewer clavicular lymph nodes in the two former trials. Although the same items of those two former trails have similar value, their difference was not significant possibly due to the small cohort patients.SLN counting and identification rates of the two former trails are shown in Fig. 99mTc-Rituximab exhibited a significantly faster injection site clearance rate and shorter half-life. The clearance rate constants for 99mTc-Rituximab ranged from 0.070 to 0.309 h− 1 and from 0.012 to 0.035 h− 1 for fTcSC. The mean injection site clearance rate constant (kc) for 99mTc-Rituximab was 0.193 ± 0.057 h− 1, which was higher (about 10 times) than the mean (kc) for fTcSC, which was 0.021 ± 0.007 h− 1. The acquired (Tc) value for 99mTc-Rituximab was 3.996 ± 1.600 h and 36.053 ± 12.495 h for fTcSC. Figure 99mTc-Rituximab and fTcSC.We have observed that 99mTc-Rituximab. All patients underwent fusion tomography to accurately locate SLNs, and it has been found that the secondary lymph nodes detected by fTcSC were mainly located in the internal mammary and clavicular basin, rather than in the axillary region. Breast lymph is mainly drained to the axillary nodes, then successively passed to subclavian and supraclavicular lymph nodes, which means that clavicular lymph nodes correspond to distal lymph nodes. The study of Cao XS et al. [SLN is the first lymph node to receive drainage from a lymphatic tumor. Ideally, only the anatomical SLNs should be excised. In fact, unnecessary removal of secondary lymph nodes can result in excessive dissection, which increases the incidence of seromas and infection . Since bS et al.  on the lThe principal mechanism of action of most targeting agents is mediated by the specific binding to ligands and their receptors, so when a SLN is not saturated, the agents are unable to drain to the secondary lymph nodes. This is due to the fact that, only under SLN saturating conditions, distal nodes will be able to uptake the agents. Consequently, under optimal dose administration, these agents can target and further identify fewer secondary lymph nodes. This phenomenon has also been demonstrated in some studies related to Lymphoseek , 14.99mTc-Rituximab+VBD or fTcSC+VBD. No significant difference between these groups was observed, in regards the directive function of operation. A preliminary clinical study, carried by Li Nan and colleagues, revealed that 99mTc-Rituximab have a high success rate (100%) and a low false negative rate (2.60%) [99mTc-Rituximab and SLNB was 2.61 and 2.95, respectively. Compared to a larger sample clinical trial [We compared breast cancer patients undergoing axillary SLNB who received  (2.60%) . In this99mTc-Rituximab have not been previously reported.Although the excision of a lower content of SLNs may reduce the surgical time and treatment costs, the detection accuracy of SLNB is essential. The minimal number of SLNs that are necessary for accurate axillary staging has not been determined yet . Many re99mTc-Rituximab exhibits a significantly faster injection site clearance rate and shorter half-life than fTcSC. This indicates low residue and scattering at the injection site, which can reduce the interference with precise SLN identification, especially when SLN is located near the injection point. In addition, the low scattering effect of 99mTc-Rituximab could potentially prevent unnecessary radiation exposure to the surgical team.Here, we demonstrate that 99mTc-Rituximab has equivalent detection rates for ALN and IMLN, as well as similar operation guidance of axillary SLNB, leading to fewer non-SLNs and faster clearance at the injection point. These features define 99mTc-Rituximab as a potential specific tracer, therefore providing a novel and clinically valuable approach for SLNB.Compared with fTcSC, the antigen-antibody binding agent


This active, open observational study aimed to investigate adverse drug reactions (ADRs) associated with six commonly used antiepileptic drugs (AEDs) in southern Chinese outpatients with epilepsy from 2003 to 2015.The Wenzhou Epilepsy Follow-Up Registry Database (WEFURD) was established by a single epilepsy center in China in January 2003 to record AED efficacy and the associated ADRs by registered outpatients diagnosed with epilepsy. We reviewed the data of outpatients who had only taken one or more of six commonly used AEDs, namely, carbamazepine (CBZ), valproate (VPA), lamotrigine (LTG), oxcarbazepine (OXC), topiramate (TPM) and levetiracetam (LEV), and were registered in the WEFURD between 2003 and 2015. We evaluated the ADRs caused by the single or combined use of the above six specific AEDs based on the WHO-UMC scale. The data of the ADRs were categorized by age, sex, number of AEDs related to ADRs, medications, seriousness of ADRs, causality levels of the WHO-UMC scale and system organ class (SOC). The unit of analysis was one ADR.p <  0.001). Eighteen SOC categories were identified, and the top three were psychiatric disorders , neurological disorders  and gastrointestinal disorders . Of the 537 SARs, skin and appendage disorders accounted for 24.4% (131/537). Among the 34 SAEs, serious allergies, fetal malformations, renal calculus and pancreatitis accounted for the majority.A total of 3069 epilepsy outpatients (1807 outpatients with 5049 eligible ADRs and 1262 outpatients without ADRs) were included. The overall ADR rate was 58.88% (1807/3069). An average of 2.79 ADRs (5049/1807) occurred per patient with an ADR; 53.8% of the 5049 ADRs were recorded in females, and 50.4% were caused by monotherapy. Of the ADRs, 10.6% (537/5049) were severe adverse reactions (SARs), including 34 serious adverse effects (SAEs). The SAR rates caused by one, two and three or more AEDs were 9.9, 10.0 and 19.6%, respectively  contains supplementary material, which is available to authorized users. Adverse drug reactions (ADRs) are a major public health concern . In receTo fill this gap, we conducted a clinical, active, open observational study to investigate the ADRs associated with six commonly used AEDs in epilepsy outpatients in southern China from 2003 to 2015.The Epilepsy Long-term Follow Up Registry Study (ELFURS) was a single-center, prospective and observational study of epilepsy outpatients in China. The ELFURS was conducted at the epilepsy center in the First Affiliated Hospital of Wenzhou Medical University (FAHWMU) in January 2003 . The stuIn this study, we reviewed the data of outpatients who only took one or more of six commonly used AEDs  and were registered in the WEFURD between 2003 and 2015. We evaluated the ADRs caused by single or combined use of the above six specific AEDs based on the WHO-UMC scale . ADRs thThe WHO-UMC criteria  were empThe 5049 ADRs were classified according to the SOCs in the WHOART . SeriousThe SARs were analyzed according to the suspected AEDs. In addition, the dosage/defined daily dose (DDD)  was usedp <  0.05 was considered significant. A Bonferroni correction was applied for the multiple comparisons, and the p-values were adjusted according to the number of tests (n = 3 for the number of AEDs received and n = 3 for the number of AEDs related to the ADR). A corrected p = 0.0167 (0.05/3) was considered significant for the number of AEDs received and the number of AEDs related to the ADR. All statistical analyses were conducted using IBM SPSS Statistics 21.0 software .Categorical/qualitative data are presented as numbers (percentages), and nonnormally distributed quantitative data are shown as the medians (ranges). The Pearson chi-square and Mann-Whitney U tests were used, as appropriate. A two-sided p <  0.001).A total of 3069 epilepsy outpatients (1807 epilepsy outpatients with 5049 eligible ADRs and 1262 epilepsy outpatients without ADRs) were included. The overall ADR rate was 58.88% (1807/3069). We compared 1807 epilepsy outpatients with ADRs and 1262 epilepsy outpatients without ADRs (Table An average of 2.79 ADRs (5049/1807) occurred per patient with an ADR. In total, 53.8% of the ADRs were recorded in females and 46.2% in males, while 50.4% were caused by a single AED (Table p <  0.001). The results showed that the rate of SARs caused by three or more AEDs was significantly higher than the rate of SARs caused by one or two AEDs.Of the 5049 ADRs, 537 were SARs. The general profiles of non-SARs and SARs in this study is displayed in Table Table n = 2, 2 drug hypersensitivity syndrome; DHS), LTG , OXC , TPM , and VPA . The other 17 SAEs were observed with combination therapy  stratified by drug is shown in Additional file ADRs contribute significantly to patient morbidity and mortality worldwide. Both safety and tolerability are determining factors in the selection of an appropriate AED . There aOur results showed that the overall ADR rate was 58.88%, which is slightly higher than the rates reported in other studies. In studies by Marson AG et al.  and Andrp = 0.006). Of the 18 SOC categories, most ADRs were categorized as psychiatric, neurological and gastrointestinal disorders, which were also the SOCs in which most ADRs to nervous system medications were found [n = 492, 30.13%); somnolence ; insomnia ; anorexia ; and irritability , which suggests that the psychiatric symptoms caused by AEDs deserve attention from clinicians.In this study, less than 2% of the ADRs occurred in children. This low percentage may have been due to selection bias, as 95% of the WEFURD patients were adults. This study showed that ADRs were more frequent in females, and some studies on AED safety have reported similar findings . Differere found , 20. As re found , 22, it re found . HoweverIn our study, 10.6% (537/5049) of the ADRs were SARs, and 0.67% (34/5049) were SAEs. These results were supported by the results of a cross-sectional study in Iran . Namazi,p <  0.001). Our study also showed that more SARs occurred with multidrug treatment. The SAR rates caused by one, two and three or more AEDs were 9.9, 10.0 and 19.6%, respectively (p <  0.001). This result suggests that clinicians should be alert for SARs, especially when three or more AEDs are used together. These findings were supported by those of other relevant studies. In the research by Horvath, L. et al. [Furthermore, we reported that the rate of SARs after a low treatment dose (dosage/DDD ≤ 0.50) was 14.5%  were assessed as conditional/unclassified, with more data needed. Active surveillance might provide another method of pharmacovigilance in China.Additional file 1:Table S1. Detailed information on 537 severe adverse reactions caused by drugs. Lists the severe adverse reactions to each drug, which may be found in the online version of this article. (DOCX 34 kb)


Neurosarcoidosis is a rare complication, and cranial neuropathy is the most frequent manifestation of this disease. However, few cohesive reports have discussed multiple cranial neuropathies in Japanese patients with sarcoidosis. The present report discusses the case of a patient with sarcoid uveitis and multiple neurological findings. We further review relevant literature regarding Japanese patients with multiple cranial nerve palsies published within the past 34 years (from January 1982 to December 2016).We report findings associated with the case of a 56-year-old Japanese woman with granulomatous pan-uveitis who was later diagnosed as having sarcoidosis by skin and transbronchial lung biopsies. She presented right-sided Bell’s palsy and was treated with orally administered prednisolone. However, while prednisolone was tapered, she developed facial (VII) and vagus (X) nerve palsies, followed by brain parenchyma lesions, which were not associated with any additional neurological symptoms. Furthermore, she exhibited increased intraocular pressure in her right eye, and she underwent trabeculectomy. Our review of the literature revealed that 64 Japanese patients with sarcoidosis experienced multiple cranial nerve palsies between 1982 and 2016. The most commonly affected cranial nerves were the facial (VII) (73.4%) and glossopharyngeal/vagus (IX/X) nerves (48.4%). Palsies of two distinct cranial nerves were found in 40.6% of the patients, followed by palsies of three (23.4%) and four (18.8%) nerves. Almost all patients (98.3%) received systemic steroid therapy, and total or partial remission was achieved in almost all patients (96.5%).According to the literature, patients with multiple cranial nerve palsies associated with sarcoidosis respond well to orally administered steroid therapy. However, our findings suggest that careful follow-up is necessary for patients with neurosarcoidosis due to potential aggravation of neuropathy. Occurring in approximately 3–15% of patients with sarcoidosis, neurosarcoidosis is a rare complication in which compression of neural tissue by granulomas or damage caused by infiltration/ischemia results in neurological symptoms –5, and mA 56-year-old Japanese woman presented with right-sided Bell’s palsy due to dysfunction of the facial nerve (VII) in October 2013. She had been diagnosed as having rheumatoid arthritis in August 2013 and treated with methotrexate orally. She had no relevant medical family history. Methotrexate was discontinued when she suffered from Bell’s palsy. Her condition improved following oral administration of 30 mg of prednisolone/day. Soon after prednisolone was discontinued in November 2013, methotrexate was resumed. In March 2014, she was admitted to a general hospital due to the appearance of bilateral floaters. Her best corrected visual acuity was 20/20 in her right eye and 20/25 in her left eye; an ophthalmological examination revealed bilateral granulomatous uveitis. Chest computed tomography (CT) revealed bilateral hilar lymphadenopathy and mediastinal lymph node swelling. Laboratory tests revealed elevated levels of serum angiotensin-converting enzyme (ACE) . She was histologically diagnosed as having sarcoidosis following skin and transbronchial lung biopsies. In February 2015, she developed dysphagia due to dysfunction of the vagus nerve (X), following which she was treated with 30 mg of prednisolone/day. Following an improvement in her symptoms, prednisolone dosage was tapered to 4 mg/day by August 2015. However, she experienced recurrence of nerve VII palsy in November 2015, despite continued treatment with 4 mg of prednisolone/day. In December 2015, magnetic resonance imaging (MRI) revealed brain parenchyma lesions  using the terms “multiple cranial neuropathies” and “neurosarcoidosis.” The most commonly affected cranial nerve was nerve VII , followed by nerves IX/X  ; three nerves, 23.4% (N = 15); four nerves, 18.8% (N = 12) (Table We reviewed 64 case reports regarding multiple cranial nerve palsies associated with sarcoidosis written in the Japanese language between January 1982 and December 2016. We searched et al. [Our patient developed palsies of cranial nerves VII and X, which are the most commonly affected nerves associated with sarcoidosis in Japan. Gascón-Bayarri et al.  reportedet al. . Howeveret al. , 4.Brain parenchyma lesions, which present as headaches and seizures, represent the second-most common manifestation of neurosarcoidosis , 4. In oet al. [Previous studies have reported that prednisolone is effective in treating neurosarcoidosis , 3, 6. Oet al.  discusseet al. . Recent et al. . Howeveret al.  and are In summary, we presented a patient with sarcoidosis with uveitis and multiple neurological findings, such as multiple cranial nerve palsies and asymptomatic brain parenchyma lesions. In the literature, it is commonly reported that patients with multiple cranial nerve palsies associated with sarcoidosis respond well to orally administered steroid therapy. However, careful follow-up is necessary for patients with neurosarcoidosis due to the potential for relapse and aggravation of neuropathy.