The ASDEU autism prevalence study in northern Spain.

The prevalence of autism spectrum disorders (ASD) was studied in children in the County of Gipuzkoa (Basque Country, Spain) as part of the European Union's Autism Spectrum Disorder in Europe project (ASDEU- https://asdeu.eu ). To identify cases in a total community sample of 7- to 9-year-old pupils (N = 14,734), a multistage approach was adopted: in the first stage, a teacher nomination (TN) form was completed by school teachers; and in the second stage, all families with a child nominated by their teachers were invited to complete the Social Communication Questionnaire (SCQ). A total of 108 (59%) schools participated fully, yielding a final sample of 9177 of 14.734 (61.9%) pupils. A total of 212 (2.3%) children were nominated via the TN form, and of these, 105 (49.5%) returned the completed SCQ. Twenty-five (23.8%) cases with SCQ scores ≥ 15 were invited to undergo a free clinical assessment, and 10 (40%) new cases of ASD were identified. The prevalence estimate included the 55 cases already being supported by the Gipuzkoa's only ASD association, the Gipuzkoa Autism Society (Asociación Guipuzcoana de Autismo/GAUTENA)), as well as the 10 new subjects identified by the ASDEU field diagnostic process. A sensitivity analysis was performed to estimate new potential ASD cases among the non-participant schools, leading to a final figure of 87 cases of ASD in this age-bracket at the date of the study. This global probabilistic estimate, including non-participating schools, would thus provide a population prevalence of 0.59% (95% CI 0.48-0.73), a result lower than those reported by some other studies. Attrition rates in cross-sectional studies are challenging and support the need for developing longitudinal ASD incidence surveillance study areas (ASD observatories).

Usefulness of matrix-assisted laser desorption ionization/time of flight mass spectrometry for the identification of Streptococcus mutans.

This study evaluated the reliability of MALDI-TOF MS coupled with statistical tools for the identification of Streptococcus mutans in comparison with PCR-based techniques. Bacterial isolates were identified and serotyped by conventional PCR, using S. mutans species and serotype-specific primers. For bacterial identification, mass spectra data from S. mutans and other streptococci were compared with Biotyper V 3.1 database and the mass peak lists were examined by cluster and principal component (PCA) analysis. Identification of potential biomarkers was performed using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and BLAST tool of the NCBI database. PCR identified 100% of the isolates as S. mutans. S. mutans strains were typed as serotypes c (85.6%), e (8.6%), k (4.8%), and f (0.9%). Although only the 70% of the strains tested were identified at species level by the Biotyper database, PCA and cluster analysis of mass peaks allowed the identification of 100% S. mutans isolates and its differentiation from the other oral and non-oral streptococci. One mass peak at m/z value of 9572.73 was identified as species-specific biomarker for S. mutans. No biomarkers were identified for S. mutans serotypes. KEY POINTS: • MALDI-TOF MS coupled with statistical tools for the identification of S. mutans. • Detection of species identifying biomarkers by MALDI-TOF MS. • PCR identification and serotyping of S. mutans from saliva samples.

High-throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I-based real-time PCR combined with melting curve analysis.

This work describes a primer pair and a high-throughput SYBR Green I-based real-time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 100 amplicon copies per assay (equivalent to 2 × 10-11  ng/µl, Cq value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non-lethal and lethal fish samples with detection levels of 0.17 × 100 gene copies in tissues artificially infected and 0.02 × 100 in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.

Phenotypic and Molecular Characterization of Lacinutrix venerupis Isolated from Atlantic Horse Mackerel Trachurus trachurus.

The aim of the present study was to characterize two gram-negative bacterial strains that were isolated from diseased Atlantic Horse Mackerel Trachurus trachurus in 2017. Based on the results obtained from the biochemical and chemotaxonomic characterization, the isolates were identified as Lacinutrix spp. The highest similarity of the 16S rRNA gene sequences was obtained with the strain L. venerupis CECT 8573T (99.1%), while other species showed similarities of 98% (L. jangbogonensis) and 97% (L. algicola and L. mariniflava). Molecular characterization by repetitive element (REP)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as proteomic characterization by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), demonstrated heterogeneity between the strains from the Atlantic Horse Mackerel and the type strain, CECT 8573T . The virulence of one of the isolates for Turbot Scophthalmus maximus, European Sea Bass Dicentrarchus labrax, Senegalese Sole Solea senegalensis, and Rainbow Trout Oncorhynchus mykiss was assessed under experimental conditions. No mortalities were recorded after intraperitoneal injections with high doses of bacteria (1 × 109  CFU/mL). Thus, further studies are necessary to elucidate the impact of this bacterial species as a fish pathogen.

Identification and typing of fish pathogenic species of the genus Tenacibaculum.

Tenacibaculosis is a major bacterial disease that causes severe fish outbreaks and losses and limits the culture of a variety of commercially valuable anadromous and marine fish species in Europe, America, Asia and Oceania. Fish affected by tenacibaculosis have external lesions and necrosis that affect different areas of the body surface, reducing their commercial value. Several species of Tenacibaculum have been identified as the causal agent of tenacibaculosis in fish, including Tenacibaculum maritimum, Tenacibaculum soleae, Tenacibaculum discolor, Tenacibaculum gallaicum, Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" (quotations marks denote species that have not been validly published). Diagnosis of tenacibaculosis is usually based on culture-dependent detection and identification techniques which are time-consuming and do not allow to differentiate closely related species. The development of reliable techniques for studying the relationships between members of the genus Tenacibaculum and for distinguishing fish-pathogenic species of Tenacibaculum genus is, therefore, a key step in understanding the diversity and incidence of tenacibaculosis in global aquaculture, designing effective prevention strategies and early implementation of infection control measures. In this review, recent advances in molecular, serological, proteomic and chemotaxonomic techniques developed for the identification and differentiation of Tenacibaculum species, as well as for the analysis of their genetic and epidemiological relationships are discussed. Key features of current diagnostic methods likely to facilitate control and prevention of tenacibaculosis and to avoid the spread of its aetiological agents are also outlined.

Comparison of serological and molecular typing methods for epidemiological investigation of Tenacibaculum species pathogenic for fish.

In the present study, the potential of serological methods, the repetitive extragenic palindromic PCR (REP-PCR) and the enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) for the typing of the species Tenacibaculum maritimum, Tenacibaculum soleae and Tenacibaculum discolor was evaluated. Moreover, molecular and proteomic techniques were used to assess variability among strains belonging to different serotypes, as well as isolated from different host species and geographical areas. Slide agglutination and dot-blot assays demonstrated the lack of immunological relationships among Tenacibaculum species analyzed. The serotype O1 was predominant within T. maritimum isolates regardless of the fish species or geographical area. Two serotypes were distinguished within T. soleae isolates and at least one within T. discolor strains. Species- and strain-specific profiles were obtained from the analysis of T. maritimum, T. soleae and T. discolor by REP-PCR and ERIC-PCR, demonstrating their potential as diagnostic tools. The genotyping analysis using both techniques showed genetic variability among the strains of each fish pathogenic Tenacibaculum species analysed. However, Tenacibaculum strains isolated from different host species or geographical areas or belonging to different serotypes produced REP and ERIC profiles with high similarity. Analysis by MALDI-TOF-MS of the T. maritimum strains could not detect any serotype-identifying biomarkers. Serotype-specific mass peaks were found for the serotypes O1 and O2 of T. soleae and for the serotype O1 of T. discolor. However, no relationships between the proteomic profiles and the source of isolation of the strains were obtained for any of the Tenacibaculum species analysed in this study.

MALDI-TOF mass spectrometry for rapid differentiation of Tenacibaculum species pathogenic for fish.

Tenacibaculosis is a fish disease that limits the culture of a variety of marine fish species of commercial value in the world. The genus Tenacibaculum includes several species, and their discrimination is of clinical interest in order to improve the management of an outbreak of the disease. In this study, a novel proteomic approach based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was evaluated for the identification and differentiation of Tenacibaculum species. The peak mass lists derived from MALDI-TOF-MS analysis were examined for the detection of potential biomarkers, similarity and cluster analysis and principal component analysis (PCA). Culture media used for bacterial growth did not affect the mass fingerprints. Eight genus-specific peaks were found in all the Tenacibaculum species analysed. Moreover, at least one species-specific peak was found in the species Tenacibaculum maritimum, Tenacibaculum soleae, Tenacibaculum dicentrarchi, Tenacibaculum litoreum and Tenacibaculum ovolyticum. These peaks could serve as biomarkers for the rapid identification of these bacterial fish pathogens. The cluster and PCA clearly separated the species T. maritimum, T. soleae, T. dicentrarchi and T. ovolyticum in different clusters. However, species of Tenacibaculum discolor and Tenacibaculum gallaicum were difficult to distinguish based on their protein fingerprints. To our knowledge, this is the first study that deals with the characterization and determination of biomarkers of Tenacibaculum species by MALDI-TOF mass spectrometry. This approach proved to be an effective and reliable technique for the discrimination of the Tenacibaculum species; therefore, it could be integrated as a routine diagnostic tool in microbiological laboratories.

Development of a SYBR green I real-time PCR assay for specific identification of the fish pathogen Aeromonas salmonicida subspecies salmonicida.

A SYBR Green I real-time polymerase chain reaction protocol for specific detection of the fish pathogen Aeromonas salmonicida subsp. salmonicida was developed and validated for rapid diagnosis of typical furunculosis. The sequence of the aopO gene of A. salmonicida subsp. salmonicida, which encodes for a serine/threonine protein kinase linked to virulence, was chosen for primer design. The selected primers amplified a 119-bp internal fragment of the aopO gene. The specificity test proved that 100 % (40/40) of the A. salmonicida subsp. salmonicida strains tested showed a positive amplification with subspecies-specific melting temperatures (Tm) of 80.75 ± 0.35 °C. Atypical A. salmonicida subspecies and other non-related bacterial fish pathogens did not amplify or showed unspecific melting profiles, except for one strain of A. salmonicida subsp. achromogenes and one strain of A. salmonicida subsp. smithia. The detection sensitivity was 21 fg of purified bacterial DNA per reaction, corresponding to 1-2 bacterial cells and 6-60 bacteria per reaction for seeded kidney and blood. The assay was highly reproducible with low variation coefficient values for intra-run and inter-run assays. The assay also allowed the specific detection of A. salmonicida subsp. salmonicida in tissues of fish naturally and experimentally infected. No amplification was detected when tissues from healthy fish or fish affected by other diseases were tested. The SYBR Green real-time PCR and melt curve analysis developed in this study is a rapid and accurate method for the specific identification of A. salmonicida subsp. salmonicida isolates and its detection on tissues of fish affected by furunculosis.

Executive functions in daily living skills: A study in adults with autism spectrum disorder.

Introduction: Adults with autism and adults with schizophrenia show difficulties in adaptive skills, especially those related to daily functioning. Some studies suggest that adaptive skills are associated with deficits in executive functions (EF), while others indicate that intelligence quotient (IQ) might also play a role. Literature suggests that autistic symptoms further affect adaptive skills. The interest of the current study, therefore, was to explore to what extent IQ, EFs as well as core autistic symptoms predict adaptive skills. Methods: To do this, 25 controls, 24 adults with autism, and 12 with schizophrenia were assessed on IQ (Wechsler Adult Intelligence Scale), and executive functioning. The EF was measured with neuropsychological tasks (inhibition, updating, and task switching) and with the Dysexecutive-Spanish Questionnaire (DEX-Sp) which assessed everyday life EF problems. Core ASD symptoms were measured using the Autism Diagnostic Observation Schedule, the Autism Spectrum Quotient-Short version (AQ-S), and the Repetitive Behavior Questionnaire - 3 (RBQ-3). Results: The results indicated EF difficulties in both, autism and schizophrenia. The IQ explained a high percentage of the variance found in adaptive skills, but only in the autism group. We can conclude, therefore, that high IQ is associated with low adaptive skills levels and EFs affect adaptive functioning in people with autism; however, this does not explain the difficulties in adaptive functioning in the schizophrenia group. Core features of autism assessed with self-report questionnaires (but not the ADOS-2) predicted low scores on the adaptive skills, only in the autism group. Discussion: Both EF measures predicted adaptive skills scores in autism, but not in schizophrenia. Our results suggest that different factors affect the adaptive functioning in each disorder. For instance, the EFs should be a central focus for improvement, especially for individuals with autism.

Determinants of satisfaction with the detection process of autism in Europe: Results from the ASDEU study.

LAY ABSTRACT: Professional guidance and support in response to first concerns appears to be an important predictor of the level of satisfaction with the detection process of autism in young children. In this study, we analyzed the views of 1342 family members, including 1278 parents, who completed an online survey form collecting information about their experience and satisfaction with the early detection of autism in their child. Specifically, we were interested in how specific experiences with the detection process relate to the satisfaction with it and whether we could identify important predictors of satisfaction. The detection process is an emotionally charged period for parents, often described as painful, chaotic, and lengthy. A better understanding of their experiences is important to take appropriate action to improve the detection process. In our sample, the level of satisfaction with the detection process varied greatly from one respondent to another. Among the different experiences we considered, whether or not respondents received professional guidance and support in response to first concerns explained most of this variation. We also found that difficulty finding information about detection services, lack of professional guidance and support in response to first concerns, having to find a diagnostic service on one's own, and longer delays between confirmation of concerns and first appointment with a specialist were experiences associated with a greater likelihood of being unsatisfied. The findings of this study highlight the importance of the parent-professional relationship in the detection process and have important practical implications for health administrations to improve the detection process.

Spanish Cultural Validation of the Modified Checklist for Autism in Toddlers, Revised.

The Modified Checklist for Autism in Toddlers-revised/follow-up (M-CHAT-R/F) was developed to reduce the number of cases requiring telephone verification. The aim of this study was to validate a Spanish version of the M-CHAT-R/F in the Spanish public health system. The M-CHAT-R/F was translated, culturally adapted, and then administered to 6625 children. Of the 39 positive screening cases, 15 children were diagnosed with autism spectrum disorder (ASD) and 24 with non-ASD disorders or delays. The sensitivity was 0.79 and specificity of 0.99. Positive and negative predictive values were 0.39 and 0.99, respectively. These results are similar to the English equivalent, though observed prevalence was lower. This study supports Spanish National Health System policy makers to consider a universal ASD screening program.

Early Detection, Diagnosis and Intervention Services for Young Children with Autism Spectrum Disorder in the European Union (ASDEU): Family and Professional Perspectives.

Early services for ASD need to canvas the opinions of both parents and professionals. These opinions are seldom compared in the same research study. This study aims to ascertain the views of families and professionals on early detection, diagnosis and intervention services for young children with ASD. An online survey compiled and analysed data from 2032 respondents across 14 European countries (60.9% were parents; 39.1% professionals). Using an ordinal scale from 1 to 7, parents' opinions were more negative (mean = 4.6; SD 2.2) compared to those of professionals (mean = 4.9; SD 1.5) when reporting satisfaction with services. The results suggest services should take into account child's age, delays in accessing services, and active stakeholders' participation when looking to improve services.

Correction to: Early Detection, Diagnosis and Intervention Services for Young Children with Autism Spectrum Disorder in the European Union (ASDEU): Family and Professional Perspectives.

The original version of this article unfortunately contained a mistake in one of the co-author's family name. The correct name should be María Victoria Martín-Cilleros instead of María Victoria Cilleros-Martín.

Use of ribosomal proteins as biomarkers for identification of Flavobacterium psychrophilum by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) is a rapid methodology for identification of bacteria that is increasingly used in diagnostic laboratories. This work aimed at evaluating the potential of MALDI-TOF-MS for identification of the main serotypes of Flavobacterium psychrophilum isolated from salmonids, and its discrimination from closely related Flavobacterium spp. A mass spectra library was constructed by analysing 70 F. psychrophilum strains representing the serotypes O1, O2a, O2b and O3, including reference and clinical isolates. Peak mass lists were examined using the Mass-Up software for the detection of potential biomarkers, similarity and cluster analysis. Fourteen species-identifying biomarkers were detected in all the F. psychrophilum isolates tested, moreover, sets of serotype-identifying biomarkers ions were selected. F. psychrophilum-specific biomarkers were identified as ribosomal proteins by matching with protein databases. Furthermore, sequence variation corresponding to amino acid exchanges in several biomarker proteins were tentatively assigned. Closely related Flavobacterium species (F. flevense, F. succinicans, F. columnare, F. branchiophilum and F. johnsoniae) could be differentiated from F. psychrophilum by defining species identifying biomarkers and hierarchical cluster analysis. These results demonstrated that MALDI-TOF spectrometry represents a powerful tool for an accurate identification of the fish pathogen F. psychrophilum as well as for epidemiological studies. BIOLOGICAL SIGNIFICANCE: The results obtained in this study demonstrated that MALDI-TOF mass spectrometry represents a powerful tool that can be used by diagnostic laboratories for rapid identification of the fish pathogen Flavobacterium psychrophilum and its differentiation from other Flavobacterium-related species. Analysis of mass peak lists revealed the potential of the MALDI-TOF technique to identify epidemiologically important serotypes affecting salmonid fish. Furthermore, this study revealed the importance of the technique in the early and fast detection of bacterial pathogens, with the subsequent improvement in the health status and animal welfare and food safety in farmed fish.

Immune-modulation of two BATF3 paralogues in rainbow trout Oncorhynchus mykiss.

Basic leucine zipper transcription factor ATF-like (BATF) -3 is a member of the activator protein 1 (AP­1) family of transcription factors and is known to play a vital role in regulating differentiation of antigen-presenting cells in mammals. In this study, two BATF3 homologues (termed BATF3a and BATF3b) have been identified in rainbow trout (Oncorhynchus mykiss). Both genes were constitutively expressed in tissues, with particularly high levels of BATF3a in spleen, liver, pyloric caecae and head kidney. BATF3a was also more highly induced by PAMPs and cytokines in cultured cells, with type II IFN a particularly potent inducer. In rIL-4/13 pre-stimulated cells, the viral PAMPS polyI:C and R848 had the most pronounced effect on BATF3 expression. BATF3 expression could also be modulated in vivo, following infection with Yersinia ruckeri, a bacterial pathogen causing redmouth disease in salmonids, or with the rhabdovirus IHNV. The results suggest that BATF3 may be functionally conserved in regulating the differentiation and activation of immune cells in lower vertebrates and could be explored as a potential marker for comparative investigation of leucocyte lineage commitment across the vertebrate phyla.

Early Detection and Intervention of ASD: A European Overview.

Over the last several years there has been an increasing focus on early detection of Autism Spectrum Disorder (ASD), not only from the scientific field but also from professional associations and public health systems all across Europe. Not surprisingly, in order to offer better services and quality of life for both children with ASD and their families, different screening procedures and tools have been developed for early assessment and intervention. However, current evidence is needed for healthcare providers and policy makers to be able to implement specific measures and increase autism awareness in European communities. The general aim of this review is to address the latest and most relevant issues related to early detection and treatments. The specific objectives are (1) analyse the impact, describing advantages and drawbacks, of screening procedures based on standardized tests, surveillance programmes, or other observational measures; and (2) provide a European framework of early intervention programmes and practices and what has been learnt from implementing them in public or private settings. This analysis is then discussed and best practices are suggested to help professionals, health systems and policy makers to improve their local procedures or to develop new proposals for early detection and intervention programmes.