Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.

Necrotizing enteritis associated with Clostridium perfringensType B in chinchillas (Chinchilla lanigera)

Four 3-4 month-old chinchillas (Chinchilla lanigera) from a commercial flock of 395 chinchillas, were found dead with evidence of previous diarrhea and prolapsed rectum. A fifth 8 month-old chinchilla died 8 hours after being found recumbent, apathetic, diarrheic and with a prolapsed rectum. Two chinchillas were necropsied and observed gross lesions consisted of extensive hemorrhagic enteritis, mild pulmonary edema and enlarged and yellow liver; this latter finding was particularly prominent in the chinchilla presenting longer clinical course. Histologically there was necrotizing enteritis associated with abundant bacterial rods aggregates in the intestinal surface epithelium and within the lamina propria. In the lungs there were small amounts of pink proteinaceous material (edema) in the interstitium and marked vacuolar hepatocellullar degeneration (lipidosis) in the liver. Anaerobic cultures from the intestinal contents of one of the affected chinchillas yielded Clostridium perfringens. Genotyping of this C. perfringens isolate was achieved by multiplex polymerase chain reaction (mPCR) as C. perfringenstype B due to detection of alpha, beta and epsilon-toxin genes. These findings suggest C. perfringens type B as an important cause of sudden or acute death in chinchillas.

Identificação e genotipagem de Mycobacterium bovis em bovinos positivos no teste intradérmico para tuberculose em Mato Grosso do Sul

Neste estudo, realizou-se genotipagem de isolados de Mycobacterium bovis, provenientes de amostras de tecidos de bovinos positivos no teste cervical comparativo (TCC) para tuberculose em Mato Grosso do Sul, por meio da técnica de spoligotyping. Tecidos de 13 bovinos positivos, oriundos de diferentes municípios do estado, foram cultivados em meio de Stonebrink. As colônias resultantes foram submetidas à coloração de Ziehl-Neelsen e todos os isolados apresentaram características tintoriais de BAAR. Os 13 isolados de BAAR foram identificados por PCR multiplex (mPCR). O gene hsp65 foi alvo para identificação de Mycobacterium spp, a sequência de inserção IS6110 foi alvo para identificação de complexo Mycobacterium tuberculosis (CMT) e a região rvd1rv2031c foi explorada para detecção de M. bovis. Os isolados micobacterianos foram genotipados pela técnica de spoligotyping. Dos 13 bovinos, sete tinham pelo menos uma lesão sugestiva de tuberculose em linfonodos retrofaríngeos, parotídeos e pulmonares ou no pulmão, e em seis não foram encontradas lesões visíveis sugestivas da doença. Na mPCR, 11/13 (84,6%) isolados foram positivos para Mycobacterium spp; 8/13 (61,5%) positivos para CMT e 7/13 (53,8%) positivos para M. bovis. Com base no spoligotyping, oito isolados de BAAR foram agrupados dentro de três diferentes agrupamentos de genótipos e uma amostra remanescente apresentou perfil único, sendo quatro isolados com padrão de espoligotipo SB0121, dois SB1145, dois SB0881 e um SB0140. A técnica de spoligotyping demonstrou que há diversidade genética entre os espoligotipos presentes no estado de Mato Grosso do Sul, embora predomine o perfil SB0121

Standardization of a multiplex PCR for the identification of coagulase-positive Staphylococcus

The enterotoxigenic species Staphylococcus aureus, S. hyicus and S. intermedius show very similar characteristics, making their identification through conventional microbiological methods difficult. This study aimed at the development of a Multiplex PCR (mPCR) for the identification of S. aureus, S. intermedius and S. hyicus using the nuc gene as the target sequence. The results obtained suggest that the set of primers used was specific for the three species of Staphylococcus evaluate with a detection limit of 10² CFU.mL-1.

Inteligência emocional: a compreensão das emoções em crianças do pré-escolar

A inteligência emocional (IE) tem sido geralmente estudada no contexto da compreensão do funcionamento do adulto (Hall, Geher, & Brackett, 2004). Como tal, muitos aspectos da IE têm sido raramente estudados em crianças (Lahaye et al., 2010). Este estudo procura estudar e medir em crianças do pré-escolar a compreensão emocional (CE), um construto complexo, considerado componente-chave da IE das crianças pequenas (Denham et al., 2003). Especificamente, o estudo analisa as relações do desenvolvimento cognitivo, temperamento, relação com os pares, género, idade, estatuto socioeconómico e número de irmãos com a CE. A amostra é constituída por 140 crianças entre os 4 e 6 anos, às quais foram administradas o Test of Emotion Comprehension (TEC, Pons, Harris, & de Rosnay, 2004), as Matrizes Progressivas Coloridas de Raven (MPCR, aferida por Simões, 2000), a Escala de Personalidade da Criança (EPC, Dibble & Cohen, 1974) e uma análise sociométrica (Peceguina, Santos, & Daniel, 2008). Os resultados indicam correlações entre a CE e o desenvolvimento cognitivo e o temperamento, não existindo uma sobreposição entre estes três construtos. A CE é um construto desenvolvimental que vai aumentando através da idade, e que influencia as relações da criança com os pares. Os resultados desten estudo fornecem pistas relevantes para intervenções que visem a promoção de competências socio-emocionais.

A relação entre inteligência emocional e o rendimento escolar em crianças do 1º Ciclo do Ensino Básico da R.A.M.

Este estudo insere-se no domínio da investigação sobre a relação entre inteligência emocional e o rendimento escolar. A pesquisa foi desenvolvida no sentido de dar resposta às questões: Será que existe alguma relação entre a IE, a IE percebida e o rendimento escolar? Será que o género, a idade, o índice de participação social, o estatuto socioeconómico dos pais e a retenção escolar influenciam a IE, a IE percebida e o rendimento escolar? Participaram neste estudo 129 crianças de ambos os géneros, feminino e masculino, que frequentavam o 1.º Ciclo do Ensino Básico, com idades compreendidas entre os seis e os treze anos. A metodologia utilizada foi a quantitativa transversal correlacional. Os instrumentos usados: Informação Sociodemográfica e Educativa, Matrizes Coloridas de Raven (MPCR, Raven et al., 2001), Questionário de Inteligência Emocional de Bar-On (Candeias & Rebocho, 2007; Candeias et al., 2008) e o Test of Emotional Comprehension (TEC, Pons, Harris, & Rosnay, 2004). Os resultados revelam que apenas alguns aspetos da IE se relacionam com algumas das variáveis do rendimento escolar não sendo, neste caso, consideradas as variáveis género, participação social e retenção, se relacionando com a IE.

Myonecrosis by Clostridium septicum in a dog, diagnosed by a new multiplex-PCR

Clostridial myositis is an acute, generally fatal toxemia that is considered to be rare in pet animals. The present report describes an unusual canine clostridial myositis that was diagnosed by a new multiplex-PCR (mPCR) designed for simultaneous identification of Clostridium sordellii, Clostridium septicum, Clostridium perfringens type A, Clostridium chauvoei, and Clostridium novyi type A. A ten-month-old male Rottweiler dog, that had displayed lameness and swelling of the left limb for 12 h, was admitted to a veterinary hospital. The animal was weak, dyspneic and hyperthermic, and a clinical examination indicated the presence of gas and edema in the limb. Despite emergency treatment, the animal died in only a few minutes. Samples of muscular tissue from the necrotic area were aseptically collected and plated onto defibrinated sheep blood agar (5%) in anaerobic conditions. Colonies suggestive of Clostridium spp. were submitted to testing by multiplex-PCR. Impression smears of the tissues, visualized with Gram and also with panoptic stains, revealed long rod-shaped organisms, and specimens also tested positive using the fluorescent antibody technique (FAT). The FAT and mPCR tests enabled a diagnosis of C. septicum myonecrosis in the dog. (C) 2012 Elsevier Ltd. All rights reserved.

Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Padronização da técnica de multiplex PCR para a detecção de Staphylococcus aureus, Streptococcus agalactiae e Escherichia coli em amostras de leite bovino, obtidas de tanques de expansão

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Five-year results of a randomized controlled clinical trial comparing zirconia and titanium abutments supporting single-implant crowns in canine and posterior regions

OBJECTIVES: To test the survival rates, and the technical and biological complication rates of customized zirconia and titanium abutments 5 years after crown insertion. MATERIAL AND METHODS: Twenty-two patients with 40 single implants in maxillary and mandibular canine and posterior regions were included. The implant sites were randomly assigned to zirconia abutments supporting all-ceramic crowns or titanium abutments supporting metal-ceramic crowns. Clinical examinations were performed at baseline, and at 6, 12, 36 and 60 months of follow-up. The abutments and reconstructions were examined for technical and/or biological complications. Probing pocket depth (PPD), plaque control record (PCR) and Bleeding on Probing (BOP) were assessed at abutments (test) and analogous contralateral teeth (control). Radiographs of the implants revealed the bone level (BL) on mesial (mBL) and distal sides (dBL). Data were statistically analyzed with nonparametric mixed models provided by Brunner and Langer and STATA (P < 0.05). RESULTS: Eighteen patients with 18 zirconia and 10 titanium abutments were available at a mean follow-up of 5.6 years (range 4.5-6.3 years). No abutment fracture or loss of a reconstruction occurred. Hence, the survival rate was 100% for both. Survival of implants supporting zirconia abutments was 88.9% and 90% for implants supporting titanium abutments. Chipping of the veneering ceramic occurred at three metal-ceramic crowns supported by titanium abutments. No significant differences were found at the zirconia and titanium abutments for PPD (meanPPD(ZrO2) 3.3 ± 0.6 mm, mPPD(T) (i) 3.6 ± 1.1 mm), PCR (mPCR(Z) (rO) (2) 0.1 ± 0.3, mPCR(T) (i) 0.3 ± 0.2) and BOP (mBOP(Z) (rO) (2) 0.5 ± 0.3, mBOP(T) (i) 0.6 ± 0.3). Moreover, the BL was similar at implants supporting zirconia and titanium abutments (mBL(Z) (rO) (2) 1.8 ± 0.5, dBL(Z) (rO) (2) 2.0 ± 0.8; mBL(T) (i) 2.0 ± 0.8, dBL(T) (i) 1.9 ± 0.8). CONCLUSIONS: There were no statistically or clinically relevant differences between the 5-year survival rates, and the technical and biological complication rates of zirconia and titanium abutments in posterior regions.

A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.

Assessment of iodine intake and related cognitive function impairments in elementary schoolchildren from Terceira Island, Azores (Portugal)

Dissertação de Mestrado, Ciências Biomédicas, 28 de Junho de 2016, Universidade dos Açores.

Competências sócio-emocionais em crianças com dificuldades de aprendizagem : Estudo de caracterização em alunos do 1º ciclo do ensino básico

As dificuldades de aprendizagem são tema de grande relevância no campo da educação, pelas repercussões que têm no desenvolvimento das crianças que as possuem, quer a nível pessoal, familiar, escolar e social. A compreensão das competências sócio-emocionais destas crianças surge como objecto de estudo desta investigação. A amostra é constituída por 74 crianças com dificuldades de aprendizagem do 1° Ciclo do Ensino Básico. Os instrumentos utilizados foram: o Inventário de Quociente Emocional Bar-On: Versão para crianças (EQ-i:YV; versão portuguesa; Candeias et al., 2008), o Teste de Resolução de Problemas lnterpessoais da Inteligência Social para Crianças (PRPI - 6/11; Candeias et al., 2008), o Teste de Competências Sociais para Crianças (PACS - Socialmente em Acção 6/11, Candeias et al., 2008), o Teste de Percepção de Emoções (Franco & Candeias, 2008) e as Matrizes Progressivas Coloridas de Raven (MPCR, Raven, 1965). Estudaram-se as relações entre a inteligência social, inteligência emocional, competência social e variáveis sócio-demográficas como a idade, o nível sócio-económico e a participação social destas crianças. Conclui-se que, quanto mais elevada é a competência emocional destes alunos, menores serão as suas dificuldades em termos de competência social. Estes achados permitem aumentar o conhecimento teórico sobre esta problemática, possibilitando a aplicação prática na intervenção psicológica e psicopedagógica. / ABSTRACT: Learning difficulties are the subject of great relevance in education, the impact they have on the development of children who have, whether for personal, familial, educational and social. Understanding of the socio-emotional skills of these children appears to be the subject of this research study. This study used a sample of 74 children with learning difficulties the first cycle of the cities of Évora and Montemor-o­ Novo. We applied: Emotional Quocient lnventory Bar-On: young version (EQ-i: YV; Candeias et al., 2008); Perception and Recognition Emotions (Franco & Candeias, 2008); Cognitive Test of Social lntelligence for Children (PRPI-6/11; Candeias et al., 2008); Social Competence Test for Children (PACS-6/11; Candeias et al., 2008) and Coloured Progressive Matrices (CPM, Raven, 1965). We studied the relationship between social intelligence, emotional intelligence, social competence and socio-demographic variables such as age, socio-economic and social participation of these children. lt was concluded that, the higher the emotional competence of these students, lower their difficulties in terms of social competence. These findings increase the theoretical knowledge on this issue, enabling the practical application in psychological and pedagogic. This study leaves open some suggestions for future work.