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A new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently emerged to cause a human pandemic. Although molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. We developed serologic assays for detection of SARS-CoV-2 neutralizing, spike protein-specific, and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed SARS-CoV-2 infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrated that most PCR-confirmed SARS-CoV-2-infected persons seroconverted by 2 weeks after disease onset. We found that commercial S1 IgG or IgA ELISAs were of lower specificity, and sensitivity varied between the 2 assays; the IgA ELISA showed higher sensitivity. Overall, the validated assays described can be instrumental for detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies.
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Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , COVID-19 , Teste para COVID-19 , Ensaio de Imunoadsorção Enzimática , Humanos , Testes de Neutralização , Pandemias , SARS-CoV-2 , Sensibilidade e Especificidade , Testes SorológicosRESUMO
Middle East respiratory syndrome coronavirus (MERS-CoV) infections in humans can cause asymptomatic to fatal lower respiratory lung disease. Despite posing a probable risk for virus transmission, asymptomatic to mild infections can go unnoticed; a lack of seroconversion among some PCR-confirmed cases has been reported. We found that a MERS-CoV spike S1 protein-based ELISA, routinely used in surveillance studies, showed low sensitivity in detecting infections among PCR-confirmed patients with mild clinical symptoms and cross-reactivity of human coronavirus OC43-positive serum samples. Using in-house S1 ELISA and protein microarray, we demonstrate that most PCR-confirmed MERS-CoV case-patients with mild infections seroconverted; nonetheless, some of these samples did not have detectable levels of virus-neutralizing antibodies. The use of a sensitive and specific serologic S1-based assay can be instrumental in the accurate estimation of MERS-CoV prevalence.
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Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Imunidade Humoral/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Infecções por Coronavirus/virologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Fatores de TempoRESUMO
We tested samples collected from camels, camel workers, and other animals in Sudan and Qatar in 2015 and 2017 for evidence of Middle East respiratory syndrome coronavirus (MERS-CoV) infection. MERS-CoV antibodies were abundant in Sudan camels, but we found no evidence of MERS-CoV infection in camel workers, other livestock, or bats.
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Camelus/virologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio , Zoonoses/epidemiologia , Zoonoses/virologia , Animais , Animais Domésticos/virologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/história , Infecções por Coronavirus/imunologia , História do Século XXI , Humanos , Testes de Neutralização , Estudos Soroepidemiológicos , Sudão/epidemiologia , Zoonoses/históriaRESUMO
Arboviruses such as West Nile Virus (WNV) and Usutu Virus (USUV) are emerging pathogens that circulate between mosquitoes and birds, occasionally spilling over into humans and horses. Current serological screening methods require access to a well-equipped laboratory and are not currently available for on-site analysis. As a proof of concept, we propose here a species-independent lateral flow microarray immunoassay (LMIA) able to quickly detect and distinguish between WNV Non-Structural 1 (NS1) and USUV NS1-specific antibodies. A double antigen approach was used to test sera collected from humans, horses, European jackdaws (Corvus monedula), and common blackbirds (Turdus merula). Optimization of the concentration of capture antigen spotted on the LMIA membrane and the amount of detection antigen conjugated to detector particles indicated that maximizing both parameters increased assay sensitivity. Upon screening of a larger serum panel, the optimized LMIA showed significantly higher spot intensity for a homologous binding event. Using a Receiver Operating Characteristics (ROC) curve, WNV NS1 LMIA results in humans, horses, and C. monedula showed good correlation when compared to "gold standard" WNV FRNT90. The most optimal derived sensitivity and specificity of the WNV NS1 LMIA relative to corresponding WNV FRNT90-confirmed sera were determined to be 96% and 86%, respectively. While further optimization is required, this study demonstrates the feasibility of developing a species-independent LMIA for on-site analysis of WNV, USUV, and other arboviruses. Such a tool would be useful for the on-site screening and monitoring of relevant species in more remote or low-income regions.
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Chronic B-cell receptor signals incited by cognate antigens are believed to play a crucial role in the pathogenesis of mucosa-associated lymphoid tissue lymphomas. We have explored the immunoglobulin variable regions (IGHV) expressed by 124 ocular adnexal MALT lymphomas (OAML) and tested the in vitro reactivity of recombinant IgM derived from 23 OAMLs. Six of 124 OAMLs (5%) were found to express a high-affinity stereotyped rheumatoid factor. OAMLs have a biased IGHV4-34 usage, which confers intrinsic super auto-antigen reactivity with poly-N-acetyllactosamine (NAL) epitopes, present on cell surface glycoproteins of erythrocytes and B cells. Twenty-one OAMLs (17%) expressed IGHV4-34-encoded B-cell receptors. Five of the 23 recombinant OAML IgMs expressed IGHV4-34, four of which bound to the linear NAL i epitope expressed on B cells but not to the branched NAL I epitope on erythrocytes. One non-IGHV4-34-encoded OAML IgM was also reactive with B cells. Interestingly, three of the 23 OAML IgMs (13%) specifically reacted with proteins of U1-/U-snRNP complexes, which have been implicated as cognate-antigens in various autoimmune diseases such as systemic lupus erythematosus and mixed connective tissue disease. The findings indicate that local autoimmune reactions are instrumental in the pathogenesis of a substantial fraction of OAMLs.
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Autoantígenos , Neoplasias Oculares , Imunoglobulina M , Linfoma de Zona Marginal Tipo Células B , Humanos , Linfoma de Zona Marginal Tipo Células B/imunologia , Linfoma de Zona Marginal Tipo Células B/genética , Autoantígenos/imunologia , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Neoplasias Oculares/imunologia , Neoplasias Oculares/genética , Feminino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Masculino , Idoso , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Idoso de 80 Anos ou mais , Epitopos/imunologia , Adulto , Fator Reumatoide/imunologiaRESUMO
Introduction: In 2020, the first Dutch West Nile virus (WNV) infected birds were detected through risk-targeted surveillance of songbirds. Retrospective testing of patients with unexplained neurological disease revealed human WNV infections in July and August 2020. Bird ringers are highly exposed to mosquito bites and possibly avian excrements during ringing activities. This study therefore investigates whether bird ringers are at higher risk of exposure to WNV and Usutu virus (USUV). Methods: Dutch bird ringers were asked to provide a single serum sample (May - September 2021) and to fill out a survey. Sera were screened by protein microarray for presence of specific IgG against WNV and USUV non-structural protein 1 (NS1), followed by focus reduction virus neutralization tests (FRNT). Healthcare workers (2009-2010), the national immunity cohort (2016-2017) and blood donors (2021) were used as control groups without this occupational exposure. Results: The majority of the 157 participating bird ringers was male (132/157, 84%) and the median age was 62 years. Thirty-seven participants (37/157, 23.6%) showed WNV and USUV IgG microarray signals above background, compared to 6.4% (6/94) in the community cohort and 2.1% (2/96) in blood donors (p < 0.01). Two seroreactive bird ringers were confirmed WNV or USUV positive by FRNT. The majority of seroreactive bird ringers travelled to EU countries with reported WNV human cases (30/37, 81%) (p = 0.07). No difference was observed between bird ringers with and without previous yellow fever vaccination. Discussion: The higher frequency of WNV and/or USUV IgG reactive bird ringers indicates increased flavivirus exposure compared to the general population, suggesting that individuals with high-exposure professions may be considered to complement existing surveillance systems. However, the complexity of serological interpretation in relation to location-specific exposure (including travel), and antibody cross-reactivity, remain a challenge when performing surveillance of emerging flaviviruses in low-prevalence settings.
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In order to assess the risk of SARS-CoV-2 infection, transmission and reservoir development in swine, we combined results of an experimental and two observational studies. First, intranasal and intratracheal challenge of eight pigs did not result in infection, based on clinical signs and PCR on swab and lung tissue samples. Two serum samples returned a low positive result in virus neutralization, in line with findings in other infection experiments in pigs. Next, a retrospective observational study was performed in the Netherlands in the spring of 2020. Serum samples (N =417) obtained at slaughter from 17 farms located in a region with a high human case incidence in the first wave of the pandemic. Samples were tested with protein micro array, plaque reduction neutralization test and receptor-binding-domain ELISA. None of the serum samples was positive in all three assays, although six samples from one farm returned a low positive result in PRNT (titers 40-80). Therefore we conclude that serological evidence for large scale transmission was not observed. Finally, an outbreak of respiratory disease in pigs on one farm, coinciding with recent exposure to SARS-CoV-2 infected animal caretakers, was investigated. Tonsil swabs and paired serum samples were tested. No evidence for infection with SARS-CoV-2 was found. In conclusion, Although in both the experimental and the observational study few samples returned low antibody titer results in PRNT infection with SARS-CoV-2 was not confirmed. It was concluded that sporadic infections in the field cannot be excluded, but large-scale SARS-CoV-2 transmission among pigs is unlikely.
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COVID-19/veterinária , SARS-CoV-2/fisiologia , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/transmissão , Doenças dos Suínos/virologia , Animais , Exposição Ambiental , Países Baixos/epidemiologia , Vigilância em Saúde Pública , Estudos Retrospectivos , SuínosRESUMO
Norovirus is a leading cause of epidemic acute gastroenteritis. More than 30 genotypes circulate in humans, some are common, and others are only sporadically detected. Here, we investigated whether serology can be used to determine which genotypes infect children. We established a multiplex protein microarray with structural and non-structural norovirus antigens that allowed simultaneous antibody testing against 30 human GI and GII genotypes. Antibody responses of sera obtained from 287 children aged < 1 month to 5.5 years were profiled. Most specific IgG and IgA responses were directed against the GII.2, GII.3, GII.4, and GII.6 capsid genotypes. While we detected antibody responses against rare genotypes, we found no evidence for wide circulation. We also detected genotype-specific antibodies against the non-structural proteins p48 and p22 in sera of older children. In this study, we show the age-dependent antibody responses to a broad range of norovirus capsid and polymerase genotypes, which will aid in the development of vaccines.
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Infecções por Caliciviridae , Gastroenterite , Imunidade Humoral , Norovirus , Infecções por Caliciviridae/imunologia , Proteínas do Capsídeo/genética , Pré-Escolar , Europa (Continente) , Gastroenterite/imunologia , Gastroenterite/virologia , Genótipo , Humanos , Lactente , Norovirus/genética , FilogeniaRESUMO
The emergence and rapid spread of SARS-CoV-2 variants may affect vaccine efficacy substantially. The Omicron variant termed BA.2, which differs substantially from BA.1 based on genetic sequence, is currently replacing BA.1 in several countries, but its antigenic characteristics have not yet been assessed. Here, we used antigenic cartography to quantify and visualize antigenic differences between early SARS-CoV-2 variants (614G, Alpha, Beta, Gamma, Zeta, Delta, and Mu) using hamster antisera obtained after primary infection. We first verified that the choice of the cell line for the neutralization assay did not affect the topology of the map substantially. Antigenic maps generated using pseudo-typed SARS-CoV-2 on the widely used VeroE6 cell line and the human airway cell line Calu-3 generated similar maps. Maps made using authentic SARS-CoV-2 on Calu-3 cells also closely resembled those generated with pseudo-typed viruses. The antigenic maps revealed a central cluster of SARS-CoV-2 variants, which grouped on the basis of mutual spike mutations. Whereas these early variants are antigenically similar, clustering relatively close to each other in antigenic space, Omicron BA.1 and BA.2 have evolved as two distinct antigenic outliers. Our data show that BA.1 and BA.2 both escape vaccine-induced antibody responses as a result of different antigenic characteristics. Thus, antigenic cartography could be used to assess antigenic properties of future SARS-CoV-2 variants of concern that emerge and to decide on the composition of novel spike-based (booster) vaccines.
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COVID-19 , SARS-CoV-2 , Animais , Linhagem Celular , Cricetinae , Humanos , Soros Imunes , SARS-CoV-2/genéticaRESUMO
BACKGROUND: In 2015-2016, a large Zika virus (ZIKV) outbreak occurred in the Americas. Although the exact ZIKV antibody kinetics after infection are unknown, recent evidence indicates the rapid waning of ZIKV antibodies in humans. Therefore, we aimed to determine the levels of ZIKV antibodies more than three years after a ZIKV infection. METHODS: We performed ZIKV virus neutralization tests (VNT) and a commercial ZIKV non-structural protein 1 (NS1) IgG ELISA in a cohort of 49 participants from Suriname who had a polymerase-chain-reaction-confirmed ZIKV infection more than three years ago. Furthermore, we determined the presence of antibodies against multiple dengue virus (DENV) antigens. RESULTS: The ZIKV seroprevalence in this cohort, assessed with ZIKV VNT and ZIKV NS1 IgG ELISA, was 59.2% and 63.3%, respectively. There was, however, no correlation between these two tests. Furthermore, we did not find evidence of a potential negative influence of DENV immunity on ZIKV antibody titers. CONCLUSIONS: ZIKV seroprevalence, assessed with two commonly used serological tests, was lower than expected in this cohort of participants who had a confirmed previous ZIKV infection. This can have implications for future ZIKV seroprevalence studies and possibly for the duration of immunological protection after a ZIKV infection.
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Anticorpos Neutralizantes/análise , Infecção por Zika virus/imunologia , Zika virus/imunologia , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Estudos de Coortes , Reações Cruzadas/imunologia , Dengue/virologia , Vírus da Dengue/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Testes de Neutralização/métodos , Estudos Soroepidemiológicos , Testes Sorológicos/métodos , Suriname , Zika virus/patogenicidade , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologiaRESUMO
Guillain-Barré syndrome (GBS) is associated with various types of preceding infections including Campylobacter jejuni and cytomegalovirus, but there is also an association with arthropod borne viruses (arboviruses), such as Zika virus, that are endemic in tropical regions. Here we present the clinical characteristics of 12 GBS patients from Suriname that were hospitalized between the beginning of 2016 and half 2018. Extensive diagnostic testing was performed for pathogens that are commonly associated with GBS, but also for arboviruses, in order to identify the preceding infection that might have led to GBS. With this extensive testing algorithm, we could identify a recent infection in six patients of which four of them had evidence of a recent Zika virus or dengue virus infection. These results suggest that arboviruses, specifically Zika virus but possibly also dengue virus, might be important causative agents of GBS in Suriname. Furthermore, we found that more accessibility of intravenous immunoglobulins or plasma exchange could improve the treatment of GBS in Suriname.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19). Little is known about the interplay between preexisting immunity to endemic seasonal coronaviruses and the development of a SARS-CoV-2-specific IgG response. We investigated the kinetics, breadth, magnitude, and level of cross-reactivity of IgG antibodies against SARS-CoV-2 and heterologous seasonal and epidemic coronaviruses at the clonal level in patients with mild or severe COVID-19 as well as in disease control patients. We assessed antibody reactivity to nucleocapsid and spike antigens and correlated this IgG response to SARS-CoV-2 neutralization. Patients with COVID-19 mounted a mostly type-specific SARS-CoV-2 response. Additionally, IgG clones directed against a seasonal coronavirus were boosted in patients with severe COVID-19. These boosted clones showed limited cross-reactivity and did not neutralize SARS-CoV-2. These findings indicate a boost of poorly protective CoV-specific antibodies in patients with COVID-19 that correlated with disease severity, revealing "original antigenic sin."
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Linfócitos B/imunologia , Linfócitos B/virologia , COVID-19/imunologia , COVID-19/virologia , Coronavirus/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Estudos de Casos e Controles , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Reações Cruzadas , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunoglobulina G/sangue , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Pandemias , Fosfoproteínas/imunologia , Estações do Ano , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
BACKGROUND: Tick-borne encephalitis (TBE) is an infectious disease endemic to large parts of Europe and Asia. Diagnosing TBE often relies on the detection of TBEV-specific antibodies in serum and cerebrospinal fluid (CSF) as viral genome is mostly not detectable once neurological symptoms occur. OBJECTIVES: We evaluated the performance of TBEV IgM and IgG ELISAs in both serum and CSF of confirmed TBEV patients and discuss the role of (CSF) serology in TBEV diagnostics. STUDY DESIGN: For the assay evaluation we collected specimen from confirmed TBEV patients. Assay specificity was assessed using sera from patients with a related flavivirus infection or other acute infection. A selected ELISA assay was used to analyze TBEV-specific antibodies in CSF and to evaluate the use in confirming TBE diagnosis. RESULTS: In this study the overall sensitivity of the IgM TBEV ELISAs was acceptable (94 -100 %). Four out of five IgM ELISA's demonstrated an excellent overall specificity from 94 -100% whereas a low overall specificity was observed for the IgG TBEV ELISAs (30-71%). Intrathecal antibody production against TBEV was demonstrated in a subset of TBE patients. CONCLUSIONS: In four out of five ELISAs, IgM testing in serum and CSF of TBE patients is specific and confirmative. The lack of IgG specificity in all ELISAs emphasizes the need of confirmatory testing by virus neutralisation, depending on the patient's background and the geographic location of exposure to TBEV. A CSF-serum IgG antibody index can support the diagnosis specifically in chronic disease or once IgM has disappeared.
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Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/diagnóstico , Imunoglobulina M/sangue , Imunoglobulina M/líquido cefalorraquidiano , Anticorpos Antivirais/sangue , Anticorpos Antivirais/líquido cefalorraquidiano , Encefalite Transmitida por Carrapatos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/líquido cefalorraquidiano , Masculino , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Diagnostic requests for both Zika virus (ZIKV) and dengue virus (DENV) infections in returning travelers have significantly increased during the recent ZIKV outbreak in the Americás. These flaviviruses have overlapping clinical syndromes and geographical distribution, but diagnostic differentiation is important because of different clinical consequences. As flaviviruses are known to have a short viremic period, diagnostics often rely on serological methods, which are challenging due to extensive cross-reactive antibodies. OBJECTIVE: To re-evaluate the performance of DENV serological assays in laboratory confirmed ZIKV-infected travelers. STUDY DESIGN: The extent of cross-reactivity of the DENV NS1 antigen, IgM and IgG ELISA was analyzed in 152 clinical blood samples collected from 69 qRT-PCR and 24 virus neutralization titer (VNT) confirmed ZIKV-infected travelers. RESULTS: The majority of travelers in the presented cohort returned to the Netherlands from Suriname and presented with symptoms of fever and rash. Twenty-three percent of the female travelers were pregnant. None of the 39 ZIKV RNA positive blood samples were cross-reactive in the DENV NS1 antigen ELISA. The rates of cross-reactivity of the DENV IgM and IgG ELISÁs were 31% and 54%, respectively, after excluding travelers with (potential) previous DENV exposure. CONCLUSIONS: Although the DENV NS1 antigen assay was highly specific in this cohort of laboratory confirmed ZIKV-infected travelers, we demonstrate high percentages of cross-reactivity of DENV IgM and IgG ELISÁs of which diagnostic laboratories should be aware. In addition, the high rate of DENV IgG background of >25% complicates a proper serological diagnosis in this group.
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Anticorpos Antivirais/sangue , Vírus da Dengue/imunologia , Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Doença Relacionada a Viagens , Infecção por Zika virus/diagnóstico , Zika virus/imunologia , Adulto , Estudos de Coortes , Reações Cruzadas , Dengue/virologia , Vírus da Dengue/genética , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Países Baixos , RNA Viral/sangue , Reação em Cadeia da Polimerase em Tempo Real , Testes Sorológicos , Suriname , Proteínas não Estruturais Virais/sangue , Proteínas não Estruturais Virais/imunologia , Zika virus/genética , Zika virus/isolamento & purificação , Infecção por Zika virus/virologiaRESUMO
Previous research has investigated caregiver attachment relationships in children with autism during early childhood, with few differences found from matched control groups. However, little is known of this relationship during middle childhood (ages 8-12 years). In this study, the aim was to establish whether there are differences in the security of attachment in children with high-functioning autism compared to typically developing children. A secondary aim was to establish whether caregivers' perceptions of their child's attachment to them accorded with the children's own reports. Twenty-one children with high-functioning autism and 17 typically developing children were administered the Kerns Security Scale and the Inventory of Parent and Peer Attachment-Revised, and caregivers completed the same questionnaires from the viewpoint of their child. There were no differences between the groups in the children's and parents' reports of attachment security. Parents' and children's reports were moderately correlated on the Kerns Security Scale but were not correlated on the Inventory of Parent and Peer Attachment-Revised. The results indicate that levels of attachment security in children with high-functioning autism are not different from those in typically developing children.
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Transtorno Autístico/psicologia , Cuidadores , Apego ao Objeto , Relações Pais-Filho , Pais , Estudos de Casos e Controles , Criança , Humanos , MasculinoRESUMO
The proposed revision for Autism spectrum disorders (ASDs) in the Diagnostic and Statistical Manual of Mental Disorders--Fifth Edition (DSM-5) represents a shift from the Diagnostic and Statistical Manual of Mental Disorders--Fourth Edition, Text Revision (DSM-IV-TR). As the proposed DSM-5 criteria require a higher minimum number of symptoms to be present compared to DSM-IV-TR, there have been some concerns about the impact that this will have on diagnostic outcomes. Therefore, the current study aimed to compare diagnostic outcomes using both DSM-IV-TR and DSM-5 criteria for 132 children. Of the 111 participants who received an ASD diagnosis under DSM-IV-TR, 26 did not meet DSM-5 criteria. The majority of these had received a DSM-IV-TR PDD-NOS diagnosis. Implications of the results and the proposed DSM-5 changes to the ASD criteria are discussed.