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.

Antifungal activity of 2-chloro-N-phenylacetamide: a new molecule with fungicidal and antibiofilm activity against fluconazole-resistant Candida spp

Abstract 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.

Resumo 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.

In silico and in vitro analysis of a new potential antifungal substance, 2-Bromo-N-phenylacetamide, against invasive candidiasis isolates

SUMMARY Introduction: invasive candidiasis is related to high rates of morbidity and mortality. There are few classes of drugs available for the treatment of this type of infection and the index of resistant strains is increasing. Such circumstances highlight that the search for new pharmacotherapeutic alternatives is increasingly necessary. This study investigated 2-Bromo-N-phenylacetamide, a substance whose antifungal activity has not yet been reported. Objective: to evaluate its activity against invasive candidiasis isolates, by determining the minimum inhibitory and fungicide concentrations. Methodology: molecular docking was performed to investigate the possible mechanism of action of the substance. The substance was also associated with fluconazole, to assess the viability of the combination in clinical practice. The minimum inhibitory concen trations ranged between 4 to 32 jig/mL, and it acts in a fungicidal way. Results: molec ular docking suggests that 2-Bromo-N-phenylacetamide possibly acts on the fungal plasma membrane. And the association of 2-Bromo-N-phenylacetamide with flucon azole against resistant strains showed an indifferent effect. Conclusion: further studies should be carried out to elucidate the potential of this substance, which may become a future drug candidate to treat invasive candidiasis and other fungal infections.

Introducción: la candidiasis invasiva está relacionada con altas tasas de morbilidad y mortalidad. Hay pocas clases de medicamentos disponibles para el tratamiento de este tipo de infección y el índice de cepas resistentes está aumentando. Tales circunstancias ponen de relieve que la búsqueda de nuevas alternativas farmacoterapéuticas es cada vez más necesaria. Este estudio investigó la 2-Bromo-N-fenilacetamida, una sustancia cuya actividad antifúngica aún no se ha informado. Objetivo: evaluar su actividad frente a aislados de candidiasis invasiva, mediante la determinación de las concentra ciones mínimas inhibitorias y fungicidas. Metodología: se realizó un acoplamiento molecular para investigar el posible mecanismo de acción de la sustancia. La sustancia también se asoció con fluconazol, para evaluar la viabilidad de la combinación en la práctica clínica. Las concentraciones mínimas inhibidoras oscilaron entre 4 a 32 µg/mL y actúa de forma fungicida. Resultados: el acoplamiento molecular sugiere que la 2-Bromo-N-fenilacetamida posiblemente actúa sobre la membrana plasmática de los hongos. Y la asociación de 2-Brorno-Ar-fenilacetamida con fluconazol contra cepas resistentes mostró un efecto indiferente. Conclusión: deben realizarse más estudios para dilucidar el potencial de esta sustancia, que puede convertirse en un futuro candi dato a fármaco para tratar la candidiasis invasiva y otras infecciones fúngicas.

Introdução: a candidíase invasiva está relacionada a altas taxas de morbidade e morta lidade. Existem poucas classes de medicamentos disponíveis para o tratamento desse tipo de infecção e o índice de cepas resistentes está aumentando. Tais circunstâncias evidenciam que a busca por novas alternativas farmacoterapêuticas é cada vez mais necessária. Este estudo investigou a 2-Bromo-N-fenilacetamida, uma substância cuja atividade antifúngica ainda não foi relatada. Objetivo: avaliar sua atividade contra isolados de candidíase invasiva, por meio da determinação das concentrações mínimas inibitórias e fungicidas. Metodologia: o docking molecular foi realizado para inve stigar o possível mecanismo de ação da substância. A substância também foi associada ao fluconazol, para avaliar a viabilidade da associação na prática clínica. As concen trações inibitórias mínimas variaram entre 4 a 32 µg/Ml e atuam de forma fungicida. Resultados: o docking molecular sugere que a 2-Bromo-N-fenilacetamida possivel mente atua na membrana plasmática do fungo. E a associação de 2-Bromo-N-fenilace-tamida com fluconazol contra cepas resistentes mostrou efeito indiferente. Conclusão: Novos estudos devem ser realizados para elucidar o potencial dessa substância, que pode se tornar uma futura droga candidata ao tratamento de candidíase invasiva e outras infecções fúngicas.

Atividade antifúngica de isolados clínicos de Candida não-albicans aos óleos essenciais de Syzygium aromaticum (cravo-da-índia) e Eucalyptus globulus (eucalipto-comum) / Antifungal activity of clinical isolates of Candida non-albicans to the essential oils of Syzygium aromaticum (cloud) and Eucalyptus globulus (eucalyptus)

RESUMO: Introdução: O gênero Candida possui alta taxa de incidência no ser humano, sendo a espécie Candida albicans a mais isolada em infecções invasivas e superficiais. Porém, tem sido relatado um aumento considerável de espécies de Candidanão-albicans em infecções fúngicas. Os óleos essenciais, por serem voláteis, podem agir como sinais de comunicação química e arma de defesa. Objetivo: Avaliar a eficácia, in vitro, dos óleos essenciais de Syzygium aromaticum e Eu-calyptus globulus na inibição do crescimento de espécies de Candida não-albicans. Métodos: Para avaliação da atividade antifúngica de S. aromaticum e de E. globulus e do efeito dos seus óleos essenciais sobre a micromorfologia das espécies Candida krusei, Candida parapsilosis e Candida glabrata, foram empregadas, nesta ordem, a técnica de difusão em ágar e de microcultivo para leveduras. Resultados: Na técnica de difusão, o óleo essencial de S. aromaticum apresentou for-mação de halo de inibição para Candida krusei, Candida parapsilosis e Candida glabrata. O óleo de E. globulus, por sua vez, não apresentou crescimento de halos de inibição em nenhuma das concentrações testadas frente às três espécies de Candida não-albicans. Todavia, com o microcultivo, ambos os óleos essenciais se provaram, in vitro, eficazes antimi-crobianos tendo apresentado estruturas indicativas de atividade antifúngica na maior concentração dos óleos e diferentes graus de destruição celular nas demais concentrações. Conclusão: Nas condições deste estudo, concluiu-se que os pro-dutos avaliados exerceram atividade antifúngica contra cepas de Candida não-albicans, destacando-se o óleo essencial de S. aromaticum que apresentou atividade antimicrobiana em ambas as metodologias. (AU)

ABSTRACT: Introduction: The genus Candida possesses a high incidence of infection in humans, with the Candida albicans species being the most isolated in invasive and superficial infections. However, a considerable increase in non-albicans Candidaspecies has been reported in fungal infections. Essential oils, for being volatile, can act as chemical communication sig-nals and defense mechanisms. Objective: Evaluate the effectiveness, in vitro, of the essential oils of Syzygium aromati-cum and Eucalyptus globulus in inhibiting the growth of non-albicans Candida species. Methods: To evaluate the antifun-gal activity of S. aromaticum and E. globulus and the effect of their essential oils on the micromorphology of the species Candida krusei, Candida parapsilosis and Candida glabrata, in this order, the agar diffusion technique and microculture for yeasts. Results: In the diffusion technique, the essential oil of S. aromaticum presented an inhibition halo for Candida krusei, Candida parapsilosis, and Candida glabrata. E. globulus oil, on the other hand, did not show inhibition halo growth in any of the concentrations tested against the three non-albicans Candida species. However, in the microculture, both essential oils have proven to be effective antimicrobials having shown structures indicative of antifungal activity in the highest concentration and different degrees of cell destruction in the other concentrations. Conclusion: In the conditions of this study, it was concluded that the products evaluated exerted antifungal activity against non-albicans Candidastrains, highlighting the essential oil of S. aromaticum that showed antimicrobial activity in both methodologies. (AU)

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.

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.

Frecuencia y susceptibilidad antifúngica de Candida spp. (no C. albicans ) aislada de pacientes de unidades de cuidados críticos de un hospital de tercer nivel del norte del Perú / Frequency and antifungal susceptibility of Candida spp. (other than C. albicans ) isolated from patients admitted to critical care units of a third-level hospital in northern Peru

RESUMEN Objetivo Determinar la frecuencia y susceptibilidad antifúngica de Candida spp. (no C. albicans) aisladas de muestras de pacientes en cuidados críticos de un hospital de tercer nivel del norte del Perú. Materiales y métodos Se realizó un estudio transversal entre febrero del 2018 y mayo del 2019 en 232 muestras de pacientes hospitalizados en unidades de cuidados críticos del Hospital Regional Lambayeque (HRL). Candida ssp. (no C. albicans) fue identificada mediante las pruebas fenotípicas. La susceptibilidad antifúngica al fluconazol y voriconazol se determinó según la interpretación de los puntos de corte del documento M44-A2 del CLSI (por sus siglas en inglés, Clinical and Laboratory Standards Institute). Resultados Se estudiaron 232 muestras. La frecuencia de Candida spp. (no C. albicans) fue de 19,82 % (46) y C. albicans de 11,20 % (26). Las especies de Candida spp. (no C. albicans) fueron las siguientes: Treintaiuna de C. tropicalis (67,39 %), ocho de C. glabrata (17,39 %), cinco de C. parapsilosis (10,86 %) y dos de C. krusei (4,34 %). Los pacientes en quienes se aisló Candida spp. (no C. albicans) se caracterizaron por ser, en su mayoría, mujeres (56,52 %), que previamente habían usado antimicrobianos (67,39 %), y de 60 años a más (36,95 %). La muestra en la que se aisló en microorganismo con mayor frecuencia fue la orina (45,65 %). Respecto a la susceptibilidad antifúngica de las especies de Candida spp. (no C. albicans), encontramos resistencia a fluconazol (19,56 %) y a voriconazol (21,73 %). Conclusiones La alta frecuencia y predominio de especies de Candida spp. (no C. albicans) en pacientes de unidades de cuidados críticos de un hospital de tercer nivel en Chiclayo, y la resistencia que muestran a la acción de fluconazol y voriconazol son características de gran importancia en la morbilidad y mortalidad en estos pacientes.

ABSTRACT Objective To determine the frequency and antifungal susceptibility of Candida spp. (other than C. albicans) isolated from samples of patients admitted to critical care units of a third-level hospital in northern Peru. Materials and methods A cross-sectional study was conducted between February 2018 and May 2019 in 232 samples of patients admitted to critical care units of the Hospital Regional Lambayeque (HRL). Candida spp. (other than C. albicans) were identified using phenotypic tests. The antifungal susceptibility to fluconazole and voriconazole was determined according to the epidemiological cut-off values of CLSI document M44-A2. Results Two hundred thirty-two (232) samples were analyzed. The frequency of Candida spp. (other than C. albicans) was 19.82 % (46/232) and C. albicans was 11.20 % (26/232). Candida spp. (other than C. albicans) were the following: C. tropicalis (67.39 %, 31/46), C. glabrata (17.39 %, 8/46), C. parapsilosis (10.86 %, 5/46) and C. krusei (4.34 %, 2/46). Patients from whom Candida spp. (other than C. albicans) were isolated were mostly females (56.52 %), had a history of antimicrobial drugs consumption (67.39 %) and were 60 years of age and older (36.95 %). Most microorganisms were isolated from urine samples (45.65 %). Regarding the antifungal susceptibility of Candida spp. (other than C. albicans), 19.56 % and 21.73 % were resistant to fluconazole and voriconazole, respectively. Conclusions The high incidence and prevalence of Candida spp. (other than C. albicans) in patients admitted to critical care units of a third-level hospital in Chiclayo, together with resistance to fluconazole and voriconazole, are significant in the morbidity and mortality of these patients.

Formación de biopelículas por Candida parapsilosis sensu stricto y su perfil de susceptibilidad en Venezuela / Biofilm-forming of Candida parapsilosis sensu stricto and its susceptibility profile in Venezuela

Una de las principales consecuencias del desarrollo de biopelículas es la resistencia a los antifúngicos. Se determinó la formación de biopelículas por Candida parapsilosis sensu stricto aisladas de sangre y su perfil de susceptibilidad. Se evaluaron 30 cepas de C. parapsilosis sensu stricto de la Red de Candidemias del Dpto. de Micología del Instituto Nacional de Higiene Rafael Rangel-Venezuela, por el método cuantitativo en microplaca con tinción de cristal violeta y el método cualitativo en agar Sabouraud dextrosa suplementado con rojo Congo. Se utilizó el método de microdilución según el documento M-27 del Clinical and Laboratory Standards Institute. La formación de biopelículas de C. parapsilosis sensu stricto por el método cuantitativo fue de 63% y por el método cualitativo de 50%. El método cualitativo presentó valores de sensibilidad, especificidad, valores predictivos positivo y negativo de 61,1%, 23%, 78,6%, 43,8% respectivamente frente al método cuantitativo. Los aislados en forma planctónica fueron 100% sensibles a anfotericina B y caspofungina, con susceptibilidad variable a los azoles. El método cuantitativo es una técnica confiable para determinar la formación de biopelículas. El método cualitativo puede usarse como método pantalla inicial. No se encontró relación entre la susceptibilidad de las formas planctónicas y la capacidad de producción de biopelículas

One of the main consequences of biofilm development is resistance to antifungals. Biofilmforming by Candida parapsilosis sensu stricto isolated from blood and its susceptibility profile was determined. Thirty strains of C. parapsilosis sensu stricto belonging to the Candidemia Network of the Mycology Department of the Instituto Nacional de Higiene Rafael Rangel-Venezuela, were evaluated by microplate quantitative method with violet crystal staining, and Sabouraud dextrose agar supplemented with Congo red qualitative method. The microdilution method was used to determine the susceptibility, according to the Clinical and Laboratory Standards Institute M-27 document. Biofilm formation of C. parapsilosis sensu stricto by both quantitative and qualitative method was 63% and 50%, respectively. Sensitivity, specificity, and positive and negative predictive values of the qualitative method were 61.1%, 23%, 78.6%, and 43.8% respectively, when compared with quantitative method. Planktonic isolates were 100% sensitive to amphotericin B and caspofungin, with variable susceptibility to azoles. Quantitative method is a reliable assay to determine biofilm formation, while qualitative method can be used as an initial screening assay. No relationship was found between susceptibility of planktonic isolates and the ability to biofilm-forming

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.

Effect of microbial immunostimulants on WSSV infection percentage and the expression of immune-related genes in white shrimp (Litopenaeus vannamei) / Efecto de los inmunoestimulantes microbianos en el porcentaje de infección con WSSV y la expresión de genes relacionados con el sistema inmune del camarón blanco (Litopenaeus vannamei) / Efeito de imunoestimulantes microbianos sobre a porcentagem de infecção por WSSV e a expressão de genes relacionados ao sistema imune em camarões brancos (Litopenaeus vannamei)

Abstract Background: The white spot syndrome virus (WSSV) causes high mortalities in aquaculture. The use of immunostimulants increases animal resistance. Objective: To evaluate the WSSV infection percentage and the immunostimulant effect of lactic acid bacteria and yeast (MI= microbial immunostimulants) on WSSV infected Litopenaeus vannamei. Methods: A bioassay was performed for 33 d, with treatments in triplicate. The MI was added to the feed at 8.5 mg/kg feed and offered to shrimp (9.9 ± 3.1 g) daily, every 2 days, or every 3 days. Shrimp were infected with WSSV at 9 and 19 days. The expression of four immune system-related genes was studied using qRT-PCR. Results: No significant differences were observed in growth and survival among treatments. At the end of the bioassay, WSSV infection percentage (low viral load) decreased 8.3 and 25% in treatments III and IV as compared to the control group. Treatments with MI showed significant differences in the relative expression of LvToll, transglutaminase, and prophenoloxidase genes when MI was offered daily as compared to the control group. The MI did not regulate the expression of the superoxide dismutase gene. The WSSV infection percentage decreased when feed with MI was offered every 3 d. Conclusion: The MI decrease WSSV infection percentage in L. vannamei infected with low viral load when it is offered every three days. The MI up-regulates LvToll, Tgase, and proPO genes when it is offered daily. Further research is needed regarding prophylactic treatment with microbial immunostimulants against WSSV in commercial shrimp farms.

Resumen Antecedentes: El virus del síndrome de la mancha blanca (WSSV) ocasiona alta mortalidad en acuacultura. El empleo de inmunoestimulantes incrementa la resistencia de los animales. Objetivo: Evaluar el porcentaje de infección de WSSV y el efecto inmunoestimulante de bacterias ácido lácticas y levadura (MI= inmunoestimulantes microbianos) en Litopenaeus vannamei infectado con WSSV. Métodos: Se realizó un bioensayo durante 33 días con tratamientos por triplicado. Se agregó MI al alimento (8,5 mg/kg de alimento), suministrandolo al camarón (9,9 ± 3,1 g) diariamente, cada 2, o cada 3 días. Los camarones se infectaron con WSSV a los 9 y 19 días. Se estudió la expresión de cuatro genes relacionados con el sistema inmune utilizando qRT-PCR. Resultados: No hubo diferencias significativas en el crecimiento y la sobrevivencia entre los tratamientos. Al final del bioensayo, el porcentaje de infección de WSSV (baja carga viral) disminuyó un 8,3 y un 25% en los tratamientos III y IV en comparación con el grupo control. Los tratamientos con MI mostraron diferencias significativas en la expresión relativa de los genes LvToll, transglutaminasa, y profenoloxidasa cuando se suministró MI diariamente en comparación con el grupo control. Los MI no regularon la expresión del gen de la superóxido dismutasa. El porcentaje de infección de WSSV disminuyó cuando el alimento con MI se aplicó cada 3 d. Conclusiones: Los MI disminuyen el porcentaje de infección de WSSV en L. vannamei infectado con baja carga viral cuando se aplican cada tres días. Además, los MI causan una sobre-expresión de los genes LvToll, Tgase y proPO cuando se administran diariamente. Se necesita más investigación sobre los inmunoestimulantes microbianos como tratamiento profiláctico contra WSSV en granjas comerciales de camarón.

Resumo Antecedentes: O vírus da mancha branca (WSSV) causa alta mortalidade na aqüicultura e o uso de imunoestimulantes aumenta a resistência em animais. Objetivo: Este trabalho avaliou a prevalência do WSSV e o efeito imunoestimulante de bactérias ácido lácticas e levadura (MI= inmunoestimulantes microbianos) em Litopenaeus vannamei infectado con WSSV. Métodos: Se realizou um bioensayo durante 33 dias com tratamientos por triplicado. Se agregou MI al alimento (8,5 mg/kg feed) e se o fornecimento de camarão (9,9 ± 3,1 g) diariamente, cada 2 e cada 3 d. Los camarones foram infectados com WSSV a los 9 y 19 d. Se estudió a expressão de quatro genes relacionados com o sistema imune, use qRT-PCR. Resultados: No hubo diferencias significativas no crescimento e na supervisão entre os tratamientos. Al final del bioensayo, a prevalência do WSSV diminuiu um 8.3 e não 25% nos tratamentos III e IV em comparação com o grupo de controle. Os tratamentos com MI mostraram diferenças significativas na expressão relativa dos genes. LvToll, transglutaminasa e prophenoloxidase quando se administraron em diários de MI em comparação com o controle de grupo. Los MI não regularam a expressão do genótipo da superóxido dismutasa. A prevalência do WSSV diminuiu quando o alimento com MI se aplicó cada 3 d. Conclusões: Los IM diminuir a prevalência de WSSV em L. vannamei infectado com baixa carga viral quando aplicável a cada três dias. Além disso, os IM causaram uma sobre-expressão dos genes LvToll, Tgase e proPO quando se administran diariamente. Há mais informações sobre imunoestimulantes microbianos como tratamento profiláctico contra WSSV en granjas comerciales de camarón.

Recurrent fungal endocarditis: one case report and literature review / 中国感染控制杂志

A 56-year-old female patient was admitted to a hospital because of repeated fatigue, anorexia, fever for more than one year, recurrence for 2 months and sudden left limb twitch for 10 days. In 2017, patient was diagnosed with infective endocarditis, then underwent aortic valve replacement, valve biopsy showed Candida parapsilosis, she took medicine regularly after operation and withdrew medicine after three consecutive negative blood culture. Fever and multiple organ infarction occurred again in 2018, Candida parapsilosis was isolated again from blood culture, it is suggested that fungal endocarditis may be latent and recurrence in human body for a long time, exfoliation of vegetation may embolize various organs and lead to corresponding clinical manifestations, surgery combined with drug therapy and subsequent maintenance of antifungal therapy are extremely important to improve the prognosis of patients.

A Case of Fungemia with Co-isolation of Candida parapsilosis and Trichosporon asahii Confirmed by MALDI-TOF MS and D2 rRNA Sequencing

Fungi are a major cause of human infections with diverse clinical manifestations. The incidence of fungal infections has increased over time, particularly in patients who have risk factors such as neutropenia, immune suppression, an intravascular catheter, parenteral nutrition, a prosthetic device, and prior broad spectrum antibiotic therapy. Here, we present an unusual case of co-infection by 2 distinct fungi, Candida parapsilosis and Trichosporon asahii, isolated from a patient who did not have any known risk factors initially, except active pulmonary tuberculosis. Despite the negative conversion of sputum acid-fast bacilli (AFB) culture test after treatment, clinical symptoms were refractory to therapy. The patient developed symptoms suggesting septic shock, and 2 distinct colonies were isolated from a blood specimen, which were identified as C. parapsilosis and T. asahii by MALDI-TOF and rRNA sequencing. Fever and hypotension were relieved after anti-fungal agent injection, and pulmonary lesions identified by imaging also improved.

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.

Analysis of patients suffering from fungemia of Candida parapsilosis adimitted in geriatric intensive care unit / 中国中西医结合急救杂志

Objective To observe the clinical features of senile patients suffering from fungemia of Candida parapsilosis, and the effect and safety of antifungal therapy in treatment of this disease in geriatric intensive care unit (GICU). Methods The clinical data of patients with fungi positive either in peripheral blood culture or catheter culture admitted to the GICU of Tianjin Medical University General Hospital from November 2012 to June 2015 were retrospectively analyzed, of them 45 cases were of infection of Candida parapsilosis (parapsilosis group) and 15 cases infection of non-Candida parapsilosis (non-parapsilosis group). The clinical features of the two groups were collected, such as sex, age, acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score, sequential organ failure assessment (SOFA) score, timing of antifungal therapy, number of patients mechanical ventilation, concomitant disease, catheter-related infection, method of catheter-indwelling, levels of creatinine (Cr), hemoglobin (Hb), platelet count (PLT), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), etc.; the differences in above indicators were compared between the two groups; multifactor Cox-regression-analysis was used to analyze the risk factors that could affect the patients' prognosis; the patients' survival rates on 7, 14 and 28-day were calculated and compared between the two groups, and the therapeutic effects of different anti-fungal drugs on patients' survival rates and liver function damage were recorded and compared. Results The non-parapsilosis group had a higher rate in mechanical ventilation than parapsilosis group [73.3% (11/15) vs. 33.3% (15/45), P < 0.05], and in the comparisons of other clinical features, there were no statistical significant differences between the two groups (all P > 0.05). There were no statistical significant differences in survival rates in the duration of 7, 14 and 28 days between the two groups[7 days: 82.2% (37/45) vs. 66.7% (10/15), 14 days: 75.6% (34/45) vs. 60.0% (9/15), 28 days: 66.7% (30/45) vs. 46.7% (7/15), all P > 0.05]. When the patients in parapsilosis group treated with echinocinomycin were compared with those treated with azolol, no statistical significant differences were found between the 2 types of therapy in the survival rates in the duration of 7, 14, and 28 days after treatment [7 days: 100.0% (23/23) vs. 82.4% (14/17), 14 days: 91.3% (21/23) vs. 76.5% (13/17), 28 days: 78.3% (18/23) vs. 70.6% (12/17), all P > 0.05]. Multifactor Cox-regression-analyses showed:diabetes [odds ratio (OR) = 0.268, 95% confidence interval (95%CI) = 0.077 - 0.928, P = 0.038), infection of Candida parapsilosis (OR = 0.260, 95%CI = 0.072 - 0.946, P = 0.041), APACHE Ⅱ score (OR = 1.241, 95%CI = 1.051 - 1.466, P = 0.011) and SOFA score (OR = 1.405, 95%CI = 1.005 - 1.966, P = 0.047) were the risk factors affecting the prognosis of the patients. When the patients in parapsilosis group treated with echinocinomycin were compared with those treated with azolol, there were no statistical significant differences in incidences of aggravation of liver damage and newly developed liver damage (aggravation of liver damage: 18.8% vs. 21.0%, newly developed liver damage: 6.2% vs. 10.5%, both P > 0.05). Conclusion The patients with fungemia in GICU are mainly the infection of Candida parapsilosis, and diabetes, infection of parapsilosis, APACHE Ⅱ score and SOFA score are the risk factors affecting the prognosis of the patients.

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.

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.

Variabilidad genética de aislados del complejo Candida parapsilosis en dos servicios de un hospital terciario de la Ciudad de México / Genetic variability of isolates of Candida parapsilosis complex in two services of a tertiary hospital in Mexico City

La infección causada por el complejo Candida parapsilosis puede presentarse esporádicamente o en forma de brotes, por lo que el estudio de la variabilidad genética de los aislados clínicos puede revelar la presencia de genotipos endémicos y la ocurrencia de transmisión horizontal. Este estudio analizó, mediante Amplificación al Azar del ADN Polimórfico (RAPD) con cuatro oligonucleótidos (M13, AP3, T3B y R108), la variabilidad genética de 11 aislados clínicos del complejo C. parapsilosis, obtenidos en los servicios de Medicina Interna y Cirugía General de un hospital de la Ciudad de México, e identificar si los pacientes fueron infectados por el mismo genotipo. La cepa ATCC® 22019™ fue incluida en el análisis. Los aislados se identificaron por VITEK 2 Compact® y PCR. Con base en los perfiles polimórficos,se construyó un dendrograma por UPGMA y se calcularon el coeficiente de correlación cofenética(CCCr), el índice de asociación (I A) y los indicadores de diversidad genética. Todos los aislados fueron identificados como C. parapsilosis sensu stricto. El dendrograma mostró dos grupos (I y II), en el I se encontraron tres genotipos integrados por la cepa 22019 y cinco aislados asociados en su mayoría a candidiasis invasiva; el II mostró seis genotipos integrados por seis aislados asociados en su mayoría a candidiasis mucocutánea. El I A y los indicadores de diversidad genética obtenidos revelaron un sistema de reproducción recombinante. El RAPD con los oligonucleótidos M13, AP3, T3B y R108 es útil en la investigación de posibles brotes causados por C. parapsilosis y en la determinación de su variabilidad genética.

The infection caused by the Candida parapsilosis complex may occur sporadically or in the form of outbreaks, so the study of the genetic variability in clinical isolates may reveal the presence of endemic genotypes and the occurrence of horizontal transmission. This study analyzed the genetic variability of 11 clinical isolates of the C. parapsilosis complex obtained from Internal Medicine and General Surgery services of a hospital in Mexico City, using Random Amplification of Polymorphic DNA (RAPD) with four oligonucleotides (M13, AP3, T3B and R108), also evaluated whether the patients were infected by the same genotype. The strain ATCC® 22019 ™ was included in the analysis. Isolates were identified by VITEK 2 Compact® and PCR. With the polymorphic profiles, a dendrogram was constructed by UPGMA and the cophenetic correlation coefficient (CCCr), the association index (I A), and the indicators of genetic diversity were calculated. All isolates were identified as C. parapsilosis sensu stricto. The dendrogram showed two groups (I and II). Three genotypes integrated by the strain 22019 and five isolates, mostly associated with invasive candidiasis, were found in the group I. The group II showed six genotypes composed of six isolates, mostly associated with mucocutaneous candidiasis. The I A and the indicators of genetic diversity obtained, revealed a recombinant reproduction system. The RAPD with the oligonucleotides M13, AP3, T3B and R108 is useful in the investigation of possible outbreaks caused by C. parapsilosis and in the determination of their genetic variability.

In vitro evaluation of hydrolytic enzyme activity and biofilm formation of Candida parapsilosis species complex from a nosocomial environment

Abstract INTRODUCTION: Candida parapsilosis complex species, frequently found in hospital environments, have gained importance as etiological agents of candidemia. METHODS: Candida parapsilosis complex isolates from a nosocomial environment were identified and their hydrolitic enzyme activity and ability to form biofilm were characterized. RESULTS: Twenty-two C. parapsilosis sensu stricto isolates produced proteinase and three produced phospholipase. Most Candida metapsilosis isolates produced proteinase and one also produced phospholipase. All 29 isolates formed biofilms. CONCLUSIONS: The nosocomial environment may act as a reservoir for C. parapsilosis complex isolates with phenotypic features that could possibly lead to nosocomial infections and health complications in hospital patients.

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