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1.
Mem. Inst. Oswaldo Cruz ; 115: e200208, 2020. tab, graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135227

ABSTRACT

Paracoccidioides spp. isolation from environmental samples is rare and hardly reproducible. Molecular techniques have facilitated the fungal detection. However, it can be still difficult. Some strategies to enhance the capacity of DNA detection have been adopted, including the analysis of soil samples belonging to the habitat of animals from which Paracoccidioides spp. have already been isolated, notably armadillo burrows. To date, the detection of Paracoccidioides spp. has not yet been reported from outbreak hotspots. Clusters and outbreaks of acute paracoccidioidomycosis (PCM), usually a more severe clinical form, have currently occurred in urban areas being associated to climate changes, deforestation, and great constructions. These occurrences potentially signalise the fungus' environmental niche, a riddle not yet solved. The authors performed an environmental investigation in a deeply disturbed area, after a highway construction in Rio de Janeiro, Brazil, where a recent outbreak of acute PCM occurred. Specific DNA sequences of Paracoccidioides brasiliensis were detected in shallow soil samples around the highway, reinforcing the association between the road construction and this PCM outbreak.


Subject(s)
Animals , Paracoccidioides/isolation & purification , Paracoccidioidomycosis/microbiology , Armadillos , DNA, Fungal/genetics , Paracoccidioides/growth & development , Paracoccidioides/genetics , Soil Microbiology , Brazil , Base Sequence , Sequence Analysis, DNA , Ecosystem
2.
J. appl. oral sci ; 28: e20190578, 2020. tab, graf
Article in English | LILACS, BBO | ID: biblio-1101256

ABSTRACT

Abstract Objective This study sought to analyze the gene expression of Candida albicans in sound root surface and root caries lesions, exploring its role in root caries pathogenesis. Methodology The differential gene expression of C. albicans and the specific genes related to cariogenic traits were studied in association with samples of biofilm collected from exposed sound root surface (SRS, n=10) and from biofilm and carious dentin of active root carious lesions (RC, n=9). The total microbial RNA was extracted, and the cDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500. Unique reads were mapped to 163 oral microbial reference genomes including two chromosomes of C. albicans SC5314 (14,217 genes). The putative presence of C. albicans was estimated (sum of reads/total number of genes≥1) in each sample. Count data were normalized (using the DESeq method package) to analyze differential gene expression (using the DESeq2R package) applying the Benjamini-Hochberg correction (FDR<0.05). Results Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) were up-regulated on SRS, and their functions are related to biofilm formation. Seven genes ( UTP20 , FDR=0.018; ITR1 , FDR=0.036; DHN6 , FDR=0.046; CaO19.7197 , FDR=0.046; CaO19.7838 , FDR=0.046; STT4 , FDR=0.046; GUT1 , FDR=0.046) were up-regulated on RC and their functions are related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. The use of alternative carbon sources, including lactate, and the ability to form hypha may be a unique trait of C. albicans influencing biofilm virulence. Conclusions C. albicans is metabolically active in SRS and RC biofilm, with different roles in health and disease.


Subject(s)
Humans , Tooth Root/microbiology , Candida albicans/genetics , DNA, Fungal/genetics , Root Caries/microbiology , Biofilms/growth & development , Candida albicans/isolation & purification , Candida albicans/growth & development , Gene Expression , Gene Expression Regulation, Fungal , Up-Regulation , Sequence Analysis, RNA , Transcriptome , Morphogenesis
3.
Rev. Soc. Bras. Med. Trop ; 53: e20190364, 2020. tab, graf
Article in English | LILACS | ID: biblio-1057277

ABSTRACT

Abstract The present report describes the first case of postpartum disseminated histoplasmosis in a 24-year-old HIV-negative woman. On the tenth day after vaginal delivery, the patient presented with dyspnea, fever, hypotension, tachycardia, and painful hepatomegaly. Yeast-like Histoplasma capsulatum features were isolated in the buffy coat. The phylogenetic analysis demonstrated that the fungal isolate was similar to other H. capsulatum isolates identified in HIV patients from Ceará and Latin America. Thus, histoplasmosis development in individuals with transitory immunosuppression or during the period of immunological recovery should be carefully examined.


Subject(s)
Humans , Female , Adult , DNA, Fungal/analysis , DNA, Ribosomal Spacer/genetics , Postpartum Period , Histoplasma/genetics , Histoplasmosis/diagnosis , Phylogeny , Polymerase Chain Reaction , Histoplasma/isolation & purification , Histoplasmosis/microbiology
4.
Rev. Soc. Bras. Med. Trop ; 52: e20180473, 2019. tab
Article in English | LILACS | ID: biblio-990445

ABSTRACT

Abstract INTRODUCTION: Candidiasis is the most frequent opportunistic mycosis in humans and can cause mortality, particularly in immunodeficient patients. One major concern is the increasing number of infections caused by drug-resistant Candidas trains, as these cannot be efficiently treated with standard therapeutics. The most common mechanism of fluconazole resistance in Candida is mutation of ERG11, a gene involved in the biosynthesis of ergosterol, a compound essential for cell integrity and membrane function. METHODS: Based on this knowledge, we investigated polymorphisms in the ERG11 gene of 3 Candida species isolated from immunocompromised and immunocompetent patients. In addition, we correlated the genetic data with the fluconazole susceptibility profile of the Candida isolates. RESULTS: A total of 80 Candida albicans, 8 Candida tropicalis and 6 Candida glabrata isolates were obtained from the saliva of diabetic, kidney transplant and immunocompetent patients. Isolates were considered susceptible to fluconazole if the minimum inhibitory concentration was lower than 8 μg/mL. The amino acid mutations F105L, D116E, K119N, S137L, and K128T were observed in C. albicans isolates, and T224C and G263A were found in C. tropicalis isolates. CONCLUSIONS: Despite the high number of polymorphisms observed, the mutations occurred in regions that are not predicted to interfere with ergosterol synthesis, and therefore are not related to fluconazole resistance.


Subject(s)
Humans , Male , Female , Adult , Aged , Polymorphism, Genetic/drug effects , Candida/drug effects , Candida/genetics , Fluconazole/pharmacology , Kidney Transplantation , Diabetes Mellitus/microbiology , Antifungal Agents/pharmacology , Reference Values , Saliva/microbiology , Candida/isolation & purification , DNA, Fungal/genetics , Microbial Sensitivity Tests , Polymerase Chain Reaction , Drug Resistance, Fungal/genetics , Immunocompetence , Middle Aged , Mutation/drug effects
5.
Article in English | WPRIM | ID: wpr-773442

ABSTRACT

OBJECTIVE@#A strain of Aspergillus niger (A. niger), capable of releasing bound phenolic acids from wheat bran, was isolated. This strain was identified by gene sequence identification. The antioxidant and anti-inflammatory capacity of ferulic acid released from wheat bran by this A. niger strain (FA-WB) were evaluated.@*METHODS@#Molecular identification techniques based on PCR analysis of specific genomic sequences were conducted; antioxidant ability was examined using oxygen radical absorbance capacity (ORAC), cellular antioxidant activity (CAA) assays, and erythrocyte hemolysis assays. RAW264.7 cells were used as a model to detect anti-inflammatory activity.@*RESULTS@#The filamentous fungal isolate was identified to be A. niger. ORAC and CAA assay showed that FA-WB had better antioxidant activity than that of the ferulic acid standard. The erythrocyte hemolysis assay results suggested that FA-WB could attenuate AAPH-induced oxidative stress through inhibition of reactive oxy gen species (ROS) generation. FA-WB could significantly restore the AAPH-induced increase in intracellular antioxidant enzyme activities to normal levels as well as inhibit the intracellular malondialdehyde formation. TNF-a, IL-6, and NO levels indicated that FA-WB can inhibit the inflammation induced by lipopolysaccharide (LPS).@*CONCLUSION@#Ferulic acid released from wheat bran by a new strain of A. niger had good anti-inflammatory activity and better antioxidant ability than standard ferulic acid.


Subject(s)
Animals , Anti-Inflammatory Agents , Metabolism , Pharmacology , Antioxidants , Metabolism , Pharmacology , Aspergillus niger , Genetics , Metabolism , Coumaric Acids , Metabolism , Pharmacology , DNA, Fungal , Dietary Fiber , Microbiology , Erythrocytes , Metabolism , Fermentation , Hep G2 Cells , Humans , Interleukin-6 , Metabolism , Lipopolysaccharides , Pharmacology , Mice , Sheep , Tumor Necrosis Factor-alpha , Metabolism
6.
Article in Chinese | WPRIM | ID: wpr-773130

ABSTRACT

The endophytic fungi from root,main stem,branch and leaf of Scrophularia ningpoensis were isolated from Zhejiang,whether these strains could yield harpagide or harpagoside were tested by HPLC and LC-MS. According to the morphological characteristic and the similarity of the nucleotide sequence of internal transcribed spacer( ITS) between r DNAs,the strains producing harpagide or harpagoside were identified. The results showed that 210 strains were isolated from the samples,which were classified into 9 orders,13 families and 17 genera by morphological study. Harpagide was detected in endogenous fungi ZJ17 and harpagoside was detected in endogenous fungi ZJ25 by HPLC coupled with LC-MS. ZJ17 was identified as Alternaria alternate and ZJ25 was identified as A.gaisen by its morphology and authenticated by ITS( ITS4 and ITS5 regions and the intervening 5. 8 S rDNA region).


Subject(s)
China , DNA, Fungal , Genetics , DNA, Ribosomal Spacer , Genetics , Endophytes , Classification , Metabolism , Fungi , Classification , Metabolism , Glycosides , Iridoid Glycosides , Metabolism , Pyrans , Metabolism , Scrophularia , Microbiology
7.
Rev. Soc. Bras. Med. Trop ; 51(4): 485-492, July-Aug. 2018. tab, graf
Article in English | LILACS | ID: biblio-957449

ABSTRACT

Abstract INTRODUCTION: This study aimed to describe cryptococcal meningitis (CM) cases and the associated demographic, clinical, and microbiological data obtained from cities in the State of Mato Grosso do Sul in the Midwestern region of Brazil. METHODS: The data from 129 patients with laboratory-confirmed CM admitted from 1997 to 2014 were retrospectively reviewed. The molecular types of Cryptococcus neoformans and Cryptococcus gattii isolated from cerebrospinal fluid were analyzed to determine their geographic distribution. RESULTS: The patients had a mean age of 37 years and consisted mostly of men (76.7%). Most of the Cryptococcus isolates were obtained from patients infected with human immunodeficiency virus (HIV) and included 105 (87.5%) and 5 (55.6%) isolates of C. neoformans and C. gattii complexes, respectively. A restriction fragment length polymorphism (RFLP) analysis of URA5 revealed that most of the isolates were C. neoformans molecular type VNI (89.1%), whereas the molecular types VGII (7%) and VNII (3.9%) were observed less frequently. Notably, 65% of the cases with a time from symptom onset to laboratory diagnosis of more than 60 days resulted in fatalities, and sequelae were observed among the patients who survived. CONCLUSIONS: The present study documents the occurrence of neurocryptococcosis, which is mainly caused by C. neoformans VNI, in Mato Grosso do Sul, Brazil, with probable autochthonous cases in the Brazilian Pantanal, the world's largest tropical wetland, and a biome where cryptococcosis has not yet been explored.


Subject(s)
Humans , Male , Female , Child , Adolescent , Adult , Young Adult , DNA, Fungal/analysis , Meningitis, Cryptococcal/epidemiology , Cryptococcus neoformans/isolation & purification , Cryptococcus gattii/isolation & purification , Rural Population , Socioeconomic Factors , Urban Population , Brazil/epidemiology , DNA, Fungal/cerebrospinal fluid , Polymerase Chain Reaction , Retrospective Studies , Meningitis, Cryptococcal/cerebrospinal fluid , Meningitis, Cryptococcal/microbiology , Cryptococcus neoformans/genetics , Cryptococcus gattii/genetics , Genotype , Middle Aged
9.
Article in English | WPRIM | ID: wpr-812354

ABSTRACT

To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.


Subject(s)
Breeding , DNA, Fungal , Genetics , DNA, Intergenic , Genetics , Genes, Mating Type, Fungal , Hypocreales , Chemistry , Classification , Genetics , Phylogeny
10.
Mem. Inst. Oswaldo Cruz ; 113(7): e170473, 2018. tab
Article in English | LILACS | ID: biblio-894939

ABSTRACT

Cryptococcus species are the causative agents of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals. Initial work on the molecular epidemiology of this fungal pathogen utilized genotyping approaches to describe the genetic diversity and biogeography of two species, Cryptococcus neoformans and Cryptococcus gattii. Whole genome sequencing of representatives of both species resulted in reference assemblies enabling a wide array of downstream studies and genomic resources. With the increasing availability of whole genome sequencing, both species have now had hundreds of individual isolates sequenced, providing fine-scale insight into the evolution and diversification of Cryptococcus and allowing for the first genome-wide association studies to identify genetic variants associated with human virulence. Sequencing has also begun to examine the microevolution of isolates during prolonged infection and to identify variants specific to outbreak lineages, highlighting the potential role of hyper-mutation in evolving within short time scales. We can anticipate that further advances in sequencing technology and sequencing microbial genomes at scale, including metagenomics approaches, will continue to refine our view of how the evolution of Cryptococcus drives its success as a pathogen.


Subject(s)
Humans , Phylogeny , Genetic Variation , Cryptococcus gattii/genetics , Multilocus Sequence Typing , Phylogeography , Phylogeny , DNA, Fungal , Cryptococcus gattii/pathogenicity , Phylogeography , Genotype
11.
Rev. chil. infectol ; 35(5): 574-579, 2018. tab, graf
Article in Spanish | LILACS | ID: biblio-978072

ABSTRACT

Resumen Introducción: El diagnóstico de aspergilosis invasora (AI) se realiza mediante criterios clínicos y microbiológicos los que incluyen marcadores séricos. Recientemente, el test inmunocromatográfico Aspergillus lateral flow device (LFD), ha sido evaluado como método para diagnóstico de AI. Objetivo: Evaluar el desempeño de este test para el diagnóstico de AI. Material y Método: Estudio transversal en que se evaluaron muestras de suero y lavado bronco-alveolar (LBA) procesadas para galactomanano provenientes de pacientes adultos con sospecha de AI, atendidos en el Hospital Clínico de Red de Salud UCCHRISTUS. Resultados: Se procesó un total de 142 muestras de 98 pacientes, correspondientes a AI probada 5,6%, AI probable 41,5%, AI posible 12,7% y ausencia de AI 40,1%. Al confrontar los resultados con las categorías diagnósticas según criterios EORTC/MSG se obtuvo una sensibilidad y especificidad de LFD para diagnóstico de AI de 70,9 y 53,5% para muestras de suero y 83,3 y 38,5% para muestras de LBA. La concordancia entre galactomanano y LFD fue de 62,4% (54,1-69,9) con un índice Kappa de 0,202 (0,03682-0,3669). Conclusiones: Aspergillus LFD presentó una adecuada sensibilidad; sin embargo, la especificidad fue baja por lo que un resultado positivo requiere ser confirmado.


Background: The incidence of invasive aspergillosis is increasing. Its diagnosis is based on clinical and microbiological criteria which include the determination of serological markers such as galactomannan. Recently, the Aspergillus lateral flow device, an inmunocromatograph assay has been described for its diagnosis. Aim: To evaluate the performance of the lateral flow device for the diagnosis of invasive aspergillosis (IA) in adult patients. Material and Method: In this cross-sectional study, frozen samples that had been previously evaluated for galactomannan from patients classified with proven/probable/possible or no AI according to the EORTC/MSG criteria were selected. Results: A total of 142 samples from 98 patients were processed, corresponding to proven AI 5.6%, probable IA 41.5%, possible IA 12.7% and no-IA 40.1%. The sensitivity and specificity of the Aspergillus lateral flow was 70.9% and 53.5% for serum samples and 83.3% and 38.5% for BAL samples. The concordance between the galactomannan and Aspergillus lateral flow was 62.4% (54.1 - 69.9) with a Kappa index of 0.202 (0.03682 - 0.3669). Conclusions: We observed a good sensitivity but low specificity, a positive result need a confirmatory test.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Aspergillosis/diagnosis , Aspergillus/genetics , Aspergillus/immunology , DNA, Fungal/analysis , Bronchoalveolar Lavage Fluid/microbiology , Mannans/analysis , Cross-Sectional Studies , Chromatography, Affinity/methods , Sensitivity and Specificity , Hospitals, University
12.
Braz. j. med. biol. res ; 51(9): e7404, 2018. graf
Article in English | LILACS | ID: biblio-951760

ABSTRACT

DNA repair pathways, cell cycle checkpoints, and redox protection systems are essential factors for securing genomic stability. The aim of the present study was to analyze the effect of Ilex paraguariensis (Ip) infusion and one of its polyphenolic components rutin on cellular and molecular damage induced by ionizing radiation. Ip is a beverage drank by most inhabitants of Argentina, Paraguay, Southern Brazil, and Uruguay. The yeast Saccharomyces cerevisiae (SC7Klys 2-3) was used as the eukaryotic model. Exponentially growing cells were exposed to gamma rays (γ) in the presence or absence of Ip or rutin. The concentrations used simulated those found in the habitual infusion. Surviving fractions, mutation frequency, and DNA double-strand breaks (DSB) were determined after treatments. A significant increase in surviving fractions after gamma irradiation was observed following combined exposure to γ+R, or γ+Ip. Upon these concomitant treatments, mutation and DSB frequency decreased significantly. In the mutant strain deficient in MEC1, a significant increase in γ sensitivity and a low effect of rutin on γ-induced chromosomal fragmentation was observed. Results were interpreted in the framework of a model of interaction between radiation-induced free radicals, DNA repair pathways, and checkpoint controls, where the DNA damage that induced activation of MEC1 nodal point of the network could be modulated by Ip components including rutin. Furthermore, ionizing radiation-induced redox cascades can be interrupted by rutin potential and other protectors contained in Ip.


Subject(s)
Rutin/pharmacology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/radiation effects , Plant Extracts/pharmacology , Antimutagenic Agents/pharmacology , Ilex paraguariensis/chemistry , Radiation Protection/methods , Mass Spectrometry , DNA, Fungal/radiation effects , Cell Survival/drug effects , Cell Survival/radiation effects , Cells, Cultured , Reproducibility of Results , Chromatography, Liquid , Mutagenesis , DNA Repair , Dose-Response Relationship, Radiation , DNA Breaks, Double-Stranded , Mutation Rate , Gamma Rays
13.
Mem. Inst. Oswaldo Cruz ; 113(10): e180340, 2018. tab, graf
Article in English | LILACS | ID: biblio-1040583

ABSTRACT

Histoplasmosis is a systemic mycosis infection caused by Histoplasma capsulatum, a heterothallic ascomycete. The sexual reproduction of this fungus is regulated by the mating type (MAT1) locus that contains MAT1-1 and MAT1-2 idiomorphs, which were identified by uniplex polymerase chain reaction (PCR). This study aimed to optimise single-step multiplex PCR for the accurate detection of the distinct mating types of H. capsulatum. Among the 26 isolates tested, 20 had MAT1-1 genotype, while six showed MAT1-2 genotype, in agreement with the uniplex PCR results. These results suggest that multiplex PCR is a fast and specific tool for screening H. capsulatum mating types.


Subject(s)
DNA, Fungal/genetics , DNA Primers/genetics , Histoplasma/genetics , Reproducibility of Results , Sequence Analysis, DNA , Multiplex Polymerase Chain Reaction , Genotype , Histoplasma/classification
14.
Article in English | WPRIM | ID: wpr-773565

ABSTRACT

To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.


Subject(s)
Breeding , DNA, Fungal , Genetics , DNA, Intergenic , Genetics , Genes, Mating Type, Fungal , Hypocreales , Chemistry , Classification , Genetics , Phylogeny
15.
J. appl. oral sci ; 25(3): 274-281, May-June 2017. tab, graf
Article in English | LILACS, BBO | ID: biblio-893617

ABSTRACT

Abstract Pulpal and periodontal tissues have similar microbiota that allows cross-contamination between the pulp and periodontal tissues. Objective The aim of this study was to investigate the prevalence of isolated Candida albicans from periodontal endodontic lesions in diabetic and normoglycemic patients, and the fungi's virulence in different atmospheric conditions. Material and Methods A case-control study was conducted on 15 patients with type 2 diabetes mellitus (G1) and 15 non-diabetics (G2) with periodontal endodontic lesions. Samples of root canals and periodontal pockets were plated on CHROMagar for later identification by polymerase chain reaction (PCR) and virulence test. Results C. albicans was identified in 79.2% and 20.8% of the 60 samples collected from diabetic and normoglycemic patients, respectively. Of the 30 samples collected from periodontal pockets, 13 showed a positive culture for C. albicans, with 77% belonging to G1 and 23% to G2. Of the 11 positive samples from root canals, 82% were from G1 and 18% from G2. Production of proteinase presented a precipitation zone Pz<0.63 of 100% in G1 and 72% in G2, in redox and negative (Pz=1), under anaerobic conditions in both groups. Hydrophobicity of the strains from G1 indicated 16.4% with low, 19.3% with moderate, and 64.3% with high hydrophobicity in redox. In G2, 42.2% had low, 39.8% had moderate, 18% had high hydrophobicity in redox. In anaerobic conditions, G1 showed 15.2% with low, 12.8% with moderate, and 72% with high hydrophobicity; in G2, 33.6% had low, 28.8% had moderate, and 37.6% had high hydrophobicity. There was statistical difference in the number of positive cultures between G1 and G2 (p<0.05) with predominance in G1. There was statistical difference for all virulence factors, except hemolysis (p=0.001). Conclusions Candida albicans was isolated more frequently and had higher virulence in diabetic patients.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Periodontal Diseases/microbiology , Candida albicans/isolation & purification , Candida albicans/pathogenicity , Dental Pulp Diseases/microbiology , Diabetes Mellitus, Type 2/microbiology , Oxidation-Reduction , Peptide Hydrolases/analysis , Periodontal Diseases/physiopathology , Periodontal Diseases/diagnostic imaging , Periodontal Pocket/microbiology , Phospholipases/analysis , Virulence , DNA, Fungal , Radiography, Dental , Case-Control Studies , Polymerase Chain Reaction , Statistics, Nonparametric , Dental Pulp Cavity/microbiology , Dental Pulp Diseases/physiopathology , Dental Pulp Diseases/diagnostic imaging , Diabetes Mellitus, Type 2/physiopathology , Electrophoresis , Hydrophobic and Hydrophilic Interactions
16.
Mem. Inst. Oswaldo Cruz ; 112(6): 403-410, June 2017. tab, graf
Article in English | LILACS | ID: biblio-841808

ABSTRACT

BACKGROUND Enterocytozoon bieneusi are the most common microsporidia associated with different clinical manifestations such as diarrhoea, respiratory tract inflammation and acalculous cholecystitis, especially in immunocompromised patients. Infection usually occurs by ingestion of food and water contaminated with spores, but can also result from direct contact with spores through broken skin, eye lesions, and sexual transmission, depending on the microsporidian species. Although there are reports of E. bieneusi found in humans and animals in Brazil, there are no published studies of environmental samples examined by molecular methods. OBJECTIVES The purpose of this study was to verify the presence of E. bieneusi in raw sewage and treated effluent from a combined system by molecular methods. METHODS Raw sewage and treated effluent samples collected from a combined system were analysed for the presence of E. bieneusi using the internal transcriber spacer (ITS) region of E. bieneusi by nested polymerase chain reaction. FINDINGS The analysis revealed E. bieneusi presence and a novel genotype (EbRB) in one raw sewage sample and one treated effluent. MAIN CONCLUSIONS The presence of E. bieneusi in final effluent indicates that the combined system may not remove microsporidian spores. This study is the first report of E. bieneusi in environmental samples in Brazil.


Subject(s)
DNA, Fungal/genetics , Polymerase Chain Reaction , Enterocytozoon/isolation & purification , Enterocytozoon/genetics , Phylogeny , Brazil , Sequence Analysis , Genotype
17.
Mem. Inst. Oswaldo Cruz ; 112(3): 214-219, Mar. 2017. tab, graf
Article in English | LILACS | ID: biblio-1040568

ABSTRACT

Since the description of Candida orthopsilosis and C. metapsilosis in 2005, several methods have been proposed to identify and differentiate these species from C. parapsilosis sensu stricto. Species-specific uniplex polymerase chain reaction (PCR) was performed and compared with sequencing of the D1/D2 region of the LSU 28S rDNA gene, microsatellite typing of C. parapsilosis sensu stricto, and PCR-restriction fragment length polymorphism patterns in the ITS1-5.8S-ITS2 region of the rDNA gene. There was agreement between results of testing of 98 clinical isolates with the four PCR-based methods, with 59 isolates identified as C. parapsilosis sensu stricto, 37 as C. orthopsilosis, and two as C. metapsilosis.


Subject(s)
Humans , Candida/isolation & purification , Mycological Typing Techniques/methods , Polymorphism, Restriction Fragment Length , Candida/classification , Candida/genetics , DNA, Fungal/analysis , Polymerase Chain Reaction , DNA Fingerprinting , Sequence Analysis, DNA , DNA, Ribosomal Spacer/genetics , Genotype
18.
Mem. Inst. Oswaldo Cruz ; 112(2): 140-145, Feb. 2017. graf
Article in English | LILACS | ID: biblio-841762

ABSTRACT

BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis.


Subject(s)
Paracoccidioides/classification , Paracoccidioides/genetics , DNA, Fungal , DNA, Ribosomal Spacer , Species Specificity , Oligonucleotide Probes , In Situ Hybridization, Fluorescence , Fluorescence , Fluorescent Dyes
19.
Article in English | LILACS | ID: biblio-842772

ABSTRACT

ABSTRACT The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species.


Subject(s)
Humans , Child , AIDS-Related Opportunistic Infections/microbiology , Candida/genetics , Candidiasis, Oral/microbiology , DNA, Fungal/genetics , Candida albicans/genetics , Candida albicans/isolation & purification , Candida/classification , Candida/isolation & purification , Genotype , Mouth Mucosa/microbiology , Mycological Typing Techniques , Phenotype , Polymerase Chain Reaction
20.
Rev. Inst. Med. Trop. Säo Paulo ; 59: e13, 2017. tab, graf
Article in English | LILACS | ID: biblio-842793

ABSTRACT

ABSTRACT Cryptococcosis, a systemic disease caused by the fungus Cryptococcus neoformans/ Cryptococcus gattii is more severe in immunocompromised individuals. This study aimed to analyze the epidemiology of the disease, the molecular characteristics and the antifungal susceptibility of C. neoformans isolated from patients treated in a Brazilian university hospital. This retrospective study was conducted in the Clinical Hospital, Federal University of Uberlândia, and evaluated cases of cryptococcosis and strains of C. neoformans isolated from 2004 to 2013. We evaluated 41 patients, 85% of whom were diagnosed with AIDS. The fungus was isolated from the cerebrospinal fluid (CSF) of 21 patients (51%); 19.5% had fungemia and in 24% the agent was isolated from the CSF and blood, concurrently. Meningoencephalitis was the most frequent (75%) manifestation of infection. Despite adequate treatment, the mortality of the disease was 58.5%. Most isolates (97.5%) presented the VNI genotype (serotype A, var. grubii) and one isolate was genotyped as C. gattii (VGI); all the isolates were determined as mating type MATa and showed susceptibility to the tested antifungals (fluconazole, voriconazole, amphotericin B and 5-flucytosine). Although AIDS detection rates remain stable, opportunistic infections such as cryptococcosis remain as major causes of morbidity and mortality in these patients.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Young Adult , Cryptococcosis/mortality , Cryptococcus neoformans/isolation & purification , AIDS-Related Opportunistic Infections/microbiology , AIDS-Related Opportunistic Infections/mortality , Antifungal Agents/pharmacology , Brazil/epidemiology , Cryptococcosis/microbiology , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/genetics , DNA, Fungal/analysis , Hospitals, University , Microbial Sensitivity Tests , Retrospective Studies
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