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- ItemAcesso aberto (Open Access)Caracterização molecular, avaliação do perfil de susceptibilidade a antifúngicos e formação de biofilme de isolados clínicos pertencentes ao complexo Candida haemulonii e Candida auris(Universidade Federal de São Paulo (UNIFESP), 2017-06-29) Lima, Soraia Lopes [UNIFESP]; Melo, Analy Salles de Azevedo [UNIFESP]; Padovan, Ana Carolina Barbosa; http://lattes.cnpq.br/7106567615656883; s http://lattes.cnpq.br/8922840487452492; http://lattes.cnpq.br/6572863047648284; Universidade Federal de São Paulo (UNIFESP)The Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) and the phylogenetically related species (C. auris and C. pseudohaemulonii) are agents of superficial and invasive mycoses, responsible for high rates of morbidity and mortality. The phenotypic methods are unable to correctly identify closely related species, such as that included in the C. haemulonii complex. It is essential to use molecular techniques for identification, and sequencing the ITS region of the ribosomal DNA is considered the gold standard method for Candida species identification. As these microorganisms can be found in hospital environments and have shown low susceptibility to antifungal agents, it is essential that more studies are performed to evaluate their biological characteristics. Objectives: (i) To identify clinical isolates stored at the Culture Collection of LEMI, identified as C. haemulonii (sensu lato) and C. auris by sequencing the ITS region of rDNA generating high quality sequences; (ii) To evaluate in vitro susceptibility profile of these isolates with 5 antifungal agents; (iii) To evaluate biofilm formation of C. haemulonii (sensu lato) isolates and related species. Material and methods: Sixty-six clinical isolates from different sites were selected from several medical centers from four Latin American countries from 2008 to 2016. For the molecular identification, ITS sequencing was performed and the sequences obtained were compared to those deposited in NCBI genomic database. Susceptibility tests with the antifungals fluconazole (FLC), voriconazole (VRC), anidulafungin (ANI), 5-fluorocytosine (5-FC) and amphotericin-B (AMB) were performed according to CLSI document M27-A3. For the biofilm assays, 96-well plates were used and biofilm quantification was performed by using violet crystal staining. Results: Among the 66 isolates studied, 37 were identified as C. auris, 11 as C. haemulonii var. vulnera, 9 as C. haemulonii (sensu stricto) and 9 as C. duobushaemuonii. No C. pseudohaemulonii isolates were found. Inter-specific variability of antifungal susceptibility was observed among four agents tested. The 4 species identified were resistant to AMB and FLC. Regarding VRC, lower MICs were obtained for the majority of C. haemulonii (sensu lato) isolates. 5-FC and ANI agents showed activity against the isolates tested. Regarding the biofilm formation, C. haemulonii (sensu stricto) and C. haemulonii var. vulnera were the species that showed higher intra-specific variability. The species that presented the highest and the lowest biofilm formation were C. auris and C. duobushaemulonii, respectively. Conclusions: The sequencing of ITS region allowed accurate identification of all isolates and generated high quality sequences. C. haemulonii complex and C. auris were resistant to AMB and FLC, but the majority of the isolates were susceptible to ANI and 5-FC. All species studied were able to form biofilm, especially C. auris, which presented the highest capability to produce biofilm.
- ItemAcesso aberto (Open Access)Complexo Candida guilliermondii: identificação molecular, prevalência de espécies de interesse clínico e susceptibilidade aos antifúngicos(Universidade Federal de São Paulo (UNIFESP), 2018-06-28) Pedoni, Diego Betto [UNIFESP]; Colombo, Arnaldo Lopes [UNIFESP]; Melo, Analy Salles de Azevedo [UNIFESP]; http://lattes.cnpq.br/8922840487452492; http://lattes.cnpq.br/4512261018429681; http://lattes.cnpq.br/6527598447733253; Universidade Federal de São Paulo (UNIFESP)Candida guilliermondii today represents a complex of emerging species, some of them of clinical importance, having been described in 1912 by Castellani et al., from sputum culture of a patient with bronchial infection. It is currently responsible for 1-25% of the bloodstream infections caused by Candida sp. in different geographic regions. Despite biological variation of this complex in the world, these isolates of C. guilliermondii are characterized as a single taxonomic unit when identified by phenotypic methods. However, the molecular analysis of genomic DNA allows the differentiation of 7 distinct species in this complex: C. guilliermondii sensu stricto (Meyerozyma guilliermondii), C. fermentati (Meyerozyma carribicca), C. smithsonii, C. athensensis, C. elateridarum, C. carpophila and C. glucosophila. To date, only three species of the complex have been associated with infections in humans: C. guilliermondii, C. fermentati and C. carpophila. There are few data on putative biological and clinical peculiarities of the infections caused by the species of this complex, and their prevalence in medical centers in Brazil and Latin America is unknown. Objectives: To determine the prevalence of different species of the C. guilliermondii complex in episodes of candidemia sequentially documented in sentinel medical centers, standardize strategies for identifying species of the complex and to assess the in vitro susceptibility profile to antifungal of these agents isolates. Material and Methods: A total of 150 isolates obtained from patients attended at 18 medical centers in Brazil and Latin America, initially identified as belonging to C. guilliermondii complex, were submitted to identification by sequencing of the ITS rDNA regions. For the RFLP a PCR of the ITS region of the rDNA was performed, and the amplified DNA fragment was digested with the enzyme Taq I. Refering to the proteomic profile of these isolates, the protein profiles were obtained on the Bruker Daltonics system and assembly of the in-house library. In vitro susceptibility tests were performed according to CLSI document M27-A3 and M27-S4, using antifungal the agents: amphotericin B (AMB), fluconazole (FLC), voriconazole (VOR) and anidulafungina (AND). Results: Among the 150 isolates studied, 135 isolates were identified as C. guilliermondii sensu stricto, 13 isolates as C. fermentati and 2 isolates as C. carpophila. Acording the RFLP assay, it was possible to verify that C. guilliermondii sensu stricto isolates presented a banding profile with 2 bands (~ 250 and 190 bp) and C. fermentati and C. carpophila with 3 bands (~ 250.190 and 150 bp). We created a robust proteomic database with 5 profiles of C. guilliermondii and C. fermentati isolates. In general, in the antifungal susceptibility tests, C. fermentati isolates presented higher geometric means of MICs for FLC, AND and AMB (3.79 μg / mL, 2.11 μg / mL and 1 μg / mL, respectively) ) compared to C. guilliermondii sensu stricto (2.70 μg / mL, 1.77 μg / mL and 0.66 μg / mL, respectively). For the MIC values for VOR, both obtained the same geometric mean of 1 μg / mL. Conclusion: Sequencing of regions of the ribosomal gene allowed accurate identification of all the isolates tested. Using the RFLP technique it was possible to discriminate C. guilliermondii sensu stricto from C. fermentati, but it was not possible to discriminate C. carpohila from C. fermentati. We created a proteomic database capable to discriminate isolates of C. guilliermondii sensu stricto and C. fermentati. In the study, the clinical isolates of C. fermentati presented lower susceptibility to the antifungal agents FLC, AND and AMB, when compared to C. guilliermondii sensu stricto isolates.
- ItemAcesso aberto (Open Access)Investigação das bases moleculares do câncer não medular familiar da tireoide(Universidade Federal de São Paulo (UNIFESP), 2018-12-20) Lima, Erika Urbano de [UNIFESP]; Rubio, Ileana Gabriela Sanchez de [UNIFESP]; http://lattes.cnpq.br/3231635049279767; http://lattes.cnpq.br/4993081281957671; Universidade Federal de São Paulo (UNIFESP)Almost 95% of thyroid cancer cases originate from follicular thyroid cells (NMTC). The majority are sporadic (90%), but 3-9% correspond to familial forms (FNMTC) characterized by three or more first-degree relatives affected by thyroid cancer. It can be divided into different subgroups: familial papillary thyroid carcinoma (PTC) with or without oxyphilia cell, familial PTC with papillary renal cell carcinoma, and familial PTC with multinodular goiter. Some chromosomal loci and genes were associated with CNMFT in families with specific phenotypes. It has been proposed that FNMTC may be a polygenic disease with autosomal dominant inheritance with variable penetrance. The objective of this study was to identify genetic variants involved in the susceptibility to FNMTC in two Brazilian families using Whole Exome Sequencing (WES) and specific bioinformatics analyses. WES of DNA samples from peripheral blood leukocyte was performed using SureSelectXT Human All Exon V6+UTR Capture Library kit (Agilent) and the Illumina® NextSeq ™ 500 platform. For the variants selection an extensive bioinformatics analysis was performed using 12 specific programs and 4 population databases (ExAc, AbraOM, dbSNP and Ensembl). In the first family from Manaus (Amazonas) and characterized as familial PTC with multinodular goiter, the c.C110A variant of the ACTL8 gene was exclusively present in all affected individuals. The analysis showed that the ACTL8 variant may impair the protein function and structure, and that this protein could interact with the cytoskeleton and epithelial differentiation proteins. The c.C623T and c.C2654T variants of the SRPX2 and EPHB1 genes were identified in the second family from Jacobina (Bahia) classified as familial PTC without oxyphilic cells. Both variants were classified as damaging in all prediction analyzes and the joint presence of these variants showed increased odds ratio to develop the disease (OR= 51; 95% CI: 1.7046 - 1525.9024; P=0.0234), with 100% of sensitivity and 88.9% of specificity. It was also was observed that both proteins (SRPX2 and EPHB1) would participate in a network of protein interactions related to MAPK pathway activation.These results suggest that the ACTL8, SRPX2 and EPBH1 genes variants identified in this study confer susceptibility to the development of FNMTC in these particular families. These results are in agreement with the literature that suggests that the genetic factors of FNMTC susceptibility are specific to each family.