Biochemical Characterization of an In-House Coccidioides Antigen: Perspectives for the Immunodiagnosis of Coccidioidomycosis

The objective of this study was to evaluate the reactivity of an in-house antigen, extracted from a strain of C. posadasii isolated in northeastern Brazil, by radial immunodiffusion and Western blotting, as well as to establish its biochemical characterization. the protein antigen was initially extracted with the use of solid ammonium sulfate and characterized by 1-D electrophoresis. Subsequently, it was tested by means of double radial immunodiffusion and Western blotting. A positive reaction was observed against the antigen by both immunodiagnostic techniques tested on sera from patients suffering from coccidioidomycosis. Besides this, two immunoreactive protein bands were observed and were revealed to be a beta-glucosidase and a glutamine synthetase after sequencing of the respective N-terminal regions. Our in-house Coccidioides antigen can be promising as a quick and low-cost diagnostic tool without the risk of direct manipulation of the microorganism.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordination for the Improvement of Higher Education PersonnelUniv Fed Ceara, Specialized Med Mycol Ctr, Sch Med, BR-60430270 Fortaleza, Ceara, BrazilUniv Estadual Ceara, Postgrad Program Vet Sci, Sch Vet Med, BR-60740000 Fortaleza, Ceara, BrazilUniv Fed Ceara, Dept Biochem & Mol Biol, BR-60455760 Fortaleza, Ceara, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04021001 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04021001 São Paulo, BrazilCNPq: 302574/2009-3CNPq: 306637/2010-3Coordination for the Improvement of Higher Education Personnel: CAPES/PNPD2103/2009Web of Scienc

Antiretroviral drugs saquinavir and ritonavir reduce inhibitory concentration values of itraconazole against Histoplasma capsulatum strains in vitro

Recent studies have shown that some drugs that are not routinely used to treat fungal infections have antifungal activity, such as protease inhibitor antiretroviral drugs. This study investigated the in vitro susceptibility of Histoplasma capsulatum var. capsulatum to saquinavir and ritonavir, and its combination with the antifungal itraconazole. The susceptibility assay was performed according to Clinical and Laboratory Standards Institute guidelines. All strains were inhibited by the protease inhibitor antiretroviral drugs. Saquinavir showed minimum inhibitory concentrations ranging from 0.125 to 1 mu g mL(-1) for both phases, and ritonavir presented minimum inhibitory concentrations ranging from 0.0312 to 4 mu g mL(-1) and from 0.0625 to 1 mu g mL(-1) for filamentous and yeast phase, respectively. Concerning the anti fungal itraconazole, the minimum inhibitory concentration values ranged from 0.0019 to 0.125 mu g mL(-1) and from 0.0039 to 0.0312 mu g mL(-1) for the filamentous and yeast phase, respectively. The combination of saquinavir or ritonavir with itraconazole was synergistic against H. capsulatum, with a significant reduction in the minimum inhibitory concentrations of both drugs against the strains (p < 0.05). These data show an important in vitro synergy between protease inhibitors and itraconazole against the fungus H. capsulatum. (C) 2016 Published by Elsevier Editora Ltda.CNPqCAPESUniv Fed Ceara, Postgrad Program Med Microbiol, Specialized Med Mycol Ctr, Fortaleza, CE, BrazilUniv Fed Ceara, Postgrad Program Med Sci, Fortaleza, CE, BrazilUniv Estadual Ceara UECE, Postgrad Program Vet Sci, Fortaleza, CE, BrazilUniv Fed Ceara, Dept Stat & Appl Math, Fortaleza, CE, BrazilUniv Fed Sao Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, Sao Paulo, SP, BrazilHosp Sao Jose, Fortaleza, CE, BrazilUniv Fed Sao Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, Sao Paulo, SP, BrazilCNPq: 303396/2014-8CNPq: 552161/2011-0CAPES: AE1 - 0052-000630100/11Web of Scienc

Evaluation of the genetic diversity of Histoplasma capsulatum var. capsulatum isolates from north-eastern Brazil

Since the beginning of the HIV epidemic, there has been a significant increase in the number of histoplasmosis cases in Ceara, a state in north-east Brazil. The lack of epidemiological data on the genotypes circulating in the north-east region shows the importance of more detailed studies on the molecular epidemiology of Histoplasma capsulatum var. capsulatum in this region. Different molecular techniques have been used to better characterize the genetic profile of H. capsulatum var. capsulatum strains. The aim of this study was to analyse the genetic diversity of H. capsulatum var. capsulatum isolates in Fortaleza, the capital of Ceara, through the sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, and establish the molecular profile of these isolates, along with strains from south-east Brazil, by RAPD analysis, featuring the different clusters in those regions. The isolates were grouped into two clusters. Cluster 1 included strains from the south-east and north-east regions with separation of isolates into three distinct subgroups (subgroups 1a, 1 b and 1 c). Cluster 2 included only samples from north-east Brazil. Sequencing of the ITS1 -5.8S-ITS2 region allowed the detection of two major clades, which showed geographical correlation between them and their subgroups. Therefore, it can be concluded that the H. capsulatum var. capsulatum isolates from Ceara have a high degree of genetic polymorphism. The molecular data also confirm that populations of this fungus are composed of different genotypes in Brazil and worldwide.National Council for Scientific and Technological Development (CNPq)[562296/2010-7, 552161/2011-0, 304779/2011-3, 473025/2012-4]Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES) [2103/2009

HEPATOTOXICIDADE DOS PESTICIDAS USADOS EM VITICULTURAS DA REGIÃO SUL DO BRASIL

Introdução: O consumo de agrotóxicos, vem crescendo de maneira exponencial. Os Etilenobisditiocarbamatos (EBDCs), tendo como representante o Mancozebe que são fungicidas utilizados, na Serra Gaúcha, no cultivo de videiras. Objetivo: Avaliar a hepatotoxicidade de pesticidas do grupo dos EBDCs em trabalhadores expostos em viticulturas e não expostos em culturas orgânicas. Métodos: Estudo transversal comparativo com avaliação de 50 trabalhadores expostos e 48 não expostos a EBDCs com entrevista e questionário, coleta de sangue e urina. Avaliação de hepatotoxicidade (exame bioquímico), genotoxicidade (dano DNA), acetilcolinestarase e indicador biológico de exposição (etilenotiouréia urinária - IBE). Vinculado ao projeto CAAE 11627319500005327. Resultados: No grupo não exposto 9 participantes afirmaram terem tido exposição a pesticida no passado. Identificou-se alterações de provas de função hepática (AST/ALT) presente em 5 amostras do grupo exposto. O IBE esteve presente em 100% das amostras, inclusive nas do grupo não exposto. Não foi identificada alteração na análise de acetilcolinestarase nos expostos. Avaliação do dano ao DNA (genotoxicidade) foi significativamente superior no grupo com exposição. Conclusão: As análises preliminares sugerem que a exposição ao mancozebe é capaz de provocar danos à saúde do trabalhador, podendo ocorrer fora do ambiente ocupacional, uma vez que a etilenotiureia, foi identificada inclusive em amostras de urina do grupo não exposto

Coccidioidomycosis and Histoplasmosis in Equines: An Overview to Support the Accurate Diagnosis

Fungal infections of the respiratory tract of horses are not as frequent as those of bacterial and viral origin, often leading to worsening of clinical conditions due to misdiagnosis and incorrect treatment. Coccidioidomycosis and histoplasmosis are systemic mycoses caused by the dimorphic fungi Coccidioides spp. and Histoplasma capsulatum, respectively, which affect humans and a variety of other animals, including equines. These systemic mycoses of chronic and progressive nature can exhibit clinical manifestations similar to other microbial infections. Thus, this article broadly discusses the epidemiology, etiology, virulence, pathogenesis, clinical presentation, treatment, and diagnostic strategies of coccidioidomycosis and histoplasmosis, to support accurate diagnosis. (C) 2016 Elsevier Inc. All rights reserved.National Scientific and Technological Research CouncilBrazil (CNPq)Univ Fed Ceara, Postgrad Program Med Microbiol, Specialized Med Mycol Ctr, Dept Pathol & Legal Med,Sch Med, BR-60430275 Fortaleza, Ceara, BrazilUniv Estadual Ceara, Postgrad Program Vet Sci, Sch Vet, Fortaleza, Ceara, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilCNPq: 445670/2014-2CNPq: 552161/2011-0Web of Scienc

A Review of the Development of Multitarget Molecules against HIV-TB Coinfection Pathogens

The human immunodeficiency virus (HIV) produces the pathologic basis of acquired immunodeficiency syndrome (AIDS). An increase in the viral load in the body leads to a decline in the number of T lymphocytes, compromising the patient’s immune system. Some opportunistic diseases may result, such as tuberculosis (TB), which is the most common in seropositive patients. Long-term treatment is required for HIV-TB coinfection, and cocktails of drugs for both diseases are used concomitantly. The most challenging aspects of treatment are the occurrence of drug interactions, overlapping toxicity, no adherence to treatment and cases of resistance. Recent approaches have involved using molecules that can act synergistically on two or more distinct targets. The development of multitarget molecules could overcome the disadvantages of the therapies used to treat HIV-TB coinfection. This report is the first review on using molecules with activities against HIV and Mycobacterium tuberculosis (MTB) for molecular hybridization and multitarget strategies. Here, we discuss the importance and development of multiple targets as a means of improving adherence to therapy in cases of the coexistence of these pathologies. In this context, several studies on the development of structural entities to treat HIV-TB simultaneously are discussed

O IMPACTO DA FORMAÇÃO DE BIOFILME NA RECORRÊNCIA DA INFECÇÃO POR CLOSTRIDIOIDES DIFFICILE: UM ESTUDO COMPARATIVO DE CEPAS TOXIGÊNICAS MLST CLADO 2

Introdução/objetivo: Clostridioides difficile é a principal causa de diarreia associada ao uso de antibióticos relacionada a assistência à saúde. Um desafio no tratamento da infecção por C. difficile é a capacidade dessa bactéria em formar biofilmes, um mecanismo de virulência crítico por promover resistência a antibióticos e, consequentemente, maior recorrência da doença. Nesse estudo in vitro, o objetivo foi comparar a capacidade de formação de biofilme de cepas MLST Clado 2: ICC-45 (ribotipo SLO231/UK[CE]821) isolada no Brasil, e duas cepas epidêmicas: NAP1/027/ST01 (LIBA5756), isolada em um surto na Costa Rica e a cepa epidêmica de referência NAP1/027/ST01 (R20291). Além disso, a cepa não toxigênica ATCC700057 foi incluída como controle. Métodos: A capacidade das cepas de formar biofilme foi avaliada por coloração com cristal violeta. Além disso, as amostras foram coradas com Film Tracer biofilm matrix (Invitrogen®) e a espessura da matriz do biofilme foi medida usando microscopia confocal. A arquitetura da matriz foi analisada por Microscopia Eletrônica de Varredura (MEV). A expressão dos genes de virulência (tcdA, tcdB, tcdC, cdtB, spo0A, slpA, cwp66 e cwp84) foi examinada por RT-qPCR. Investigou-se ainda o efeito dos antibióticos Metronidazol (MTZ) e Vancomicina (VAN) no crescimento do biofilme. Resultados: Todas as cepas testadas mostraram capacidade de formar biofilmes moderados (1,13,5). Após 72h, a biomassa do biofilme das cepas epidêmicas NAP1/027/ST01 (LIBA5756 e R20291) foi significativamente maior do que os biofilmes ICC-45 e ATCC 700057, o que foi confirmado por MEV e confocal. As cepas R20291 e LIBA 5756 apresentaram uma expressão mais elevada dos genes tcdA, tcdB, tcdC, cdtA, slpA e spo0A em comparação com a cepa ICC-45. Não foram observadas diferenças significativas na expressão de cdtB, cwp66 e cwp84. Quanto ao efeito dos antibióticos, tanto a VAN quanto o MTZ inibiram a formação de biofilme nas cepas epidêmicas. No entanto, na linhagem ICC-45, as concentrações MIC de VAN e MIC e 4MIC de MTZ não inibiram a formação de biofilme. Conclusão: Os três isolados MLST Clado 2, de diferentes ribotipos, são bactérias formadoras de biofilmes competentes, indicando suas capacidades de induzir a recorrência da infecção por C. difficile, tornando o tratamento desafiador. Esses dados evidenciam a importância da vigilância epidemiológica voltada para a emergência de cepas resistentes e causadoras de recidivas diante de um mundo globalizado

Histoplasmosis in HIV-positive patients in Ceara, Brazil: clinical-laboratory aspects and in vitro antifungal susceptibility of Histoplasma capsulatum isolates

This study contains a descriptive analysis of histoplasmosis in AIDS patients between 2006 and 2010 in the state of Ceara, Brazil. Additionally, the in vitro susceptibility of Histoplasma capsulatum isolates obtained during this period was assessed. We report 208 cases of patients with histoplasmosis and AIDS, describing the epidemiological, clinical, laboratory and therapeutic aspects. the in vitro antifungal susceptibility test was carried out by the microdilution method, according to Clinical and Laboratory Standards Institute, with H. capsulatum in the filamentous and yeast phases, against the antifungals amphotericin B, fluconazole, itraconazole, voriconazole and caspofungin. in 38.9% of the cases, histoplasmosis was the first indicator of AIDS and in 85.8% of the patients the CD4 cell count was lower than 100 cells/mm(3). the lactate dehydrogenase levels were high in all the patients evaluated, with impairment of hepatic and renal function and evolution to death in 42.3% of the cases. the in vitro susceptibility profile demonstrated there was no antifungal resistance among the isolates evaluated. There was a significant increase in the number of histoplasmosis cases in HIV-positive patients during the period surveyed in the state of Ceara, northeastern Brazil, but no antifungal resistance among the recovered isolates of H. capsulatum. (C) 2012 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Fed Ceara, Specialized Med Mycol Ctr, Fortaleza, Ceara, BrazilUniv Fed Ceara, Postgrad Program Med Microbiol, Fortaleza, Ceara, BrazilUniv Fed Ceara, Postgrad Program Med Sci, Fortaleza, Ceara, BrazilUniv Estadual Ceara, Postgrad Program Vet Sci, Fortaleza, Ceara, BrazilUniv Fed Ceara, Dept Stat & Appl Math, Fortaleza, Ceara, BrazilHosp Sao Jose, Fortaleza, Ceara, BrazilUniversidade Federal de São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo, São Paulo, BrazilCNPq: 562296/2010-7CNPq: 552161/2011-0CNPq: 304779/2011-3CAPES: 2103/2009Web of Scienc