Risk factors for open angle glaucoma; a clinical and molecular genetic study

Glaucoma, optic neuropathy with excavation in the optic nerve head and corresponding visual field defect, is one of the leading causes for blindness worldwide. However, visual disability can often be avoided or delayed if the disease is diagnosed at an early stage. Therefore, recognising the risk factors for development and progression of glaucoma may prevent further damage. The purpose of the present study was to evaluate factors associated with visual disability caused by glaucoma and the genetic features of two risk factors, exfoliation syndrome (ES) and a positive family history of glaucoma. The present study material consisted of three study groups 1) deceased glaucoma patients from the Ekenäs practice 2) glaucoma families from the Ekenäs region and 3) population based families with and without exfoliation syndrome from Kökar Island. For the retrospective study, 106 patients with open angle glaucoma (OAG) were identified. At the last visit, 17 patients were visually impaired. Blindness induced by glaucoma was found in one or both eyes in 16 patients and in both eyes in six patients. The cumulative incidence of glaucoma caused blindness for one eye was 6% at 5 years, 9% at 10 years, and 15% at 15 years from initialising the treatment. The factors associated with blindness caused by glaucoma were an advanced stage of glaucoma at diagnosis, fluctuation in intraocular pressure during treatment, the presence of exfoliation syndrome, and poor patient compliance. A cross-sectional population based study performed in 1960-1962 on Kökar Island and the same population was followed until 2002. In total 965 subjects (530 over 50 years) have been examined at least once. The prevalence of exfoliation syndrome (ES) was 18% among subjects older than 50 years. Seventy-five of all 78 ES-positives belonged to the same extended pedigree. According to the segregation and family analysis, exfoliation syndrome seemed to be inherited as an autosomal dominant trait with reduced penetrance. The penetrance was more reduced for males, but the risk for glaucoma was higher in males than in females. To find the gene or genes associated with exfoliation syndrome, a genome wide scan was performed for 64 members (28 ES affected and 36 controls) of the Kökar pedigree. A promising result was found: the highest two-point LOD score of 3.45 (θ=0.04) in chromosome18q12.1-21.33. The presence of mutations in glaucoma genes TIGR/MYOC (myocilin) and OPTN (optineurin) was analysed in eight glaucoma families from the Ekenäs region. An inheritance pattern resembling autosomal dominant mode was detected in all these families. Primary open angle glaucoma or exfoliation glaucoma was found in 35% of 136 family members and 28% were suspected to have glaucoma. No mutations were detected in these families.

Clinical Phenotype and Genetic Epidemiology of Schizophrenia in a Finnish Isolate

This study is part of the joint project "The Genetic Epidemiology and Molecular Genetics of schizophrenia in Finland" between the Departments of Mental Health and Alcohol Research, and Molecular Medicine at the National Public Health Institute. In the study, we utilized three nationwide health care registers: 1) the Hospital Discharge Register, 2) the Free Medication Register, and 3) the Disability Pension Register, plus the National Population Register, in order to identify all patients with schizophrenia born from 1940 to 1976 (N=33,731) in Finland, and their first degree-relatives. 658 patients with at least one parent born in a homogeneous isolate in northeastern Finland were identified, as well as 4904 familial schizophrenia patients with at least two affected siblings from the whole country. The comparison group was derived from the Health 2000 Study. We collected case records and reassessed the register diagnosis. Were contacted the isolate patients and a random sample of patients from the whole country to make diagnostic clinical interviews and to assess the negative and positive symptoms and signs of schizophrenia. In addition to these patients, we interviewed siblings who were initially healthy according to the Hospital Discharge Register. Of those with a register diagnosis of schizophrenia, schizoaffective or schizophreniform disorder, 69% received a record-based consensus diagnosis and 63% an interview-based diagnosis of schizophrenia. Patients with schizophrenia having first-degree relatives with psychotic disorder had more severe affective flattening and alogia than those who were the only affected individuals in their family. The novel findings were: 1) The prevalence of schizophrenia in the isolate was relatively high based on register (1.5%), case record (0.9-1.3%), and interview (0.7-1.2%) data. 2) Isolate patients, regardless of their familial loading for schizophrenia, had less delusions and hallucinations than the whole country familial patients, which may be related to the genetic homogeneity in the isolate. This phenotype encourages the use of endophenotypes in genetic analyses instead of diagnoses alone. 3) The absence of register diagnosis does not confirm that siblings are healthy, because 7.7% of siblings had psychotic symptoms already before the register diagnoses were identified in 1991. For genetic research, the register diagnosis should therefore be reassessed using either a structured interview or a best- estimate case note consensus diagnosis. Structured clinical interview methods need be considered also in clinical practice.

Molecular markers for discriminating Streptococcus pyogenes and S.dysgalactiae subspecies equisimilis

Given the increasing aetiological importance of Streptococcus dysgalactiae subspecies equisimilis in diseases which are primarily attributed to S. pyogenes, molecular markers are essential to distinguish these species and delineate their epidemiology more precisely. Many clinical microbiology laboratories rely on agglutination reactivity and biochemical tests to distinguish them. These methods have limitations which are particularly exacerbated when isolates with mixed properties are encountered. In order to provide additional distinguishing parameters that could be used to unequivocally discriminate these two common pathogens, we assess here three molecular targets: the speB gene, intergenic region upstream of the scpG gene (IRSG) and virPCR. Of these, the former two respectively gave positive and negative results for S. pyogenes, and negative and positive results for S. dysgalactiae subsp. equisimilis. Thus,a concerted use of these nucleic acid-based methods is particularly helpful in epidemiological surveillance to accurately assess the relative contribution of these species to streptococcal infections and diseases.

Epidemiology of candidemia in Finland

Candida species are an important cause of nosocomial bloodstream infections in hospitalized patients worldwide, with associated high mortality, excess length of stay and costs. Main contributors to candidemias is profound immunosuppression due to serious underlying condition or intensive treatments leading to an increasing number of susceptible patients. The rank order of causative Candida species varies over time and in different geographic locations. The aim of this study was to obtain information on epidemiology of candidemia in Finland, to identify trends in incidence, causative species, and patient populations at risk. In order to reveal possible outbreaks and assess the value of one molecular typing method, restriction enzyme analysis (REA), in epidemiological study, we analyzed C. albicans bloodstream isolates in Uusimaa region in Southern Finland during eight years. The data from the National Infectious Disease Register were used to assess the incidence and epidemiological features of candidemia cases. In Helsinki University Central Hospital (HUCH) all patients with blood culture yielding any Candida spp. were identified from laboratory log-books and from Finnish Hospital Infection Program. All the patients with a stored blood culture isolate of C. albicans were identified through microbiology laboratory logbooks, and stored isolates were genotyped with REA in the National Institute for Health and Welfare (former KTL). The incidence of candidemia in Finland is globally relatively low, but increased between between 1990s and 2000s. The incidence was highest in males >65 years of age, but incidence rates for patients <1-15 years were lower during 2000s than during 1990s. In HUCH the incidence of candidemia remained low and constant during our 18 years of observation, but a significant shift in patient-populations at risk was observed, associated with patients treated in intensive care units, such as premature neonates and surgical patients. The predominating causative species in Finland and in HUCH is C. albicans, but the proportion of C. glabrata increased considerably. The crude one-month case fatality was constantly high between 28-33%. REA differentiated efficiently between C. albicans blood culture isolates and no clusters were observed in the hospitals involved, despite of abundant transfer of patients among them. Candida spp. are an important cause of nosocomial blood stream infections in Finland, and continued surveillance is necessary to determine the overall trends and patient groups at risk, and reduce the impact of these infections in the future. Molecular methods provide an efficient tool for investigation of suspected outbreak and should be available in the future in Finland, also.

Caracterização e diversidade molecular do transposon Tn1546, carreador do gene vanA, responsável pela expressão de resistência à vancomicina, em amostras clínicas de diferentes espécies de Enterococcus

Enterococcus resistentes à vancomicina (VRE) são reconhecidos como importantes patógenos causadores de infecções nosocomiais, configurando um grave problema de saúde pública, principalmente pela escassez de opção terapêutica eficaz. O fenótipo de resistência VanA é o mais frequente, sendo definido pela resistência a altos níveis de vancomicina e teicoplanina. VanA é caracterizado por um conjunto gênico (vanRSHAXYZ) localizado no elemento genético móvel denominado transposon Tn1546. A diversidade de Tn1546 resulta de alterações estruturais promovidas por deleções ou integração de sequências de inserção (IS) que, exercem papel chave na evolução do elemento VanA, modificando os aspectos relacionados à sua transferência e expressão do fenótipo. O objetivo deste estudo foi caracterizar e avaliar o polimorfismo de elementos Tn1546 presentes em amostras de diferentes espécies de Enterococcus isoladas em instituições hospitalares do Estado do Rio de Janeiro no período de 2000 a 2012. Foram incluídas neste estudo 70 amostras VRE que foram caracterizadas quanto ao gênero, espécies e genótipo de resistência aos glicopeptídeos por métodos convencionais e PCR multiplex. A susceptibilidade a 17 antimicrobianos foi avaliada pelo método de difusão em ágar, e concentração inibitória mínima (CIM) para vancomicina e teicoplanina foi determinada por microdiluição em caldo. O tranposon foi obtido após lise das células bacterianas e amplificação por PCR longo, utilizando-se oligonucleotídeos específicos para a região repetida e invertida que flanqueia este elemento genético. A diversidade dos elementos Tn1546 foi avaliada por um conjunto de métodos moleculares que incluiu a análise do polimorfismo do tamanho de fragmentos de restrição (restriction fragment lenght polymorphism, RFLP), utilizando-se a endonuclease ClaI, amplificação de segmentos internos por PCR de sobreposição de oligonucleotídeos (overlapping PCR) e detecção de sequências de inserção (ISs). A caracterização em espécies considerada para as demais análises foi obtida pela metodologia de PCR de acordo com a seguinte distribuição: E. avium (N=6), E. faecalis (N=12), E. faecium (N=46), E. gallinarum (N=4) e E. raffinosus (N=2). Todas as amostras apresentaram o genótipo vanA. Nos testes de susceptibilidade aos antimicrobianos foi observado que todas as amostras foram multirresistentes, sendo resistente de 6 a 13 dentre os 17 antimicrobianos testados. A presença de elementos semelhantes ao arquétipo de Tn1546 foi observada em 61,5% das amostras; entretanto, 27 amostras apresentaram perfis variantes de Tn1546. Foram identificados nove perfis de RFLP, dentre 66 avaliadas, sendo o perfil I, prevalente e semelhante ao arquétipo de Tn1546. Não foi possível analisar quatro amostras por RFLP. Os produtos de amplificação de Tn1546 alterados, obtidos pela overlapping PCR e pelo rastreamento de IS, levaram à classificação de 15 tipos polimórficos, nomeados de A a O. A maioria dos Tn1546 polimórficos teve suas regiões de ORF1 e/ou ORF2 deletadas; e IS1542 juntamente com IS1216V foram as inserções mais frequentes, que em muitas situações compartilhavam a mesma região de inserção. IS19 foi detectada apenas na região vanS-vanH. Os dados apresentados neste estudo indicam que o polimorfismo de Tn1546 pode ser explorado no rastreamento de rotas de transmissão, acompanhamento da dispersão de elementos VanA e investigação da evolução de amostras VRE.

Caracterização molecular de isolados de Enterobacteriaceae produtores de beta-lactamases de espectro alargado (ESBLs) provenientes de suiniculturas de produção intensiva e extensiva de Portugal

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Epidemiology and comparative analysis of Yersinia in Ireland

Yersiniosis is an acute or chronic enteric zoonosis caused by enteropathogenic Yersinia species. Although yersiniosis is predominantly associated with gastroenteric forms of infection, extraintestinal forms are often reported from the elderly or patients with predisposing factors. Yersiniosis is often reported in countries with cold and mild climates (Northern and Central Europe, New Zealand and North of Russian Federation). The Irish Health Protection Surveillance Centre (HPSC) currently records only 3-7 notified cases of yersiniosis per year. At the same time pathogenic Yersinia enterocolitica is recovered from pigs (main source of pathogenic Y. enterocolitica) at the levels similar to that observed in Yersinia endemic countries. Introduction of Yersinia selective culture procedures may increase Yersinia isolation rates. To establish whether the small number of notifications of human disease was an underestimate due to lack of specific selective culture for Yersinia we carried out a prospective culture study of faecal samples from outpatients with diarrhoea, with additional culture of appendix and throat swabs. Higher levels of anti-Yersinia seroprevalence than yersiniosis notification rates in endemic countries suggests that most yersiniosis cases are unrecognised by culture. Subsequently, in addition to a prospective culture study of clinical specimens, we carried out serological screening of Irish blood donors and environmental screening of human sewage. Pathogenic Yersinia strains were not isolated from 1,189 faeces samples, nor from 297 throat swabs, or 23 appendix swabs. This suggested that current low notification rates in Ireland are not due to the lack of specific Yersinia culture procedures. Molecular screening detected a wider variety of Y. enterocolitica-specific targets in pig slurry than in human sewage. A serological survey for antibodies against Yersinia YOP (Yersinia Outer Proteins) proteins in Irish blood donors found antibodies in 25%, with an age-related trend to increased seropositivity, compatible with the hypothesis that yersiniosis may have been more prevalent in Ireland in the recent past. Y. enterocolitica is a heterogeneous group of microorganisms that comprises strains with different degree of pathogenicity. Although non-pathogenic Y. enterocolitica lack conventional virulence factors, these strains can be isolated from patients with diarrhoea. Insecticidal Toxin Complex (ITC) and Cytolethal Distending Toxins can potentially contribute to the virulence of non-pathogenic Y. enterocolitica in the absence of other virulence factors. We compared distribution of ITC and CDT loci among pathogenic and non-pathogenic Y. enterocolitica. Additionally, to demonstrate potential pathogenicity of non-pathogenic Y. enterocolitica we compared their virulence towards Galleria mellonella larvae (a non-mammalian model of human bacterial infections) with the virulence of highly and mildly pathogenic Y. enterocolitica strains. Surprisingly, virulence of pathogenic and non-pathogenic Y. enterocolitica in Galleria mellonella larvae observed at 37°C did not correlate with their pathogenic potential towards humans. Comparative phylogenomic analysis detects predicted coding sequences (CDSs) that define host-pathogen interactions and hence providing insights into molecular evolution of bacterial virulence. Comparative phylogenomic analysis of microarray data generated in Y. enterocolitica strains isolated in the Great Britain from humans with diarrhoea and domestic animals revealed high genetic heterogeneity of these species. Because of the extensive human, animal and food exchanges between the UK and Ireland the objective of this study was to gain further insight into genetic heterogeneity and relationships among clinical and non-clinical Y. enterocolitica strains of various pathogenic potential isolated in Ireland and Great Britain. No evidence of direct transfer of strains between the two countries was found.

Welfare epidemiology as a tool to assess the welfare impact of inherited defects on the pedigree dog population

The effect that breed standards and selective breeding practices have on the welfare of pedigree dogs has recently come under scrutiny from both the general public and scientific community. Recent research has suggested that breeding for particular aesthetic traits, such as tightly curled tails, highly domed skulls and short muzzles predisposes dogs with these traits to certain inherited defects, such as spina bifida, syringomyelia and brachycephalic airway obstruction syndrome, respectively. Further to this, there is a very large number of inherited diseases that are not related to breed standards, which are thought to be prevalent, partly as a consequence of inbreeding and restricted breeding pools. Inherited diseases, whether linked to conformation or not, have varying impact on the individuals affected by them, and affect varying proportions of the pedigree dog population. Some diseases affect few breeds but are highly prevalent in predisposed breeds. Other diseases affect many breeds, but have low prevalence within each breed. In this paper, we discuss the use of risk analysis and severity diagrams as means of mapping the overall problem of inherited disorders in pedigree dogs and, more specifically, the welfare impact of specific diseases in particular breeds.

Staphylococcus aureus: towards a comprehensive view on epidemiology and clonal spread

Dissertation presented to obtain a PhD degree in Biology/ Molecular Biology by the Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica

Epidemiology of multiple congenital anomalies in Europe: a EUROCAT population-based registry study.

BACKGROUND: This study describes the prevalence, associated anomalies, and demographic characteristics of cases of multiple congenital anomalies (MCA) in 19 population-based European registries (EUROCAT) covering 959,446 births in 2004 and 2010. METHODS: EUROCAT implemented a computer algorithm for classification of congenital anomaly cases followed by manual review of potential MCA cases by geneticists. MCA cases are defined as cases with two or more major anomalies of different organ systems, excluding sequences, chromosomal and monogenic syndromes. RESULTS: The combination of an epidemiological and clinical approach for classification of cases has improved the quality and accuracy of the MCA data. Total prevalence of MCA cases was 15.8 per 10,000 births. Fetal deaths and termination of pregnancy were significantly more frequent in MCA cases compared with isolated cases (p < 0.001) and MCA cases were more frequently prenatally diagnosed (p < 0.001). Live born infants with MCA were more often born preterm (p < 0.01) and with birth weight < 2500 grams (p < 0.01). Respiratory and ear, face, and neck anomalies were the most likely to occur with other anomalies (34% and 32%) and congenital heart defects and limb anomalies were the least likely to occur with other anomalies (13%) (p < 0.01). However, due to their high prevalence, congenital heart defects were present in half of all MCA cases. Among males with MCA, the frequency of genital anomalies was significantly greater than the frequency of genital anomalies among females with MCA (p < 0.001). CONCLUSION: Although rare, MCA cases are an important public health issue, because of their severity. The EUROCAT database of MCA cases will allow future investigation on the epidemiology of these conditions and related clinical and diagnostic problems.

Enfermedad de Creutzfeldt-Jakob esporádica: estudio clínico, patológico y molecular de un caso

Generalidades. Las encefalopatías espongiformes transmisibles son enfermedades neurodegenerativas ocasionadas por la acumulación anormal de una variante mal plegada de la proteína priónica, lo cual induce la formación de conglomerados proteicos resistentes a la degradación. Además, son responsables de la disfunción sináptica, daño neuronal y de la sintomatología clásica acompañante. Esta proteína de membrana es codificada por el exón 2 del gen PRNP, ubicado en el brazo corto del cromosoma 20 y parece estar involucrada en la trasmisión sináptica, la transducción de señales, la actividad antioxidante de la superoxidodismutasa, neuroplasticidad y sobrevida celular. Un polimorfismo en el codón 129 se asocia con una susceptibilidad diferencial a la enfermedad Creutzfeldt-Jakob esporádica. Objetivo. Estudio clínico, patológico y molecular de un caso de una mujer de 58 años con diagnóstico de enfermedad de Creutzfeldt- Jakob esporádica. Métodos y resultados. Se presenta el caso de una mujer en quien aparece un trastorno depresivo del afecto con demencia progresiva y sintomatología general. Al final de la enfermedad, el cuadro progresó a un déficit neurológico focalizado en el área visual. La RMN mostró hiperintensidades inespecíficas córtico-subcorticales en el núcleo estriado; en el EEG se encontró pérdida de ritmos de fondo, patrón de descargas periódicas generalizadas y complejos trifásicos; en la biopsia cerebral postmorten se evidenció pérdida severa de la población neuronal en todas las capas, vacuolas en el neuropil, en el soma neuronal y en la glía. El análisis de secuencia del gen PRNP, a partir de extracción de DNA de sangre periférica, identificó homocigosis para metionina en el codón 129. La paciente fallece a los 3 meses del inicio de la sintomatología. Conclusión. Por epidemiología, curso clínico y exámenes paraclínicos se confirma el diagnóstico de enfermedad de Creutzfeldt- Jakob esporádica.La determinación del genotipo para los polimorfismos de riesgo se convierte en una herramienta útil para complementar por medios moleculares el diagnóstico y para profundizar la comprensión de la fisiopatología de la enfermedad de Creutzfeldt-Jakob, tanto para formas esporádicas como para la nueva variante.

Molecular tools to investigate Rhizoctonia solani distribution in soil

Real-time PCR protocols were developed to detect and discriminate 11 anastomosis groups (AGs) of Rhizoctonia solani using ribosomal internal transcribed spacer (ITS) regions (AG-1-IA, AG-1-IC, AG-2-1, AG-2-2, AG-4HGI+II, AG-4HGIII, AG-8) or beta-tubulin (AG-3, AG-4HGII, AG-5 and AG-9) sequences. All real-time assays were target group specific, except AG-2-2, which showed a weak cross-reaction with AG-2tabac. In addition, methods were developed for the high throughput extraction of DNA from soil and compost samples. The DNA extraction method was used with the AG-2-1 assay and shown to be quantitative with a detection threshold of 10-7 g of R. solani per g of soil. A similar DNA extraction efficiency was observed for samples from three contrasting soil types. The developed methods were then used to investigate the spatial distribution of R. solani AG-2-1 in field soils. Soil from shallow depths of a field planted with Brassica oleracea tested positive for R. solani AG-2-1 more frequently than soil collected from greater depths. Quantification of R. solani inoculum in field samples proved challenging due to low levels of inoculum in naturally occurring soils. The potential uses of real-time PCR and DNA extraction protocols to investigate the epidemiology of R. solani are discussed.

Development of molecular assays for the identification of the 11 Eimeria species of the domestic rabbit (Oryctolagus cuniculus)

Coccidiosis are the major parasitic diseases in poultry and other domestic animals including the domestic rabbit (Oryctolagus cuniculus). Eleven distinct Eimeria species have been identified in this host, but no PCR-based method has been developed so far for unequivocal species differentiation. In this work, we describe the development of molecular diagnostic assays that allow for the detection and discrimination of the 11 Eimeria species that infect rabbits. We determined the nucleotide sequences of the ITS1 ribosomal DNAs and designed species-specific primers for each species. We performed specificity tests of the assays using heterologous sets of primers and DNA samples, and no cross-specific bands were observed. We obtained a detection limit varying from 500 fg to 1 pg, which corresponds approximately to 0.8-1.7 sporulated oocysts, respectively. The test reported here showed good reproducibility and presented a consistent sensitivity with three different brands of amplification enzymes. These novel diagnostic assays will permit population surveys to be performed with high sensitivity and specificity, thus contributing to a better understanding of the epidemiology of this important group of coccidian parasites. (C) 2010 Elsevier B.V. All rights reserved.

Molecular characterisation of Bartonella species in cats from Sao Luis, state of Maranhao, north-eastern Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular typing and antifungal susceptibility of clinical sequential isolates of Cryptococcus neoformans from São Paulo State, Brazil

The antifungal susceptibility profiles and the genetic variability of 83 sequential clinical isolates of Cryptococcus neoformans, including four Cryptococcus gattii isolates, obtained from 38 São Paulo AIDS patients with cryptococcal meningitis were assessed by electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis. The majority of the Cryptococcus neoformans isolates were highly susceptible to amphotericin B and fluconazole. Twenty percent of the minimum inhibitory concentration values for amphotericin B varied from 0.5 to 1 mu g mL(-1). For fluconazole, 22% occurred in the range 8-16 mu g mL(-1). Sequential isolates from nine patients showed a trend towards lower susceptibility to fluconazole, flucytosine, itraconazole and amphotericin B. The results of molecular typing by electrophoretic karyotyping and RAPD analysis showed the presence of 22 electrophoretic karyotypes (EK) and 15 RAPD profiles that were highly correlated. Our results provided evidence for the occurrence of genetic changes in some strains associated with microevolution during the course of infection. We also observed both microevolution and simultaneous coinfection with two distinct Cryptococcus neoformans strains in one patient. In some patients, we found changed EK- and RAPD patterns in association with increased MIC values.