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Aplicação de metodologias de preservação e caracterização de fungos na coleção de culturas do Instituto de Medicina Tropical de São Paulo; Application of preservation methods and characterization of fungal isolates from the Instituto de Medicina Tropical de São Paulo culture collection

Cavalcanti, Sarah Desirée Barbosa
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 11/02/2011 PT
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A coleção de fungos do Instituto de Medicina Tropical de São Paulo/USP compreende 1047 isolados de fungos, algas e actinomicetos de interesse médico e veterinário, que até o início deste estudo eram mantidos sob repiques sucessivos a cada três meses. Criada em 1925 pelo Prof. Dr. Floriano Paulo de Almeida e, a partir de 1953, mantida pelo Prof. Dr. Carlos da Silva Lacaz, a coleção necessitava de adequação de seu espaço físico e atualização nos métodos de preservação dos isolados. A reforma física foi realizada de acordo com as normas de biossegurança recomendadas pelo manual de procedimentos para manipulação de patógenos. Diversos estudos têm demonstrado que repiques sucessivos propiciam alterações nas características morfológicas, antigênicas, genéticas e de virulência de isolados fúngicos. Deste modo, foram aplicadas metodologias de preservação de longo prazo, como: liofilização, água destilada esterilizada, congelamento a -80ºC e em nitrogênio líquido (-196ºC). Foram selecionados 386 isolados, entre fungos dimórficos (213), filamentosos (hialinos e melanizados - 106) e leveduras (67), relacionados às infecções fúngicas mais prevalentes em nosso meio. Foram utilizados dois métodos de preservação para cada grupo de fungos: as leveduras foram submetidas à liofilização e congelamento em freezer -80ºC; e os fungos dimórficos e filamentosos preservados em água destilada esterilizada e no vapor de nitrogênio líquido. Esses isolados foram inicialmente avaliados quanto à caracterização morfológica e...

Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

Motta, Teresa R.; Moreira-Filho, Carlos A.; Mendes, Rinaldo P.; Souza, Lenice R.; Sugizak, Maria F.; Baueb, Sonia; Calich, Vera L.G.; Vaz, Celideia A.C.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 151-157
ENG
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Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

COX24 codes for a mitochondrial protein required for processing of the COX1 transcript

Barros, Mario H.; Myers, Alan M.; Van Driesche, Sarah; Tzagoloff, Alexander
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 3743-3751
ENG
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In most strains of Saccharomyces cerevisiae the mitochondrial gene COX1, for subunit 1 of cytochrome oxidase, contains multiple exons and introns. Processing of COX1 primary transcript requires accessory proteins factors, some of which are encoded by nuclear genes and others by reading frames residing in some of the introns of the COX1 and COB genes. Here we show that the low molecular weight protein product of open reading frame YLR204W, for which we propose the name COX24, is also involved in processing of COX1 RNA intermediates. The growth defect of cox24 mutants is partially rescued in strains harboring mitochondrial DNA lacking introns. Northern blot analyses of mitochondrial transcripts indicate cox24 null mutants to be blocked in processing of introns aI2 and aI3. The dependence of intron aI3 excision on Cox24p is also supported by the growth properties of the cox24 mutant harboring mitochondrial DNA with different intron compositions. The intermediate phenotype of the cox24 mutant in the background of intronless mitochondrial DNA, however, suggests that in addition to its role in splicing of the COX1 pre-mRNA, Cox24p still has another function. Based on the analysis of a cox14-cox24 double mutant, we propose that the other function of Cox24p is related to translation of the COX1 mRNA. © 2006 by The American Society for Biochemistry and Molecular Biology...

Disseminated Amphotericin-Resistant Fusariosis in Acute Leukemia Patients: Report of Two Cases

Pereira, Graziella Hanna; de Angelis, Derlene Attili; Brasil, Roosecelis Araujo; dos Anjos Martins, Marilena; de Matos Castro e Silva, Dulcilena; Szeszs, Maria Walderez; de Souza Carvalho Melhem, Marcia
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 107-114
ENG
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Disseminated fusariosis has emerged as a significant, usually fatal infection in immunocompromised hosts despite antifungal treatment. We describe here two patients with acute leukemia who developed disseminated amphotericin-resistant fusariosis, and review of six studies of cases series in the literature. Two Fusarium solani strains were isolated from blood and skin cultures of one patient, and one strain from the blood culture of the second patient. Both patients died despite antifungal treatment. Strains were identified by sequencing of ITS1 and ITS4 regions. Random amplified polymorphic DNA analysis of the three F. solani isolates showed a low degree of similarity. Screening for Fusarium spp. contaminants within our facility was negative. Using the CLSI M-38-A2 broth dilution method and E tests®, we found that the MICs were low for voriconazole (0. 12 and 0. 5 mg/L, respectively), unexpectedly high for amphotericin B (≥8 and ≥32 μg/mL, respectively) and itraconazole (≥16 mg/ml). Patients with leukemia or persistent neutropenia should be assessed for disseminated fungal infections, including biopsy and skin cultures. Antifungal susceptibility tests are important due to the possibility of the strains being amphotericin resistant. Treatments must be aggressive...

Filamentous fungi from the Atlantic marine sponge Dragmacidon reticulatum

Passarini, Michel R. Z.; Santos, Cledir; Lima, Nelson; Berlinck, Roberto G. S.; Sette, Lara D.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 99-111
ENG
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Dragmacidon reticulatum is a marine sponge of wide occurrence in the Eastern and Western Atlantic. Little is known about D. reticulatum fungal diversity. Filamentous fungi recovered from D. reticulatum were assessed in the present study using a polyphasic taxonomic approach, including classical morphology, molecular biology and MALDI-TOF ICMS. Ninety-eight fungal strains were isolated from two D. reticulatum samples by using six different culture media, which were identified up to the genus level. Sixty-four distinct fungal ribotypes were obtained, distributed among twenty-four different genera belonging to the Ascomycota and Zygomycota. Representatives of Penicillium and Trichoderma were the most diverse and abundant fungi isolated. Amongst Penicillium spp. three isolates belonged to the same ribotype can be considered as a putative new species. Data derived from the present study highlight the importance of using a polyphasic approach to get an accurate identification in order to structure a reliable culture collection. © 2012 Springer-Verlag Berlin Heidelberg.

The population genetic structure of Rhizoctonia solani AG-3PT from potato in the Colombian Andes

Ferrucho, Rosa L.; Ceresini, Paulo C.; Ramirez-Escobar, Ursula M.; McDonald, Bruce A.; Cubeta, Marc A.; García-Domínguez, Celsa
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 862-869
ENG
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The soilborne fungus Rhizoctonia solani anastomosis group 3 (AG-3PT) is a globally important potato pathogen. However, little is known about the population genetic processes affecting field populations of R. solani AG-3PT, especially in the South American Colombian Andes, which is near the center of diversity of the two most common groups of cultivated potato, Solanum tuberosum and S. phureja. We analyzed the genetic structure of 15 populations of R. solani AG-3PT infecting potato in Colombia using 11 simple-sequence repeat (SSR) markers. In total, 288 different multilocus genotypes were identified among 349 fungal isolates. Clonal fractions within field populations were 7 to 33%. R ST statistics indicated a very low level of population differentiation overall, consistent with high contemporary gene flow, though moderate differentiation was found for the most distant southern populations. Genotype flow was also detected, with the most common genotype found widely distributed among field populations. All populations showed evidence of a mixed reproductive mode, including both asexual and sexual reproduction, but two populations displayed evidence of inbreeding. © 2013 The American Phytopathological Society.

Polymerase Chain Reaction Screening for Fungemia and/or Invasive Fungal Infections in Patients with Hematologic Malignancies

Ribeiro, P; Costa, F; Monteiro, A; Caldas, J; Silva, M; Ferreira, G; Veiga, J; Sousa, MO; Viegas, MP; Santos, E; Gonçalves, AJ; Sousa, AB
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
Publicado em //2006 ENG
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INTRODUCTION: Invasive fungal infections (IFIs) are a life-threatening complication in patients with hematologic malignancies, mainly in acute leukemia patients, following chemotherapy. IFI incidence is increasing, and associated mortality remains high due to unreliable diagnosis. Antifungal drugs are often limited by inadequate antimicrobial spectrum and side effects. Thus, the detection of circulating fungal DNA has been advocated as a rapid, more sensitive diagnostic tool. PATIENTS AND METHODS: Between June 01 and January 03, weekly blood samples (1,311) were screened from 193 patients undergoing intensive myelosuppressive or immunosuppressive therapy. IFI cases were classified according to European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Fungal DNA was extracted from whole blood and amplified using polymerase chain reaction (PCR) published primers that bind to the conserved regions of the fungal 18S rRNA gene sequence. In our study, two or more consecutive positive samples were always associated with fungal disease. RESULTS: PCR screening predicted the development of IFI to be 17 days (median). This test had a specificity of 91.1% and a sensitivity of 75%. IFI incidence was 7.8%. DISCUSSION: Therefore...

Polymerase Chain Reaction Screening for Fungemia and/or Invasive Fungal Infections in Patients with Hematologic Malignancies

Ribeiro, P; Costa, F; Monteiro, A; Caldas, J; Silva, M; Ferreira, G; Veiga, J; Sousa, MO; Viegas, MP; Santos, E; Gonçalves, AJ; Sousa, AB
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
Publicado em //2006 ENG
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INTRODUCTION: Invasive fungal infections (IFIs) are a life-threatening complication in patients with hematologic malignancies, mainly in acute leukemia patients, following chemotherapy. IFI incidence is increasing, and associated mortality remains high due to unreliable diagnosis. Antifungal drugs are often limited by inadequate antimicrobial spectrum and side effects. Thus, the detection of circulating fungal DNA has been advocated as a rapid, more sensitive diagnostic tool. PATIENTS AND METHODS: Between June 01 and January 03, weekly blood samples (1,311) were screened from 193 patients undergoing intensive myelosuppressive or immunosuppressive therapy. IFI cases were classified according to European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Fungal DNA was extracted from whole blood and amplified using polymerase chain reaction (PCR) published primers that bind to the conserved regions of the fungal 18S rRNA gene sequence. In our study, two or more consecutive positive samples were always associated with fungal disease. RESULTS: PCR screening predicted the development of IFI to be 17 days (median). This test had a specificity of 91.1% and a sensitivity of 75%. IFI incidence was 7.8%. DISCUSSION: Therefore...

Comparison of DNA extraction protocols for microbial communities from soil treated with biochar

Leite,D.C.A.; Balieiro,F.C.; Pires,C.A.; Madari,B.E.; Rosado,A.S.; Coutinho,H.L.C.; Peixoto,R.S.
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2014 EN
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Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit MiniprepTM (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits...

A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies

Burgdorf,R.J.; Laing,M.D.; Morris,C.D.; Jamal-Ally,S.F.
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2014 EN
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Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed...

Toll-Like Receptor 9-Dependent Immune Activation by Unmethylated CpG Motifs in Aspergillus fumigatus DNA▿ †

Ramirez-Ortiz, Zaida G.; Specht, Charles A.; Wang, Jennifer P.; Lee, Chrono K.; Bartholomeu, Daniella C.; Gazzinelli, Ricardo T.; Levitz, Stuart M.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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Phagocytic defenses are critical for effective host defenses against the opportunistic fungal pathogen Aspergillus fumigatus. Previous studies found that following challenge with A. fumigatus, Toll-like receptor 9 (TLR9) knockout mice survived longer than wild-type mice. However, the mechanism responsible was not defined. Here we demonstrate that A. fumigatus contains unmethylated CpG sequences, the natural ligands for TLR9. A. fumigatus DNA and synthetic CpG-rich oligodeoxynucleotides (ODNs) containing sequences found in the A. fumigatus genome potently stimulated the production of proinflammatory cytokines in mouse bone marrow-derived dendritic cells (BMDCs) and human plasmacytoid dendritic cells. The response was decreased when the fungal DNA was treated with a CpG methylase or with CpG-specific endonucleases. A role for TLR9 was demonstrated as cytokine production was abolished in BMDCs from TLR9-deficient mice. Moreover, transfection of HEK293 cells with human TLR9 conferred responsiveness to synthetic CpG-rich ODNs containing sequences found in A. fumigatus DNA. Taken together, these data demonstrate that TLR9 detects A. fumigatus DNA, resulting in the secretion of proinflammatory cytokines, which may contribute to the immune response to the pathogen.

Multicenter Comparison of Serum and Whole-Blood Specimens for Detection of Aspergillus DNA in High-Risk Hematological Patients

Springer, Jan; Morton, C. O.; Perry, Michael; Heinz, Werner J.; Paholcsek, Melinda; Alzheimer, Mona; Rogers, T. R.; Barnes, Rosemary A.; Einsele, Hermann; Loeffler, Juergen; White, P. Lewis
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2013 EN
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Samples from patients at high risk for invasive aspergillosis (IA) were prospectively collected and analyzed for the presence of molecular markers of fungal infection. Serum specimens were screened for galactomannan and Aspergillus DNA, and whole-blood specimens were screened only for Aspergillus DNA. Fungal infections were categorized according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group, National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria. Forty-seven cases (proven and probable IA) and 31 controls (no evidence of IA) were selected retrospectively for this case-control study, comprising 803 samples, in order to determine the performance of whole-blood PCR, serum PCR, and serum galactomannan testing. Although no single assay was able to detect every case of IA, a combination of different assays provided the best performance. There was no significant difference between the use of whole-blood and serum specimens for PCR-based diagnosis of IA, but there was a trend for whole blood to be more sensitive (85% versus 79%) and to yield an earlier positive result (36 days versus 15 days) than for serum. However, DNA extraction from serum specimens is easier and faster than that from whole-blood specimens...

An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR

Karakousis, A.; Tan, L.W.; Ellis, D.; Alexiou, H.; Wormald, P.J.
Fonte: Elsevier Science BV Publicador: Elsevier Science BV
Tipo: Artigo de Revista Científica
Publicado em //2006 EN
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To date, no single reported DNA extraction method is suitable for the efficient extraction of DNA from all fungal species. The efficiency of extraction is of particular importance in PCR-based medical diagnostic applications where the quantity of fungus in a tissue biopsy may be limited. We subjected 16 medically relevant fungi to physical, chemical and enzymatic cell wall disruption methods which constitutes the first step in extracting DNA. Examination by light microscopy showed that grinding with mortar and pestle was the most efficient means of disrupting the rigid fungal cell walls of hyphae and conidia. We then trialled several published DNA isolation protocols to ascertain the most efficient method of extraction. Optimal extraction was achieved by incorporating a lyticase and proteinase K enzymatic digestion step and adapting a DNA extraction procedure from a commercial kit (MO BIO) to generate high yields of high quality DNA from all 16 species. DNA quality was confirmed by the successful PCR amplification of the conserved region of the fungal 18S small-subunit rRNA multicopy gene.; http://www.elsevier.com/wps/find/journaldescription.cws_home/506034/description#description; A. Karakousis, L. Tan, D. Ellis, H. Alexiou and P.J. Wormald

Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

Wang, H.; Kong, F.; Sorrell, T.; Wang, B.; McNicholas, P.; Pantarat, N.; Ellis, D.; Xiao, M.; Widmer, F.; Chen, S.
Fonte: BioMed Central Ltd. Publicador: BioMed Central Ltd.
Tipo: Artigo de Revista Científica
Publicado em //2009 EN
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BACKGROUND Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. RESULTS The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D...

Residual soil DNA extraction increases the discriminatory power between samples

Young, J.M.; Weyrich, L.S.; Clarke, L.J.; Cooper, A.
Fonte: Springer Publicador: Springer
Tipo: Artigo de Revista Científica
Publicado em //2015 EN
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Forensic soil analysis relies on capturing an accurate and reproducible representation of the diversity from limited quantities of soil; however, inefficient DNA extraction can markedly alter the taxonomic abundance. The performance of a standard commercial DNA extraction kit (MOBIO PowerSoil DNA Isolation kit) and three modified protocols of this kit: soil pellet re-extraction (RE); an additional 24-h lysis incubation step at room temperature (RT); and 24-h lysis incubation step at 55°C (55) were compared using high-throughput sequencing of the internal transcribed spacer I ribosomal DNA. DNA yield was not correlated with fungal diversity and the four DNA extraction methods displayed distinct fungal community profiles for individual samples, with some phyla detected exclusively using the modified methods. Application of a 24 h lysis step will provide a more complete inventory of fungal biodiversity, and re-extraction of the residual soil pellet offers a novel tool for increasing discriminatory power between forensic soil samples.; Jennifer M. Young, Laura S. Weyrich, Laurence J. Clarke, Alan Cooper

Nachweis von Histoplasma capsulatum-DNA in Blutproben von Mäusen und humanen Gewebeproben mittels PCR-Verfahren; Detection of Histoplasma capsulatum DNA in murine models and human tissue by nested PCR assays

Feucht, Antje
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
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IInvasive Mykosen sind lebensbedrohliche Erkrankungen vor allem bei Patienten mit Defekten des zellulären Immunsystems. Auch bei der Histoplasmose, verursacht durch den dimorphen Pilz Histoplasma capsulatum, ist ein disseminierter Befall nach Inhalation der Erreger bei Immungeschwächten möglich. Nur die frühzeitige Diagnostik einer Histoplasmose bietet die Möglichkeit einer rechtzeitigen antimykotischen Therapie. Diagnostische Methoden sollten sensitiv und spezifisch und möglichst innerhalb eines Arbeitstages durchführbar sein. Sowohl Verfahren zum Direktnachweis wie die kulturelle Anzucht der Pilzzellen oder die mikroskopische Identifizierung in Patientenmaterial als auch serologische Methoden mit Antikörper- und Antigen-Nachweis erfüllen diese Anforderungen jedoch nur teilweise. Bereits 2001 war von Bialek et al. eine 18 S DNA-PCR-Methode zum Nachweis von Histoplasma capsulatum-spezifischer DNA im Tiermodell aufgebaut worden. In der vorliegenden Arbeit zeigten ergänzende Untersuchungen, dass der Nachweis der Pilz-DNA auch im Blut infizierter Mäuse möglich ist. Dabei stellte sich jedoch heraus, dass es aufgrund der hoch konservierten Zielregion innerhalb der 18 S rDNA häufig zu einer Kreuzreaktion der Primer kommt. Wir entwickelten daher eine neue PCR mit Zielregion in einem Gen...

Etablierung und Evaluierung einer vollautomatisierten Extraktionsmethode für DNA aus humanpathogenen Pilzen; Establishment and Evaluation of a fully automated extraction method for DNA from pathogenic fungi

Schmidt, Kathrin Dorothee
Fonte: Universität Tübingen Publicador: Universität Tübingen
Tipo: Dissertation; info:eu-repo/semantics/doctoralThesis
DE_DE
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Die Anwendung von MagNA Pure LC (TM) und das entwickelte Protokoll zur Extraktion von Pilz-DNA erwies sich als schnelle, sensitive und reproduzierbare Methode zur molekularen Diagnostik invasiver Mykosen. Sie leistet damit einen Beitrag zur Standardisierung molekularer Nachweisverfahren von Pilzinfektionen. In dieser Arbeit wurde eine automatisierte Methode zur Extraktion von fungalen Nukleinsäuren entwickelt und an klinischen Proben evaluiert. Die Methode basiert auf dem Extraktionsautomaten MagNA Pure LC (TM) (Roche Diagnostics) und wurde zur Aufspaltung der rigiden Pilzzellwände durch die Anwendung von Glasperlen erweitert. Mit dieser Methode konnte die DNA von insgesamt 23 verschiedenen Hefe- und Schimmelpilzen erfolgreich isoliert werden. Serielle Verdünnungen von Aspergillus fumigatus Konidien und Candida albicans Zellen (jeweils von 106 bis 100 CFU / ml) wurden zur Evaluierung der Sensitivität des Verfahrens eingesetzt. Außerdem wurde die DNA aus 68 klinischen Proben von 31 hämatologischen Patienten (57 Blutproben und 11 BALs), extrahiert und die PCR-Ergebnisse mit denen der manuellen, etablierten Methode verglichen. Die mit der neu entwickelten Methode isolierte DNA war reproduzierbar bis zu einem Detektionslimit von 101 CFU nachweisbar. Für C. albicans und in 9 / 28 Extraktionsläufen auch für A. fumigatus waren 100 CFU detektierbar. Insgesamt ist die Sensitivität der entwickelten Methode mit der des Routineprotokolls vergleichbar und ausreichend...

Vergleich der Light-cycler-PCR mit anderen molekularbiologischen Nachweisverfahren zum Nachweis von Aspergillus- und Candida-DNA im Blut von Patienten nach allogener Knochenmarkstransplantation; Comparison between Light-cycler-PCR and other molecularbiological methods for detection of Aspergillus- and Canida-DNA in bloodsamples of bone marrow transplanted patients

Hunecken, Nicole Michaela
Fonte: Universität Tübingen Publicador: Universität Tübingen
Tipo: Dissertation; info:eu-repo/semantics/doctoralThesis
DE_DE
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Patienten mit hämatologisch-onkologischen Erkrankungen oder Patienten nach Transplantationen sind gefährdet, sich eine lebensbedrohliche Pilzinfektion zuzuziehen. Mit einer möglichst früh eingeleiteten antimykotischen Therapie kann die Morbidität und die Mortalität dieser Patienten herabgesetzt werden. Dazu ist eine rasche, sensitive und speziesidentifizierende Diagnostik von großer Bedeutung. Das Ziel dieser Arbeit war es in erster Linie die LightCycler-Methode und die PCR-Elisa-Methode, aber auch andere Methoden, für die Routinediagnostik von Pilzen so anwendbar zu machen, dass der Verlust an Sensitivität und Spezifität möglichst gering gehalten wird, trotzdem aber am Tag der Blutentnahme ein Ergebnis vorliegen kann. Die Versuche zur Extraktion haben gezeigt, dass die Methode mit RCLB, WCLB und Lytikase, mit niedrigerer Zentrifugalkraft, dafür aber mit größeren Ausgangsvolumina die besten Ergebnisse lieferte. Diese Methode wurde zur Extraktion der Pilz-DNA für den PCR-Elisa als auch für das LightCycler-Verfahren angewendet. Im Hauptteil dieser Arbeit wurde die LightCycler- mit der PCR-Elisa-Methode anhand von 846 Blutproben von Patienten nach Knochenmarks- oder Stammzelltransplantation verglichen. Im Vergleich der Ergebnisse dieser Studie wird deutlich...

Toll-Like Receptor 9-Dependent Activation of Bone Marrow-Derived Dendritic Cells by URA5 DNA from Cryptococcus neoformans

Tanaka, Misuzu; Ishii, Keiko; Nakamura, Yuri; Miyazato, Akiko; Maki, Atsuko; Abe, Yuzuru; Miyasaka, Tomomitsu; Yamamoto, Hideki; Akahori, Yukiko; Fue, Misaki; Takahashi, Yurie; Kanno, Emi; Maruyama, Ryoko; Kawakami, Kazuyoshi
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2012 EN
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Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis in immunocompromised patients. Recently, we reported that Toll-like receptor 9 (TLR9) is involved in host defense against C. neoformans: specifically, it detects the pathogen's DNA. In the present study, we aimed to elucidate the mechanisms underlying TLR9-mediated activation of innate immune responses by using the URA5 gene, which encodes a virulent component of this fungal pathogen. A PCR-amplified 345-bp URA5 gene fragment induced interleukin-12 p40 (IL-12p40) production by bone marrow-derived dendritic cells (BM-DCs) in a TLR9-dependent manner. Similar activity was detected in the 5′ 129-bp DNA fragment of URA5 and in a synthesized oligodeoxynucleotide (ODN) with the same sequence. Shorter ODN fragments, which contained GTCGGT or GACGAT but had only 24 or 21 bases, induced IL-12p40 production and CD40 expression by BM-DCs, but this activity vanished when the CG sequence was replaced by GC or when a phosphorothioate modification was introduced. IL-12p40 production caused by active ODN was strikingly enhanced by treatment with DOTAP, a cationic lipid that increases the uptake of DNA by BM-DCs, though DOTAP failed to induce IL-12p40 production by inactive ODN and did not affect the activity of an ODN-containing canonical CpG motif. There was no apparent difference in intracellular trafficking between active and inactive ODNs. Finally...

Culturing and direct DNA extraction find different fungi from the same ericoid mycorrhizal roots

Allen, Tamara R; Millar, Anthony; Berch, Shannon M; Berbee, Mary L
Fonte: Cambridge University Press Publicador: Cambridge University Press
Tipo: Artigo de Revista Científica
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• This study compares DNA and culture-based detection of fungi from 15 ericoid mycorrhizal roots of salal (Gaultheria shallon), from Vancouver Island, BC Canada. • From the 15 roots, we PCR amplified fungal DNAs and analyzed 156 clones that included t