Antifungal activity of 2-chloro-N-phenylacetamide: a new molecule with fungicidal and antibiofilm activity against fluconazole-resistant Candida spp / Atividade antifúngica de 2-cloro-N-fenilacetamida: uma nova molécula com atividade fungicida e anti-biofilme contra Candida spp. resistentes a fluconazol

In the current context of emerging drug-resistant fungal pathogens such as Candida albicans and Candida parapsilosis, discovery of new antifungal agents is an urgent matter. This research aimed to evaluate the antifungal potential of 2-chloro-N-phenylacetamide against fluconazole-resistant clinical strains of C. albicans and C. parapsilosis. The antifungal activity of 2-chloro-N-phenylacetamide was evaluated in vitro by the determination of the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), inhibition of biofilm formation and its rupture, sorbitol and ergosterol assays, and association between this molecule and common antifungal drugs, amphotericin B and fluconazole. The test product inhibited all strains of C. albicans and C. parapsilosis, with a MIC ranging from 128 to 256 µg.mL-1, and a MFC of 512-1,024 µg.mL-1. It also inhibited up to 92% of biofilm formation and rupture of up to 87% of preformed biofilm. 2-chloro-N-phenylacetamide did not promote antifungal activity through binding to cellular membrane ergosterol nor it damages the fungal cell wall. Antagonism was observed when combining this substance with amphotericin B and fluconazole. The substance exhibited significant antifungal activity by inhibiting both planktonic cells and biofilm of fluconazole-resistant strains. Its combination with other antifungals should be avoided and its mechanism of action remains to be established.

No atual contexto de patógenos fúngicos resistentes emergentes tais como Candida albicans e Candida parapsilosis, a descoberta de novos agentes antifúngicos é uma questão urgente. Esta pesquisa teve como objetivo avaliar o potencial antifúngico da 2-cloro-N-fenilacetamida contra cepas clínicas de C. albicans e C. parapsilosis resistentes a fluconazol. A atividade antifúngica da substância foi avaliada in vitro através da determinação da concentração inibitória mínima (CIM), concentração fungicida mínima (CFM), ruptura e inibição da formação de biofilme, ensaios de sorbitol e ergosterol, e associação entre esta molécula e antifúngicos comuns, anfotericina B e fluconazol. O produto teste inibiu todas as cepas de C. albicans e C. parapsilosis, com uma CIM variando de 128 a 256 µg.mL-1, e uma CFM de 512-1,024 µg.mL-1. Também inibiu até 92% da formação de biofilme e causou a ruptura de até 87% de biofilme pré-formado. A 2-cloro-N-fenilacetamida não promoveu atividade antifúngica pela ligação ao ergosterol da membrana celular fúngica, tampouco danificou a parede celular. Antagonismo foi observado ao combinar esta substância com anfotericina B e fluconazol. A substância exibiu atividade antifúngica significativa ao inibir tanto as células planctônicas quanto o biofilme das cepas resistentes ao fluconazol. Sua combinação com outros antifúngicos deve ser evitada e seu mecanismo de ação deve ser estabelecido.

Optimization of the ellagic acid synthesis process at the bioreactor level using non-conventional yeasts

Abstract Ellagic acid (EA) is a phenolic biomolecule. For its biosynthesis, a source of ellagitannins is required, such as strawberries and yeasts, as precursors of the tannase and ß-glucosidase enzymes responsible for hydrolysis of ellagitannins. Two experimental mixture designs were applied., varying the yeast concentration and the number of ellagitannins in the culture medium, evaluating the enzymatic activity and ellagic acid biosynthesis. Aiming to find the optimal compositions of the non-conventional yeasts assessed in the research to biosynthesize ellagic acid feasibly and efficiently using a response surface performing the statistical analysis in the StatGraphics® program for obtaining a higher yield and optimizing the ellagic acid synthesis process, the results indicate that the strains Candida parapsilosis ITM LB33 and Debaryomyces hansenii ISA 1510 have a positive effect on the synthesis of ellagic acid, since as its concentration increases in the mixture the concentration of ellagic acid in the medium also increases; on the other hand, the addition of Candida utilis ITM LB02 causes a negative effect, resulting in the compositions of 0.516876, 0.483124 and 2.58687E-9 respectively, for a treatment under the same conditions, an optimal value of ellagic acid production would be obtained. With an approximate value of 7.33036 mg/mL

Biological activities of essential oils from six genotypes of four Ocotea species

Abstract Essential oils from four Ocotea species collected in southern Brazil were evaluated for chemical composition using gas chromatography coupled with mass spectrometry. The primary compound identified in O. acutifolia essential oil was an unsaturated tetracyclic diterpene, phyllocladene (67.7%), followed by a sesquiterpene hydrocarbon, ß-selinene (18.0%). The sesquiterpene fraction was predominant in oils from two collections of O. puberula; ß-caryophyllene (25.2%) and globulol (22.6%) were the major compounds identified in collections 1 and 2, respectively. O. silvestris essential oil contained predominantly germacrene D and bicyclogermacrene. These compounds were also predominant in essential oil from O. indecora leaves collected from shady habitats. By contrast, essential oil extracted from O. indecora grown under direct sunlight contained mainly oxygenated sesquiterpenes, such as guaiol (30.2%), α-eudesmol (27.6%), and ß-eudesmol (12.7%). Chemotaxis assays showed that Ocotea essential oils had no significant inhibitory activity on leukocyte migration compared with a chemotactic stimulant (lipopolysaccharide from Escherichia coli). However, the oils exhibited antifungal activity against Candida parapsilosis, with a minimum inhibitory concentration of 500 µg/mL. To our knowledge, this is the first study to investigate the in vitro antifungal and antichemotactic activities of essential oils from Ocotea species native to southern Brazil

Candida parapsilosis Infection after Lumbosacral Arthrodesis with a PEEK TLIF Interbody Fusion Device: Case Report / Infecção por Candida parapsilosis em pós-operatório de artrodese lombossacra com dispositivo de fusão intersomático TLIF em PEEK: Relato de caso

Abstract Spondylodiscitis is an uncommon but serious complication after spine surgeries, and its main etiologic agent is Staphylococcus aureus. Fungal infections are rare and mostly caused by Candida albicans. We report the clinical case of a 69-year-old male patient who underwent a L2-S1 arthrodesis for degenerative scoliosis correction. He presented an infection 2.5 months after the procedure, a spondylodiscitis at L5-S1 levels, caused by Candida parapsilosis. The treatment consisted of surgical material removal, tricortical iliac graft placement in an anterior approach (L5-S1), lumbopelvic fixation (from T10 to the pelvis) in a posterior approach, and drug treatment with anidulafungin and fluconazole. This last medication was administered for 12 months, with good clinical outcomes.

Resumo As espondilodiscites são complicações infrequentes, porém graves em pós-operatórios de cirurgias da coluna vertebral, tendo como principal agente etiológico o Staphylococcus aureus. As infecções fúngicas são raras, sendo a Candida albicans a principal representante desse grupo. Relatamos o caso clínico de um paciente do sexo masculino, 69 anos, operado com artrodese de L2 a S1 para correção de escoliose degenerativa. O paciente apresentou quadro clínico infeccioso 2 meses e meio após o procedimento, relacionado à espondilodiscite L5-S1, causada por Candida parapsilosis. O tratamento consistiu na remoção do material cirúrgico, colocação de enxerto tricortical de ilíaco pela via anterior (L5-S1) e fixação lombopélvica (de T10 à pelve) pela via posterior, além de iniciar o tratamento medicamentoso com anidulafungina e fluconazol, mantendo essa última medicação por 12 meses, com boa evolução clínica.

Heterogeneidad del tratamiento para cándida parapsilosis en endocarditis micótica de válvula nativa / Types of treatments for native-valve endocarditis due to candida parapsilosis

Introducción: las infecciones por cándida en el ámbito intrahospitalario han ido en ascenso en las últimas décadas en especial en las unidades de cuidado intensivo (UCI), dado el uso cada vez más frecuente de antibióticos de amplio espectro y de procedimientos invasivos tanto diagnósticos como terapéuticos. Hoy se consideran las especies de cándida como la cuarta causa de infección del torrente sanguíneo en los Estados Unidos de Norteamérica, con alto riesgo de complicaciones que incluyen endocarditis, trombosis y embolismo séptico al sistema nervioso central (SNC) entre otros. En relación con la endocarditis por especies de cándida, se ha considerado de mal pronóstico por el alto riesgo de afectación al SNC, por lo que se ha recomendado el manejo quirúrgico como piedra angular de su tratamiento. Presentación del caso: se describe el caso clínico de un paciente con endocarditis y con candidemia por Candida parapsilosis a quien se le realizó manejo médico.

Nosocomial Candida infections have increased in the last decades particularly in the intensive care units (ICU) due to the rise in broad-spectrum antibiotics usage and invasive diagnostic and therapeutic procedures usage. Today, Candida species are recognized as the fourth causative organisms of endovascular infection in the United States of America posing a high risk of endocarditis, thrombosis and septic embolization to the central nervous system (CNS). Endocarditis due to Candida species is associated with a poor prognosis because of the increased risk of CNS involvement for which surgical approach has been recommended as the cornerstone of therapy. We hereby report a patient with endocarditis and candidemia due to Candida parapsilosis, who received medical management.

Antimicrobial activity of the edible mushroom Pleurotus eryngii(DC. ) Quél grown in liquid medium

Edible mushrooms have a number of medicinal properties and this study aimed to investigate the antimicrobial activity of Pleurotus eryngiiDPUA1816 in metabolic broths after being grown in submerged cultivation. Mycelial fragments of pure P. eryngiiculture was inoculated in sweet potato culture medium and incubated at 150 rpm for 15 days at 25°C. Pleurotuseryngiiwas also cultivated for 18 days under the same conditions, the mycelial biomass was separated by filtration for quantification. The supernatant was used in the diffusion test in agar and performed against Escherichia coliCCCD-E005, Staphylococcus aureus CCCD-S009, Pseudomonas aeruginosaCCCD-P004, Candida albicansCCCD-CC001, Candida parapsilosis CCCD-CC004 and Candida tropicalisCCCD-CC002. The samples showed no inhibitory activity against bacteria, however they showed some activity againstC. albicans(12.17 mm), C. parapsilosis(27.67 mm) and C. tropicalis(13.67 mm). After being cultivated for 18 days, P. eryngiiwas able to inhibit all yeasts after 12 days of culture, with an inhibition halo of 29.33 mm at 16 days against C. parapsilosis. This study demonstrates the antifungal potential filtered liquids from P.eryngiicultivated in purple-skinned sweet potato culture medium, which suggests the possibility of the use of this species by the pharmaceutical industry as a natural source of biological action.

Caracterización de Candida spp. aisladas a partir de urocultivos en la ciudad de Medellín / Characterization of Candida spp isolated from urine cultures of Medellín

Resumen Candida spp. es un agente etiológico importante en infecciones del tracto urinario, principalmente en población con terapia antimicótica de amplio espectro y con catéteres urinarios. Candida albicans es la especie más frecuente, pero otras especies han surgido como patógenos emergentes. En este trabajo se recolectaron aislamientos de Candida spp. de urocultivos de pacientes que consultaron en Dinamica IPS entre enero 2016 y noviembre 2017. Para estimar la frecuencia de las especies y observar los patrones de sensibilidad, se realizó la identificación fenotípica y su perfil de sensibilidad con el sistema comercial Vitek 2® (BioMérieux, Inc.), adicionalmente se evaluaron mediante análisis de las secuencia y filogenética ITS1-5.8S-ITS2. En el estudio se incluyeron 78 aislamientos de Candida spp. Las frecuencias de especies de Candida identificadas empleando las herramientas moleculares fueron: C. albicans (38,5%), C. tropicalis (23,1%), C. glabrata (21,8%), C. parapsilosis (10,3%), C. metapsilosis y C. krusei (2,5%) y C. guillermondi (1,3%). La identificación por métodos moleculares y por el sistema Vitek 2 fue: C. albicans (93,3%), C. glabrata (94,1%), C. tropicalis (83,3%), C. parapsilosis (75%) C. guilliermondii y C. krusei (100%). La sensibilidad de todos los aislamientos al fluconazol fue 93,6%.

Abstract Candida spp is an important etiologic agent in urinary tract infections, mainly in patients in broad-spectrum antifungal therapy, with urinary catheters. Candida albicans is the most frequent specie; but other species have arised as emerging pathogens. In this study, isolates of Candida spp. of urine cultures from patients who consulted in Dinamica IPS between January 2016 and November 2017 were evaluated. To estimate the frequency of the species and to observe the sensitivity patterns, the phenotypic identification and its sensitivity profile was performed employed the Vitek 2® commercial system. (BioMérieux, Inc) In addition the isolates were evaluated by sequence analysis and phylogenetics ITS1-5.8S-ITS2. This study included 78 isolates of Candida spp. The frequencies of Candida species identified using the molecular tools were: C. albicans (38.5%), C. tropicalis (23.1%), C. glabrata (21.8%), C. parapsilosis (10.3%), C. guillermondi (1.3%) and C. metapsilosis and C. krusei (2.5%). The identification by molecular methods and by Vitek 2 system were: C. albicans (93.3%), for C. glabrata (94.1%), C. tropicalis (83.3%), C. parapsilosis (75%) and 100% for C. guilliermondii and C. krusei.. fluconazole sensitivity of all isolates was 93.6%

Evaluación de susceptibilidad en cándidas spp por colorimetría obtenida en gestantes de un hospital obstétrico / Susceptibility evaluation in candida spp by colorimetry obtained in pregnant women from an obstetric hospital / Avaliação da suscetibilidade em cândida spp por colorimetria obtida em gestantes de um hospital obstétrico

INTRODUCCIÓN: las infecciones fúngicas ocasionadas por levaduras del género Cándida son extremadamente comunes en mujeres de edad reproductiva, y constituyen un motivo de atención medica de salud. OBJETIVO: evaluar la susceptibilidad de Cándidas spp, mediante el método colorimétrico (Integral Yeast System Plus). MÉTODO: fue de tipo descriptivo, transversal; se recopiló información mediante observación directa en campo y el análisis documental para obtener información bibliográfica de tipo secundaria. RESULTADOS: de los 72 casos encontrados de Cándida Albicans revela que son susceptibles a la anfotericina B (2ug/ml); de los 5 casos encontrados de Cándida Krusei revela que son sensibles a la Anfotericina B (2ug/ml); De 1 caso encontrado de Cándida Parapsilosis revela sensibilidad en la Nistatina (1.25ug/ml). En este estudio la prevalencia de la infección por Cándida fue del (44.98%). CONCLUSIONES: Cándida Albicans fue la especie más común aislada en las mujeres embarazadas representando un 72%, En la evaluación de la susceptibilidad a través del kit Integral System Yeast Plus se obtuvo que Cándida Albicans es susceptible a Anfotericina B, Flucitosina entre otros, en Cándida Glabrata se obtuvo que es sensible a la Nistatina, Anfotericina B, susceptible entre otros, en Cándida Krusei se obtuvo que es sensible a la Anfotericina B, Clotrimazol, Miconazol, susceptibles a la Nistatina, Voriconazol y resistente a la Flucitosina, Ketoconazol, Itraconazol y Fluconazol.

INTRODUCTION: fungal infections caused by yeast of the genus Candida are extremely common in women of reproductive age, and constitute a reason for medical health care. OBJECTIVE: to evaluate the susceptibility of Candida spp, using the colorimetric method (Integral Yeast System Plus). METHOD: it was descriptive, transversal; Information was collected through direct observation in the field and documentary analysis to obtain secondary bibliographic information. RESULTS: of the 72 cases found, Candida Albicans reveals that they are susceptible to amphotericin B (2ug / ml); of the 5 cases found, Candida Krusei reveals that they are sensitive to Amphotericin B (2ug / ml); Of 1 case found of Candida Parapsilosis reveals sensitivity in Nystatin (1.25ug / ml). In this study, the prevalence of Candida infection was (44.98%). CONCLUSIONS: Candida Albicans was the most common species isolated in pregnant women, representing 72%. In the evaluation of susceptibility through the Integral System Yeast Plus kit it was obtained that Candida Albicans is susceptible to Amphotericin B, Flucytosine among others, in Candida Glabrata was obtained that it is sensitive to Nystatin, Amphotericin B, susceptible among others, in Candida Krusei it was obtained that it is sensitive to Amphotericin B, Clotrimazole, Miconazole, susceptible to Nystatin, Voriconazole and resistant to Flucytosin, Ketoconazole, Itraconazole and Fluconazole.

INTRODUÇÃO: as infecções fúngicas causadas por leveduras do gênero Candida são extremamente comuns em mulheres em idade reprodutiva e constituem motivo de cuidados médicos. OBJETIVO: avaliar a suscetibilidade de Candida spp, por meio do método colorimétrico (Integral Yeast System Plus). MÉTODO: foi descritivo, transversal; as informações foram coletadas por meio de observação direta em campo e análise documental para obtenção de informações bibliográficas secundárias. RESULTADOS: Dos 72 casos encontrados, Cândida Albicans revelou ser suscetíveis à anfotericina B (2ug /ml); dos 5 casos encontrados, Candida Krusei revela que são sensíveis à Anfotericina B (2ug / ml); de 1 caso encontrado de Candida Parapsilosis revela sensibilidade na Nistatina (1,25ug / ml). Neste estudo, a prevalência de infecção por Candida foi (44,98%). CONCLUSÕES: Cândida Albicans foi a espécie mais comum isolada em gestantes, representando 72%. Na avaliação da susceptibilidade através do kit Integral System Yeast Plus foi obtido que Candida Albicans é suscetível à Anfotericina B, Flucitosina entre outras, em Cândida Glabrata foi obtido que é sensível a Nistatina, Anfotericina B, suscetível entre outras, em Candida Krusei foi obtido que é sensível a Anfotericina B, Clotrimazol, Miconazol, suscetível a Nistatina, Voriconazol e resistente a Flucitosina, Cetoconazol, Itraconazol e Fluconazol.

Estudo do potencial antifúngico e do mecanismo de ação do timol contra cepas de Candida parapsilosis resistentes ao fluconazol e a anfotericina B / Study of the antifungal potential and mechanism of action of thymol against strains of Candida parapsilosis resistant to fluconazole and amphotericin B

Objetivo: investigar a suscetibilidade de cepas fúngicas de Candida parapsilosis isoladas de sangue humano frente ao timol, bem como seu mecanismo de ação. Metodologia: foram utilizadas técnicas de microdiluição em placas de 96 poços para determinar a concentração inibitória mínima (CIM) e concentração fungicida mínima (CFM). Além disso, foram realizados testes com o sorbitol e o ergosterol para investigar a ação do timol na parede e na membrana celular fúngica respectivamente. Resultados: nos testes de CIM e CFM, foi observado que as cepas de C. parapsilosis são resistentes ao fluconazol e a anfotericina B, no entanto, o timol desempenhou efeito fungicida com razão CFM/CIM entre 1 e 2. Além disso, a CIM do timol não aumentou quando o sorbitol ou o ergosterol foi adicionado no meio, sugerindo fortemente que este monoterpeno não age na parede celular fúngica ou por ligação ao ergosterol na membrana plasmática. Conclusão: portanto, esses resultados contribuem para a elucidação do mecanismo de ação do timol, sugerindo outros possíveis alvos de interação fármaco-receptor. No entanto, mais investigações de caráter enzimático e molecular em modelos in vitro são necessários para que se possa elucidar completamente o modo de ação desse promissor monoterpeno.

Objective: to investigate the susceptibility of fungal strains of Candida parapsilosis isolated from human blood against thymol, as well as its mechanism of action. Methodology: microdilution techniques were used in 96-well plates to determine minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). In addition, tests were performed with sorbitol and ergosterol to investigate the action of thymol on the wall and on the fungal cell membrane respectively. Results: in the CIM and CFM tests, it was observed that C. parapsilosis strains are resistant to fluconazole and amphotericin B, however, thymol had a fungicidal effect with MFC/MIC ratio between 1 and 2. In addition, thymol MIC did not increase when sorbitol or ergosterol was added in the medium, strongly suggesting that this monoterpene does not act on the fungal cell wall or by binding to ergosterol on the plasma membrane. Conclusion: therefore, these results contribute to the elucidation of the mechanism of action of thymol, suggesting other possible targets of drug-receptor interaction. However, further investigations of enzymatic and molecular character in in vitro models are necessary to fully elucidate the mode of action of this promising monoterpene.

Fungal keratitis management in a referral cornea center in Brazil / Manejo de ceratites fúngicas em centro de referência no Brasil

Abstract Purpose: To report etiological diagnosis, predisposing risk factors, therapeutic strategies and visual outcome of patients treated at the Department of Ophthalmology of Federal University of São Paulo. Methods: This is a retrospective, descriptive, and observational study from medical and laboratory records of the Department of Ophthalmology of Federal University of São Paulo, including all patients with culture proven fungal keratitis in 5 years, from October 2012 through October 2017. Results: There were 2260 fungi microbiologic test requests. Of these, 140 samples had positive cultures for fungi and sixty-six patients were followed at our clinic. Forty-five patients (68.2%) were men, and the mean age was 48.06 (±17.39) years. Fusarium spp. was the most frequently isolated fungus (32 cases; 48.5%), followed by Candida parapsilosis (12 cases; 18.2%). Thirty-four patients (51.5%) underwent intracameral injection of amphotericin B (5 µg per 0.1 ml). In 11 patients (32.3%), infection was eradicated after intracameral amphotericin B associated to topical antifungal treatment and, in 23 patients (67.7%), therapeutic keratoplasty was needed. No complication related to intracameral amphotericin B injection was observed in this series. Forty-three patients (65.1%) ended up with therapeutic keratoplasty. Three patients (4.5%) evolved to evisceration or enucleation. At the last follow-up visit, 53 patients (80.3%) had visual acuity worse than 20/200. Conclusion: Despite current antifungals drugs and distinct administration strategies, fungal keratitis remains challenging. Delayed antifungal therapy may explain poor clinical outcomes. Intracameral amphotericin B associated to topical antfungal treatment seems to be a safe and helpful alternative for non-responsive fungal keratitis. But it is important to formulate other treatment strategies, hence to improve patients' outcomes, since most patients ended-up with significant visual impairment even after current treatment.

Resumo Objetivo: Descrever diagnósticos etiológicos, fatores de risco, estratégias terapêuticas e resultados visuais de pacientes com ceratite fúngica tratados no Departamento de Oftalmologia da Universidade Federal de São Paulo. Métodos: Trata-se de um estudo retrospectivo, descritivo e observacional, a partir da análise de prontuários médicos e laboratoriais do Departamento de Oftalmologia da Universidade Federal de São Paulo, incluindo todos os pacientes com ceratite fúngica comprovada por cultura no período de outubro de 2012 a outubro de 2017. Resultados: Foram realizadas 2260 solicitações de testes microbiológicos. Destas, 140 amostras apresentaram culturas positivas para fungos, e 66 pacientes foram acompanhados em nosso serviço. Quarenta e cinco pacientes (68,2%) eram do sexo masculino, e a média de idade foi de 48,06 (± 17,39) anos. Fusarium spp. foi o fungo mais freqüentemente isolado (32 casos; 48,5%), seguido por Candida parapsilosis (12 casos; 18,2%). Trinta e quatro pacientes (51,5%) foram submetidos à injeção intracameral de anfotericina B (5 µg por 0,1 ml). Destes, 11 pacientes (32,3%) tiveram a infecção erradicada. Nos outros 23 pacientes (67,7%), o transplante terapêutico foi necessário. Nenhuma complicação relacionada à injeção intracameral de anfotericina B foi observada neste estudo. No total, 43 pacientes (65,1%) evoluíram para transplante terapêutico, e 3 pacientes (4,5%) foram submetidos à evisceração ou enucleação. Cinquenta e três pacientes (80,3%) apresentaram acuidade visual final pior que 20/200. Conclusões: Apesar dos diversos medicamentos antifúngicos atuais e vias de administração, o tratamento das ceratites fúngicas permanece desafiador. O atraso no início do tratamento adequado pode justificar o desfecho clínico desfavorável de grande parte dos pacientes. A injeção intracameral de anfotericina B mostrou-se uma alternativa terapêutica segura para ceratites fúngicas refratárias. Mas outras estratégias de tratamento devem ser formuladas, visando melhorar os resultados visuais dos pacientes.

In vitro characterization of virulence factors among species of the Candida parapsilosis complex

Abstract INTRODUCTION: Candida parapsilosis complex species differ from each other with regard to their prevalence and virulence. METHODS: The hydrolytic enzyme activity, biofilm production, and adhesion to epithelial cells were analyzed in 87 C. parapsilosis complex strains. RESULTS: Among the studied isolates, 97.7%, 63.2%, and 82.8% exhibited very strong proteinase, esterase, and hemolysin activity, respectively. All the C. parapsilosis complex isolates produced biofilms and presented an average adherence of 96.0 yeasts/100 epithelial cells. CONCLUSIONS: Our results show that Candida parapsilosis complex isolates showed different levels of enzyme activity, biofilm production, and adhesion to epithelial cells.

Nitrogen source and pH interact and modulate lipase secretion in a non-clinical strain of Candida parapsilosis

Lipases (E.C. 3.1.1.3) are serine-hydrolases, and act on long chain fatty acid ester bonds. They exhibit specific and enantioselective activities, which are desirable for many industrial applications. This study aimed at screening and optimizing the production of lipases by wild yeast strains from a variety of substrates, as well as characterizing the enzyme. An initial selection was made in oxygenated oil-supplemented minimum medium, and the enzymatic activity of the supernatant was tested over p-nitrophenyl palmitate. One-hundred and twenty-four yeast strains from different substrates were tested, and twenty-three showed significantly higher lipolytic activity (p<0.01). One yeast in particular, QU110, showed best lipase production and therefore was selected for the optimization and characterization processes. This yeast exhibits enzyme secretion in initial pH 6.0, with olive oil and tryptone as carbon and nitrogen sources, respectively. There was a strong interaction between nitrogen source and initial pH, and pH 9.0seems to inhibit enzyme secretion. The crude enzyme (cell-free supernatant) shows stability in surfactants and n-hexane, but not in ethanol or methanol. A Response Surface Model was created and optimal enzyme activity conditions were observed at 36°C and pH 8.0. The lipase is appropriate for transesterification reactions, as the enzyme is more stable in strong apolar solvents than moderately apolar ones. Also, secretion by pH was not reported elsewhere, which should be further investigated and contribute for other yeast bioprocesses as well.

Biofilm formation by blood isolates of candida parapsilosis sensu stricto in the presence of a hyperglycidic solution at comparable concentrations of total parenteral nutrition

Abstract INTRODUCTION: Administration of total parenteral nutrition (TPN) via catheters increases the risk for candidemia from Candida parapsilosis. METHODS: C. parapsilosis sensu stricto blood isolates were evaluated for ability total biomass biofilm formation and morphogenesis in presence of glucose at TPN equivalent concentrations. RESULTS: Biofilms were increased at high glucose concentrations (25-30%) compared to the control medium. Significant increase in filamentous forms was observed in cultures with 30% glucose. CONCLUSIONS: Biofilm formation by C. parapsilosis sensu stricto in hyperglycidic medium may contribute to its pathogenic potential for fungemia related to TPN catheters.

Complicaciones de la artroplastía de hombro: reporte de un caso / Shoulder arthroplasty complications: case report

Resumen: Antecedentes: La infección e inestabilidad se encuentran entre las complicaciones de más difícil manejo en la cirugía protésica de hombro. La inestabilidad acumula hasta la mitad de los casos, mientras que la infección por hongos representa menos de 1% del total de infecciones y pueden causar daño severo a nivel óseo y de partes blandas. Métodos: En este caso clínico se presenta una infección fúngica por Candida parapsilosis en una hemiartroplastía de hombro indicada por una fractura de húmero proximal. Se dio tratamiento quirúrgico en dos tiempos y administración de fluconazol durante ocho semanas previo al segundo tiempo quirúrgico. En la revisión se colocó una prótesis reversa como tratamiento definitivo; sin embargo, el paciente sufrió varios episodios de luxación en el postoperatorio. Fue necesaria una segunda cirugía de revisión protésica para sustituir por componentes de mayor estabilidad. Durante la misma, se extrajeron muestras que fueron analizadas, obteniéndose resultados negativos para infección. Resultados: Actualmente tras tres años de seguimiento, el paciente se encuentra bien, sin limitaciones en su labor diaria y tiene un Constant Score de 50.5. Discusión: Reportamos nuestra experiencia en esta situación excepcional. A nuestro conocer, éste es uno de los primeros casos en los que la infección fúngica y la luxación protésica coinciden en un mismo paciente. Esta situación es un reto para el cirujano, el cual tiene que tratar las dos complicaciones prácticamente a la vez. No se dispone de una evidencia científica para establecer un criterio unificado para el tratamiento de las complicaciones tras cirugía de revisión de artroplastía de hombro, especialmente en la prótesis reversa o megaprótesis.

Abstract: Background: Infection and instability are the complications of prosthetic shoulder surgery of more difficult management. Instability builds up to half of the cases, while the fungal infection accounts for less than 1% of all and can cause severe damage to bone and soft tissue. Methods: In this case clinical fungal infection by Candida parapsilosis is presented in a shoulder hemiarthroplasty indicated by a fracture of the proximal humerus. He received surgical treatment in two-stages and administration of fluconazole for eight weeks prior to the second surgical time. In revision surgery was placed a reverse prosthesis as definitive treatment; however, the patient suffered several episodes of dislocation in the postoperative period; a second revision prosthetic surgery was necessary to use more stable components. During this surgery, we extracted samples that were analyzed, obtaining negative results for infection. Results: Now after three years of follow-up, the patient is well, no limitations in their daily work and has a Constant Score of 50.5. Discussion: We report our experience in this exceptional situation. We know, this is one of the first cases where the fungal infection and dislocation prosthetics in the same patient meet. This situation is a challenge for the surgeon, which has to treat complications two practically at the same time; we do not have scientific evidence to establish a criterion unified for the treatment of complications after surgery for revision of arthroplasty of the shoulder, especially in reverse or mega-denture prosthesis.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification

Abstract In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.

Capacidad de formación de biopelículas en especies del género Candida de procedencia clínica / Capability of biofilm formation in Candida genus species of clinical origin

El objetivo de la investigación fue evaluar la capacidad de formación de biopelículas en especies del género Candida provenientes de muestras clínicas con procesos infecciosos. La muestra estuvo constituida por 70 aislados obtenidos de laboratorios clínicos privados. Para la identificación de las especies se utilizó la Prueba de tubo germinativo y la técnica de microcultivo en agar harina de maíz con tween 80, mientras que la cuantificación de la capacidad de formación de biopelículas se realizó por la técnica de microplaca de poliestireno. Entre los resultados obtenidos en la investigación las especies aisladas fueron: Candida glabrata (34%), Complejo Candida albicans (30%), Complejo Candida parapsilosis (27%) y Candida tropicalis (9%), de las cuales 41 aislados (59%) formaron biopelículas, resultando el Complejo Candida albicans y Candida glabrata las especies con mayor grado de formación de biopelículas con 29% cada una, seguida de Complejo Candida parapsilosis con 27% y Candida tropicalis con 15%. Las cepas que tuvieron mayor formación de biopelículas provenían de orina, seguidas de secreción ótica, secreción vaginal y sangre, sin embargo al aplicar la prueba de Chi-cuadrado (x2) de Pearson se determinó que no existe asociación estadísticamente significativa entre la capacidad de formación de biopelículas y el grado de formación con la procedencia clínica así como tampoco hay asociación entre la capacidad de formación de biopelículas y grado de formación con la especie involucrada (p > 0,05)

The aim of this research was to evaluate the capability of biofilm formation in Candida genus species from clinical samples with infectious conditions. The sample consisted of 70 isolates obtained from private clinical laboratories. Germ tube test and agar cornmeal with tween 80 microculture technique were used as species identification, meanwhile, quantification of biofilm formation capacity was performed by the polystyrene microplate technique. Among the results obtained in the research, isolated species were: Candida glabrata (34%), Candida albicans Complex (30%), Candidaparapsilosis Complex (27%) and Candida tropicalis (9%), of which 41 isolates (59%) formed biofilms, resulting Candida albicans and Candida glabrata Complex species with the highest degree of biofilms with 29% each, followed by Candida parapsilosis Complex with 27% and Candida tropicalis with 15%. Strains with more biofilm formation came from urine, followed by ear discharge, vaginal secretion and blood. However, by applying the Chi squaretest the Pearson, it was determined that there was no statistically significant association between thecapability of biofilm formation and the formation degree with clinical origin, neither there is association between the ability of biofilm formation and the formation degree with the species involved (p> 0.05).

Activador tisular del plasminógeno recombinante para el tratamiento de la vegetación intracardíaca en un lactante de muy bajo peso al nacer / Recombinant tissue plasminogen activator treatment for intracardiac vegetation in a very low birth weight infant

Las nuevas opciones de tratamiento prolongan la hospitalización y aumentan las infecciones intrahospitalarias bacterianas y fúngicas, pero también mejoran la sobrevida de los recién nacidos hospitalizados en la unidad de cuidados intensivos neonatales. Las infecciones fúngicas invasivas en neonatos están asociadas con una morbimortalidad significativa. También pueden diseminarse a órganos específicos y causar endocarditis, endoftalmitis, artritis séptica, nefropatía obstructiva y meningitis. En el caso de la endocarditis, se recomiendan tratamientos antimicóticos sistémicos agresivos y, en algunos casos, la intervención quirúrgica del neonato. Informamos el caso de un lactante prematuro, de bajo peso al nacer, con vegetación intracardíaca. Esta es una complicación rara y potencialmente mortal de infecciones fúngicas invasivas. El paciente recibió tratamiento con caspofungina y un activador tisular del plasminógeno recombinante, en vez de una intervención quirúrgica.

Developing treatment options have resulted in prolonged admission and increased bacterial and fungal nosocomial infections as well as improved survival in neonatal intensive care unit. Invasive fungal infections in newborns are associated with significant morbidity and mortality and can cause endorgan dissemination such as endocarditis, endophthalmitis, septic arthritis, obstructive nephropathy and meningitis. Endocarditis requires aggressive systemic antifungal therapy and sometimes surgical intervention in neonates. We report a low birth weight premature infant with intracardiac vegetation that is rare and a life-threatening complication of invasive fungal infections. He was treated with caspofungin and recombinant tissue plasminogen activator in stead of surgical intervention.

Hand hygiene adherence according to World Health Organization Recommendations in a Neonatal Intensive Care Unit / Aderência à higiene das mãos através das recomendações da Organização Mundial de Saúde em uma Unidade de Terapia Intensiva Neonatal

Abstract Objectives: to evaluate the application of hand hygiene technique, according to the World Health Organization (WHO) recommendations, in the neonatal intensive care unit, at a Maternity in Salvador de Bahia, Brazil. Methods: cross-sectional study. Hand hygiene technique by professional category and alcohol solution consumption were systematically registered. For this task an adapted instrument created by the WHO was used and applied using factsheets. The sample was taken from medical physicians, physiotherapists, nurses and nursing technicians. Results: hand hygiene adherence regarding WHO recommendations was deficient in terms of technique and in terms of frequency (adequate technique ranged from 0% to 13.3% between professional categories). Hand hygiene was frequently ignored (27% between physicians and 51.8% between nursing technicians). The moment right after touching surfaces next to patients was the most ignored one. Alcohol gel solution monthly use was only 35% of the expected value for the unit. Conclusions: despite the international investigations and efforts for better results, the adherence and compliance to the hand hygiene guidelines is still deficient and continues to be a major problem.

Resumo Objetivos: avaliar aplicação da técnica de higienização das mãos numa unidade de terapia intensiva neonatal em uma maternidade de Salvador, Bahia, Brasil. Métodos: estudo transversal no qual foi avaliada a técnica de higienização das mãos com água e sabão e álcool gel e o consumo de solução de álcool gel e por categoria profissional. Um instrumento adaptado da OMS foi usado e aplicado usando questionários. A amostra foi composta por médicos, fisioterapeutas, enfermeiros e técnicos de enfermagem. Resultados: a aderência à higienização das mãos foi deficiente em termos de técnica e de frequência (a execução correta da técnica variou de 0 a 13,3% entre as categorias profissionais). A higienização das mãos foi ignorada por 27% dos médicos e 51,8% dos técnicos de enfermagem. Depois de tocar superfícies próximas aos pacientes foi o momento mais ignorado. O uso mensal de álcool gel foi 35% do valor esperado. Conclusões: apesar das investigações internacionais e esforços para melhores resultados, a adesão e o cumprimento das diretrizes de higienização das mãos ainda é deficiente.

Cell surface hydrophobicity and biofilm formation of candida yeast species in different culture media / Hidrofobicidade da superfície celular e formação de biofilme de Candida sp em diferentes meios de cultura

Cell surface adhesion is considered an essential step in the spread, infection and persistence of Candida yeasts in the host. Their ability to adhere on biotic and abiotic surfaces depends on several factors, including hydrophobicity. Once attached, these yeasts are capable of growing in biofilms, which are constituted of structured communities of encapsulated cells within an extracellular matrix, resistant to antifungal agents. In this context, this study aimed to analyze the cell surface hydrophobicity and specific biofilm formation of six Candida strains in different culture media: Sabouraud dextrose broth (SDB), artificial saliva (AS), Roswell Park Memorial Institute medium (RPMI 1640) and N-acetylglucosamine-yeast nitrogen base proline (NYP). Six yeasts of the genus Candida were studied: three C. albicans (Ca): Ca ATCC 10231 and the clinical isolates Ca34 and Ca05 and C. parapsilosis (Cp): Cp ATCC 22019 and the clinical isolates Cp120 and Cp38. Hydrophobicity was calculated as the percentage reduction in turbidity of the aqueous phase, due to the retention of the hydrophobic cells in hydrocarbon by the hydrocarbon-water biphasic assay following the MATH (Microbial Adhesion to Hydrocarbon) method. The biofilm formation index was calculated as the optical density obtained by the growth of the yeasts in the culture media in a polystyrene microtiter plate, subsequently stained with 1% violet crystal. The results showed that hydrophobicity varied according to the media and the yeasts studied, and two of these (Ca34 and Ca10231) presented significant variation between the media. A more hydrophobic character was observed in yeasts grown on RPMI-1640 medium, and those grown on Sabouraud dextrose broth appeared more hydrophilic. The specific biofilm formation index was more intense for RPMI 1640 than in other media, which was expected, due to its ability to induce the transition between yeast-hyphae morphology, which is one of the key factors involved in the adhesion of C. albicans on different surfaces. RPMI 1640 was the best medium for obtaining biofilm in vitro, due to its greater hydrophobicity, which can enhance cell adhesion to the polystyrene plate, and due to its nutrient content, necessary for complete cell growth and biofilm formation.

A adesão é considerada um passo essencial para a disseminação, infecção e persistência das leveduras do gênero Candida nos hospedeiros. A habilidade dessas leveduras em se aderir a superfícies bióticas e abióticas depende de fatores, incluindo a hidrofobicidade da superfície celular. Uma vez aderidas, estas leveduras são capazes de crescer como biofilmes, os quais são caracterizados como comunidades estruturadas de células encapsuladas dentro de uma matriz extracelular, resistentes a drogas antifúngicas. Neste contexto, objetivou-se analisar a hidrofobicidade da superfície celular e a formação específica de biofilme de Candida spp. em diferentes meios de cultura: caldo Sabouraud dextrose (CSD), saliva artificial (SA), Roswell Park Memorial Institute medium (RPMI 1640) e N-acetylglucosamineyeast nitrogen base-proline (NYP). Foram estudadas seis leveduras do gênero Candida, sendo três C. albicans (Ca): Ca ATCC 10231 e os isolados clínicos Ca34 e Ca05 e C. parapsilosis (Cp): Cp ATCC 22019 e os isolados clínicos Cp120 e Cp38. A hidrofobicidade foi calculada como a porcentagem da redução da turbidez da fase aquosa, devido a retenção das células hidrofóbicas no hidrocarboneto pelo método bifásico água-hidrocarboneto MATH (Teste de Adesão Microbiana a Hidrocarbonetos). O índice de formação de biofilme foi calculado com as densidades ópticas obtidas por meio do crescimento das leveduras nos meios de cultura em poços de placa de microtitulação de poliestireno e posteriormente corados com cristal violeta a 1%. Os resultados demonstraram que a hidrofobicidade variou de acordo com os meios e as leveduras estudadas, sendo que duas (Ca34 e Ca10231) apresentaram variação significativa entre os meios. Foi verificado um caráter mais hidrofóbico das células crescidas em RPMI 1640 e mais hidrofílico nas células crescidas em caldo Sabouraud dextrose. A formação específica de biofilme apresentou-se mais intensa em RPMI 1640 do que nos outros meios, o que já era esperado pela sua capacidade de induzir o processo de transição levedura-hifa, o qual é considerado um dos fatores cruciais envolvidos na adesão de leveduras. Com os resultados obtidos, infere-se que o RPMI 1640 é o melhor meio para obtenção de biofilme in vitro, pois as células foram mais hidrofóbicas, o que pode aumentar a adesão das

Identificación molecular de los complejos cryptococcus neoformans y gattii por PCR fingerprinting y PCR-RFLP / Molecular identification of cryptococcus neoformans and gattii complexes by PCR fingerprinting and PCR-RFLP

La criptococosis es una micosis de curso subagudo o crónico causada por levaduras oportunistas del género Cryptococcus, principalmente por las especies de los Complejos C. neoformans y C. gattii. La criptococosis pasó de ser considerada una micosis de aparición esporádica, a posicionarse como una de las primeras afecciones oportunistas asociadas al SIDA con una tasa de infección elevada en estos pacientes quienes con frecuencia desarrollan una meningoencefalitis. En Venezuela poco se conoce sobre los serotipos, susceptibilidad y virulencia de las cepas circulantes. El propósito de este trabajo fue realizar la caracterización molecular de los Complejos Cryptococcus neoformans y gattii, se empleó la PCR-RFLP siguiendo el protocolo descrito por Escandon y col. Se utilizaron 80 cepas, 67 del complejo C.neoformans y 13 del complejo C.gattii, conservadas en la micoteca del INHRR. Se encontró que el tipo molecular más prevalente es el VNI con un 77,2% similar a lo reportado a nivel mundial, en segundo lugar se encuentra el tipo molecular VGII (12,7%), que concuerda con lo reportado para Latinoamérica. Este estudio aporta información epidemiológica sobre los tipos moleculares circulantes y permite de esta manera profundizar en el conocimiento de esta micosis oportunista en Venezuela.

Cryptococcosis is a fungal infection of subacute or chronic course caused by opportunistic yeast genus Cryptococcus, mainly species of Complex C neoformans and C. gattii. Cryptococcosis went from being considered a fungal infection of sporadic occurrence, to position itself as one of the first opportunistic diseases associated with AIDS with a high rate of infection in these patients who often develop meningoencephalitis. In Venezuela little is known about the serotypes, susceptibility and virulence of circulating strains. The purpose of this study was to perform the molecular characterization of the complexes Cryptococcus neoformans and gattii, PCR-RFLP was used following the protocol described by Escandon et al. 80 strains were used, 67 of C. neoformans and 13 of C.gattii complex, preserved in mycology library of INHRR. It was found that the most prevalent molecular type is the VNI with 77.2% similar to that reported worldwide, in second place is the molecular type VGII (12.7%), consistent with that reported for Latin America. This study provides epidemiological information on circulating molecular types and allows deeper understanding of this opportunistic fungal infections in Venezuela.