Antifungal activity testing of curry leaf ethanol extract on the growth of Pityrosporum ovale and Candida albicans fungus

Background: The curry leaf is an Indonesian plant commonly utilized as a spice. Curry leaves are abundant in secondary metabolites, which endow this plant with numerous advantages, including antibacterial and antifungal properties, as well as the ability to reduce blood sugar levels and blood pressure. The objective of this study is to assess the efficacy of an ethanol extract derived from curry leaves in suppressing the proliferation of Pityrosporum ovale and Candida albicans fungus. Methods: The symbiotic properties and phytochemical composition of curry leaf simplisia were examined. The antifungal efficacy of the ethanol extract derived from curry leaves was evaluated against Pityrosporum ovale and Candida albicans using the disc diffusion method. Calculate the precise values of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Results: The simplisia of curry leaf fulfills the criteria for simplisia characterization. Curry leaves possess a variety of secondary metabolite chemicals, including alkaloids, flavonoids, tannins, saponins, glycosides, steroids, and triterpenoids. The activity against the inhibition of fungal growth of Pityrosporum ovale and Candida albicans can suppress fungal growth with inhibition zone diameters measuring 6.93±0.15 mm and 7.27±0.47 mm, respectively. The minimum lethal concentration of leaf ethanol extract for Pityrosporum ovale fungi is 8.75%, resulting in a decrease of 98.25%. For Candida albicans fungi, the minimum lethal concentration is 12.5%, resulting in a reduction of 98.37%. Conclusions: The ethanol extract derived from curry leaves has the ability to hinder the proliferation of Pityrosporum ovale and Candida albicans fungus.

COMPARATIVE ANTIFUNGAL STUDY OF FLUCONAZOLE AND FLUCONAZOLE ENHANCED WITH SILVER METAL COLLOID CREAMS: A 3² FACTORIAL DESIGN OPTIMIZATION APPROACH

Antifungal resistance remains a critical health challenge within dermatology and pharmaceutical research. This study aimed to enhance antifungal creams by investigating the effectiveness of fluconazole (FLZ) and fluconazole combined with silver metal colloid (FLZ-AgMC). Employing a 3² factorial design, systematic exploration was conducted to assess the influence of metal colloid concentration and stearic acid content on crucial cream attributes: viscosity, spreadability, and zone of inhibition ratio. Viscosity ranged from 56132 to 58700 cP, spreadability from 28.7 to 27.8 gm.cm/sec, and the zone of inhibition increased with metal colloid concentration. Optimized cream formulations were identified using Stat-Ease Design Expert version 7. Various FLZ and AgMC concentrations were evaluated for antifungal activity against Candida albicans, with FLZ-AgMC exhibiting significantly enhanced efficacy, as indicated by a larger inhibition zone compared to FLZ alone. The inhibitory zone ratio demonstrated a 35 to 40% improvement, indicating enhanced fungal growth inhibition. Skin permeation and ex-vivo studies confirmed that the optimized Fluconazole formulation followed the Higuchi Model (R2 = 0.9847). Silver metal colloid-containing formulations demonstrated superior antifungal efficacy against C. albicans. The impact of silver metal colloid and stearic acid on viscosity and spreadability was established, revealing key factors influencing the cream’s physical properties. This optimization approach highlights the potential for innovative antifungal formulations, contributing to improved patient care, user acceptability, and clinical application.

Study on Mechanism of Sanhuang Decoction in Alleviating Ulcerative Colitis in Mice under Candida albicans Colonization Based on Dectin-1-Syk-CARD9 Signaling Pathway / 中国中医药信息杂志

Objective To study the mechanism of Sanhuang Decotion in the treatment of ulcerative colitis(UC)under Candida albicans colonization in mice based on Dectin-1-Syk-CARD9 signaling pathway.Methods Mice model of UC with fungal colonization were established with dextran sodium sulfate free drinking and C.albicans intragastric administration.Mice were divided into normal control group,model group,sulfasalazine group,fluconazole group,and Sanhuang Decotion low-and high-dosage groups,and receive corresponding drug interventions.General state of mice were observed,and the disease activity index(DAI)score of mice were calculated.The load of C.albicans in intestine was detected,the length of the colon was measured,and pathological scoring of the colon tissue was performed.The ultrastructural changes of colon epithelium were observed under transmission electron microscopy.The contents of TNF-α,IL-6 and IL-12 in serum and colon tissues were detected by ELISA.The mRNA and protein expression of Dectin-1,Syk,CARD9,NF-κBp65 and inflammation factors in intestinal epithelial cells and colon tissues were detected by qPCR,Western blot and immunohistochemistry.Results Compared with the normal control group,the model group mice showed reduced activity,decreased food intake,accompanied by loose stools,significantly increased DAI score,increased load of C.albicans in the intestine,shortened colon length,and increased histopathological score,with widening of gap between colon epithelial cells,cytoplasmic dissolution,mitochondrial swelling;TNF-α,IL-6 and IL-12 in serum and colon tissue increased,the expressions of Dectin-1 and CARD9 mRNA and protein in colon epithelial cells increased,p-Syk,p-NF-κBp65,CARD9,TNF-α,IL-1β,IL-6 protein expression in colon tissue increased(P<0.01,P<0.05).Compared with the model group,the Sanhuang Decotion high-dosage group mice showed a significant decrease in DAI score,decreased intestinal C.albicans load,increased colon length,decreased histopathological score,more complete and orderly arrangement of microvilli in colon epithelial cells,mild mitochondrial swelling,TNF-α,IL-6 and IL-12 in serum and colon tissue decreased,and the mRNA and protein expression of Dectin-1 and CARD9 in colon tissue increased,the expression of p-Syk,p-NF-κBp65,CARD9,TNF-α,IL-1β,IL-6 protein in colon tissue decreased(P<0.01,P<0.05).Conclusion Sanhuang Decotion may exert an anti C.albicans colonization UC effect by inhibiting the Dectin-1-Syk-CARD9 signaling pathway and reducing the release of inflammatory factors.

Study on antifungal effect of demethylzelamaldehyde in vitro / 药学实践与服务

Objective To study the antifungal effect of demethylzelamaldehyde in vitro. Methods The minimum inhibitory concentrations （MIC） of demethylzeylasteral and fluconazole against 23 fungal strains were determined by micro liquid dilution method. The synergistic index （FICI） of the two drugs was determined using a checkerboard micro liquid dilution method. The synergistic effect of the combination of the two drugs was visually verified by paper diffusion experiments. Finally, the cytotoxicity of demethylzelamaldehyde was determined by CCK-8 method. Results Demethylzelamaldehyde showed a broad spectrum of antifungal activity when used alone, with MICs ranging from 4 g/L to 32 g/L. When combined with fluconazole, the effective concentration of fluconazole could be reduced from over 64 g/L to 0.25 g/L, with FICI values ranging from 0.129 to 0.254, indicating the synergistic effect of the two drugs. The CCK-8 results showed that demethylzeylasteral exhibited cytotoxicity only at concentrations four times higher than the MIC value. Conclusion Demethylzelamaldehyde exhibited good antifungal effect and synergistic effect with fluconazole, and its toxicity was low.

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.

Evaluación in vitro de la actividad antifúngica de extractos fluidos de Dichrostachys cinerea L. (marabú) frente a Candida albicans

Introducción: La Dichrostachys cinerea L. (marabú) es una planta que crece en Cuba, de la que se estudian propiedades medicinales. La resistencia de levaduras del género Candida a los antifúngicos sintéticos disponibles en la actualidad es cada vez mayor, por lo que se buscan nuevos compuestos de origen vegetal que puedan ser eficaces en el tratamiento de infecciones causadas por este germen. Objetivo: Evaluar la actividad antifúngica in vitro de Dichrostachys cinerea L. contra una cepa de Candida albicans. Métodos: Se realizó un estudio observacional analítico transversal in vitro para evaluar la actividad antifúngica de extractos fluidos de hojas y de tallos de D. cinerea L mediante el método de macrodilución en caldo y como sustancia de referencia el alcohol. Resultados: A través del proceso se mostró la actividad antifúngica del extracto fluido de tallos de D. cinerea L. al 50 % hasta la dilución 1/32, determinada como la concentración mínima inhibitoria; el extracto fluido de hojas al 30 % no logró inhibir el crecimiento de la cepa de Candida albicans ATCC 10231. Conclusiones: La actividad antifúngica del extracto fluido al 50 % de las hojas de Dichrostachys cinerea L. fue efectiva, no así el preparado farmacéutico al 30 %. Se determinó la concentración mínima inhibitoria del extracto fluido de hojas al 50 % y se demostró que ésta superó a la del alcohol al 50 % en tres diluciones contra la Candida albicans.

Introduction: Dichrostachys cinerea L. (marabou) is a plant that grows in Cuba, medicinal properties of this plant are being studied. The resistance of yeasts of the Candida genus to current available synthetic antifungals is increasingly greater. This is why, new compounds of plant origin are being searched for the effective treatment of infections caused by this germ. Objective: To evaluate the in vitro antifungal activity of Dichrostachys cinerea L against a strain of Candida albicans. Methods: An in vitro a cross-sectional analytic observational study was carried out to evaluate the antifungal activity of fluid extracts of D. cinerea L leaves and stems using the broth macro-dilution method and alcohol as the reference substance. Results: Through the process was shown the antifungal activity of the fluid extract of Dichrostachys cinerea L. stems at 50% up to the 1/32 dilution; it was determined as the minimum inhibitory concentration. The leaves fluid extract at 30% failed to inhibit the growth of the Candida albicans ATCC 10231 strain. Conclusions: The antifungal activity of the fluid extract at 50% of the leaves was effective, but not the pharmaceutical preparation at 30%. The minimum inhibitory concentration of the fluid extract of leaves at 50% was determined and it was shown that it exceeded that of alcohol at 50% in three dilutions against Candida albicans.

Atividade antifúngica de nanofibras de policaprolactona/nistatina: estudo in vitro e in vivo / Antifungal activity of polycaprolactone/nystatin nanofibers: in vitro and in vivo study

Este estudo avaliou a eficácia in vitro e in vivo de mantas de nanofibras (NF) de policaprolactona (PCL) incorporadas com nistatina (NIS) no tratamento da estomatite protética (EP) em modelos animais. NF foram sintetizadas com diferentes concentrações de NIS, totalizando quatro soluções: PCL puro, PCL/NIS 0,045 g, PCL/NIS 0,090 g e PCL/NIS 0,225 g. A liberação da NIS foi analisada por espectroscopia Ultravioleta-Visível. A capacidade das mantas de inibirem o biofilme de Candida albicans, principal fator etiológico da EP, dividindo-se cinco grupos (N=5) compostos por um grupo com controle de células de C. albicans e com PCL puro, além das três concentrações de NIS. A seguir, foi analisada a viabilidade celular em queratinócitos humanos (HaCat) por meio do teste colorimétrico de resazurina. Cinco grupos foram divididos (N=10): controle celular, PCL puro e as três concentrações de NIS. Em modelos animais de ratos Wistar albinos (N=18), dispositivos palatinos (DP) de resina acrílica foram confeccionados simulando próteses totais e utilizados para a indução da EP. Para isso, DP contaminados com C. albicans foram cimentados na região molar da cavidade bucal dos animais e permaneceram em boca por 48 h. Após esse período, os DP foram removidos e os animais foram divididos em três grupos: (C) controle; (B1) com tratamento por mantas de PCL/NIS 0,045 g e (B2) PCL/NIS 0,225 g, com N=6. Então novos DP, livres de contaminação, foram cimentados na cavidade oral dos animais e permaneceu por mais 48 h. Após esse período, os animais foram eutanasiados, a contagem de UFC/ mL foi realizada e os palatos foram coletados para a análise histológica. A curva padrão de NIS obtida apresentou R2 de 0,99. As três concentrações de NF apresentaram liberação de NIS, com pico no tempo de 6 h e valores de 66,26 µg/ mL para PCL/NIS 0,045 g, de 333,87 µg/ mL para PCL/NIS 0,090 g e 436,51 µg/ mL para PCL/NIS 0,225 g, constantes até o fim do experimento. Os grupos com NIS reduziram em 2,5 log10 de crescimento do biofilme fúngico em relação aos grupos sem tratamento, Controle e PCL, sem diferença estatística significativa. Não foi observada citotoxicidade nas células HaCat, com viabilidade celular de 93,7% para PCL/NIS 0,045 g, 72,6% para PCL/NIS 0,090 g e 72,4% para PCL/NIS 0,225 g. A indução da EP nos três grupos foi possível e, porém, sem redução significativa na contagem de UFC/ mL de C. albicans nos grupos B1 e B2. Na análise histológica do grupo C pôde-se observar infiltração de hifas de Candida na camada queratinizada, presença de células inflamatórias formando micro abscessos e um discreto infiltrado inflamatório no tecido conjuntivo subjacente ao epitélio infectado. Nos grupos B1 e B2 não foram encontradas alterações epiteliais, concluindo-se que as NF demonstraram atividade antifúngica in vitro e foram efetivas na prevenção da penetração de hifas no tecido palatino de animais com DP (AU)

This study evaluated the in vitro and in vivo efficacy of nanofiber (NF) mats of polycaprolactone (PCL) incorporated with nystatin (NIS) in the treatment of denture stomatitis (DS) in animal models. NFs were synthesized with different concentrations of NIS, totaling four solutions: pure PCL, PCL/NIS 0.045 g, PCL/NIS 0.090 g, and PCL/NIS 0.225 g. The release of NIS was analyzed by Ultraviolet-Visible spectroscopy. The ability of the mats to inhibit Candida albicans biofilm, the main etiological factor of DS, was assessed by dividing five groups (N=5) composed of a group with C. albicans cell control and with pure PCL, in addition to the three concentrations of NIS. Next, cell viability in human keratinocytes (HaCat) was analyzed using the resazurin colorimetric test. Five groups were divided (N=10): cell control, pure PCL, and the three concentrations of NIS. In albino Wistar rat animal models (N=18), palatal devices (PD) made of acrylic resin were fabricated to simulate total prostheses and used to induce DS. For this, PD contaminated with C. albicans were cemented in the molar region of the animals' oral cavity and remained in the mouth for 48 hours. After this period, the PDs were removed, and the animals were divided into three groups: (C) control; (B1) treated with PCL/NIS 0.045 g mats, and (B2) PCL/NIS 0.225 g, with N=6. Then new, uncontaminated PDs were cemented in the animals' oral cavity and remained for another 48 hours. After this period, the animals were euthanized, UFC/ mL counts were performed, and the palates were collected for histological analysis. The standard NIS curve obtained showed an R2 of 0.99. The three concentrations of NF showed NIS release, with a peak at 6 h and values of 66.26 µg/ mL for PCL/NIS 0.045 g, 333.87 µg/ mL for PCL/NIS 0.090 g, and 436.51 µg/ mL for PCL/NIS 0.225 g, remaining constant until the end of the experiment. The groups with NIS reduced fungal biofilm growth by 2.5 log10 compared to the untreated groups, Control and PCL, with no significant statistical difference. No cytotoxicity was observed in HaCat cells, with cell viability of 93.7% for PCL/NIS 0.045 g, 72.6% for PCL/NIS 0.090 g, and 72.4% for PCL/NIS 0.225 g. Induction of DS in the three groups was possible; however, there was no significant reduction in UFC/ mL counts of C. albicans in groups B1 and B2. Histological analysis of group C revealed infiltration of Candida hyphae in the keratinized layer, presence of inflammatory cells forming micro abscesses, and a discreet inflammatory infiltrate in the connective tissue underlying the infected epithelium. No epithelial alterations were found in groups B1 and B2, concluding that NFs demonstrated in vitro antifungal activity and were effective in preventing hyphal penetration into palatal tissue in animals with PD.(AU)

Comparative in vitro analysis of the antifungal activity of different calcium silicate-based endodontic sealers

Aim: This study aimed to perform an in vitro comparative analysis of the antifungal activity of different calcium silicate-based endodontic sealers against three fungal species. Methods: The antifungal properties of three calcium silicate-based sealers were tested: Bio-C Sealer, Cambiar a Sealer Plus BC, and MTA-Fillapex. Two commonly used sealers were used as controls: AH Plus and Endomethasone. An agar diffusion test was performed to analyze the antifungal activity of the sealers against Candida albicans, Candida glabrata, Candida tropicalis, and a mixed microbial culture medium. The results were analyzed using ANOVA (p <0.05). Results: Endomethasone exhibited the highest inhibition against all strains examined, maintaining a consistent level of inhibition throughout 7 days. MTA-Fillapex demonstrated the best performance among the calcium silicate-based sealers for the three fungal species (p < 0.05), maintaining stable values over the 7 days, surpassing that of Endomethasone. Nevertheless, MTA-Fillapex only exhibited antimicrobial effect against the mixed culture for the first 24 hours, and no antimicrobial activity was observed at 48 hours, being surpassed by all tested sealers (p < 0.05). Conclusion: Of all silicate-based sealers tested, only MTA-Fillapex exhibited promising antifungal activity. Nevertheless, care must be taken when extrapolating these results, as MTA-Fillapex exhibited poor antimicrobial activity when tested in mixed microbial cultures

Efeitos dos líquidos ativados com plasma atmosférico sobre Candida albicans / Application of plasma activated liquids on Candida albicans

O incremento no número de casos refratários aos tratamentos convencionais e a limitação de opções terapêuticas são alguns dos desafios encontrados no tratamento da candidose bucal, apontando para a necessidade de terapias alternativas. A utilização da tecnologia de plasma de forma indireta, pela exposição prévia de líquidos ex situ, tem mostrado resultados promissores, trazendo inúmeras vantagens para a aplicação clínica. Até o momento, pouco se conhece sobre a atividade antifúngica do líquido ativado com plasma (LAP) e não foram detectados relatos sobre sua aplicabilidade no tratamento da candidose bucal. Com base neste cenário, o objetivo deste projeto foi avaliar a atividade do líquido ativado com plasma sobre Candida albicans, principal agente etiológico da candidose bucal. Para tanto, foram determinadas as condições de obtenção do LAP com maior efeito antifúngico frente a C. albicans. O LAP foi gerado em um reator de plasma tipo arco deslizante (gliding arc). Os gases empregados incluíram argônio, ar comprimido seco e suas misturas em diversas concentrações, ajustando-se o fluxo de gás e a potência conforme necessário. Avaliou-se a eficácia antifúngica de diferentes líquidos ativados contra C. albicans, tanto em estado planctônico quanto em biofilmes, visando identificar o mais efetivo. As espécies reativas dos LAP foram caracterizadas utilizando técnicas espectrofotométricas, juntamente com a avaliação dos parâmetros físico-químicos. Os resultados dos ensaios foram submetidos a análise estatística, estabelecendo-se um nível de significância de 5% para a interpretação dos dados. Observou-se que a solução salina 0,9% ativada com plasma de argônio (S1), água destilada ativada com plasma de argônio (D1) e água destilada ativado com a mistura dos gases argônio e ar comprimido (S2) apresentaram a maior atividade antifúngica sobre células planctônicas de C. albicans quando expostas por 30 minutos ao LAP. O grupo D1 apresentou maior ação frente aos biofilmes de 24 e 48 horas e o S1 frente a biofilmes de 48 horas apenas quando exposto por 30 minutos ao LAP. Ambos os LAPs apresentaram ação antifúngica após terem sido congelados e armazenados por 1 dia após a ativação. Os grupos D1 e S1 não apresentaram perfil citotóxico nos ensaios realizados. Pode-se concluir que os LAPs apresentaram ação inibitória sobre células planctônicas e sobre biofilmes de C. albicans, sem citotoxicidade para células de mamíferos, sugerindo seu potencial como adjuvante às terapias para o controle da candidose.(AU)

The increase in the number of cases refractory to conventional treatments and the limitation of therapeutic options is due to some two challenges encountered in the treatment of oral candidiasis, pointing to the need for alternative therapies. The use of plasma technology indirectly, for the exposition of liquids ex situ, has shown promising results, providing numerous advantages for clinical application. Currently, little is known about the antifungal activity of plasma-activated liquid (LAP) and there are no reports on its applicability in oral candidiasis treatment. Based on this scenario, the objective of this project is to validate the application of plasma-activated liquid as an adjuvant in the treatment of oral candidiasis. Therefore, certain conditions for obtaining LAP have greater antifungal effect against Candida albicans. The LAP was generated in a gliding arc type plasma reactor. The gases used include argon, dry compressed and their mixtures in various concentrations, adjusting the gas flow and power as necessary. The antifungal efficacy of different liquids activated against C. albicans is evaluated, both in the planktonic state and in biofilms, aiming to identify the most effective. The relative species of LAP were characterized using spectrophotometric techniques, together with the evaluation of two physical-chemical parameters. The results of two tests were submitted to statistical analysis, establishing a significance level of 5% for the interpretation of the data. It was observed that the groups that presented the greatest antifungal activity in planktonic cells of C. albicans were the groups of 0.9% saline solution activated with argonium plasma (S1), or of distilled water activated with argonium plasma (D1). e or distilled water activated with a mixture of two argon gases and compressed air (S2). The D1 group presented against biofilms of 24 and 48 hours and the S1 against biofilms of only 48 hours. Both LAPs are presented with antifungal coating and have been frozen and stored for 1 day after activation. The groups D1 and S1 do not present a cytotoxic profile in the tests carried out. It can be concluded that the LAPs have antifungal activity on planktonic cells and on biofilms and do not present a toxicity profile for human cells, being potent adjuvants in therapies for or controlling infections caused by C. albicans (AU)

Efeito de extratos vegetais de Hamamelis virginiana, Persea americana, Cynara scolymus L e Stryphnodendron barbatiman Msobre a expressão fenotípica dos fatores de virulência em biofilmes de Candida albicans / Effect of Hamamelis virginiana, Persea americana, Cynara scolymus L and Stryphnodendron barbatiman M plant extracts on the phenotypic expression of virulence factors in biofilms of the Candida albicans

Objetivo: Analisar a expressão fenotípica de fatores de virulência em biofilmes de Candida albicans frente a extratos glicólicos de plantas. Material e Métodos: Os biofilmes de Candida albicans (ATCC 18804) obtidos a partir de incubação de 48 horas foram expostos por 5 minutos e 24 horas a diferentes concentrações de extratos glicólicos de Hamamelis virginiana e Persea americana, Cynara scolymus L e Stryphnodendron barbatiman M, a fim de verificar a ação antifúngica da proteinase, fosfolipase e hemolisina. Resultados: Todos os extratos foram eficazes na redução do biofilme. Em contato por 5 minutos. os extratos reduziram 50% do biofilme. Após 24 horas. o extrato de Persea americana apresentou o biofilme em 90%, seguido de Cynara scolymus, que o interrompeu em 85%. Houve mudança na intensidade da proteinase após 5 minutos e 24 horas, com uma atividade enzimática média de 0,69 em comparação com o controle de 0,49. Cynara scolymus foi o extrato com maior concentração média de 100 mg/ml; a intensidade da fosfolipase foi alterada com Stryphnodendron barbatiman sendo mais efetivo em 24 horas em relação ao controle (p< 0,0001). A secreção de hemolisina foi modificada por Hamamelis virginiana (12,5 mg/ml) após 5 minutos de exposição e em 24 horas. todos os extratos foram capazes de causar alterações na secreção. Conclusão: Os extratos testados apresentam potencial antifúngico em biofilmes de Candida albicans, implicando em redução significativa dos fatores de virulência. Assim, estes podem ser indicados como uma ferramenta terapêutica alternativa para reduzir a morbidade dessas infecções, já que em ambos os tempos de exposição investigados, eles foram capazes de reduzir a secreção enzimática do fungo (AU)

Objective: Analyze the phenotypic expression of virulence factors in Candida albicans biofilms against plant glycolicextracts. Material and Methods: The biofilms of Candida albicans (ATCC 18804) obtained from incubation for 48 hours were exposed for 5 minutes and 24 hours to different concentrations of glycolic extracts of Hamamelis virginiana and Persea americana, Cynara scolymus L and Stryphnodendron barbatiman M, in order to verify the antifungal activity of the proteinase, phospholipase and hemolysin. Results: All extracts were effective in reducing biofilm. In contact for 5 minutes. the extracts reduced 50% of the biofilm. After 24 hours, the Persea americanaextract showed the biofilm at 90%, followed by Cynara scolymus, which interrupted it at 85%, There was a change in proteinase intensity after 5 minutes and 24 hours. with an average enzymatic activity of 0.69 compared to the control of 0.49. Cynara scolymus was the extract with the highest mean concentration of 100 mg/ml; the phospholipase intensity was changed with Stryphnodendron barbatiman being more effective in 24 hours compared to the control (p< 0.0001). The hemolysin secretion was modified by Hamamelis virginiana (12.5 mg/ml) after 5 minutes of exposure, and in 24 hours. all extracts were capable to cause changes in secretion. Conclusion: The tested extracts have antifungal potential in Candida albicans biofilms, implying a significant reduction in virulence factors. Thus, these can be indicated as an alternative therapeutic tool to reduce the morbidity of these infections, as in both investigated exposure times. they were able to reduce theenzymatic secretion of the fungus (AU)

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.

Comparative analysis of hydrophobicity and dentin adhesion ability in Candida albicans strains

Abstract Adhesion to dentin is a first step for a successful microbial root canal colonization. Cell hydrophobicity seems to have some influence in the Candida species adhesion to surfaces. Objective to measure cell surface hydrophobicity and to investigate the adherence ability to human dentin among Candida albicans strains isolated from root canal and lingual dorsum via an in vitro study. Methodology adhesion was quantified in function of dentin area covered by blastospores and/or hyphae presence detected by epifluorescence microscope. Cell surface hydrophobicity was estimated by assessing the percentage migration of cells from an aqueous phase to a hydrocarbon phase. Contact angles were measured by the sessile drop technique on the dentin surface using a contact angle measurements apparatus. We also examined the correlation between adhesion ability and hydrophobicity. Results although there was some intra-species variation in cell surface hydrophobicity, most isolates were characterized by moderate hydrophobicity. There was no significant difference in this parameter when the isolation niche was considered. Both root canal and lingual dorsum yeasts were able to adhere to dentin. No association was found between the strains' site of isolation and adhesion. Moreover, cell surface hydrophobicity and adhesion ability were not correlated. Conclusion although hydrophobicity can influence Candida albicans virulence in many ways, this study suggests that this parameter by itself was not a good predictor of adhesion to dentin.

Abscesos renales bilaterales multifocales por Candida albicans / Bilateral multifocal kidney abscess due to Candida al bicans

Resumen Los abscesos renales son una complicación poco frecuente de las infecciones del tracto urinario y suelen asociarse con un aumento de la morbi-mortalidad. La mayoría de los casos ocurre en pacientes con factores predisponentes como la inmunosupresión. El diagnóstico requiere de una elevada sospecha clínica y el trata miento consiste en el uso de antibióticos y antifúngicos parenterales asociados o no a intervenciones quirúrgicas como nefrostomía y nefrectomía. Son pocos los casos publicados en la literatura médi ca de abscesos renales bilaterales multifocales y menos aún por Candida albicans. Se presenta el caso de una mujer de 20 años de edad con diabetes mellitus tipo 1 diagnosticada a los 8 años, múltiples internaciones por cetoacidosis diabética y reciente internación por can didemia (Candida albicans) completando tratamiento con fluconazol por 23 días. A los 18 días de su externación, consulta por dolor en flancos de tipo sordo y síntomas ge nerales; se realizó tomografía de abdomen con contraste que mostró abscesos multifocales bilaterales. Aislándose Candida albicans en una de las muestras obtenidas de las lesiones; recibió tratamiento con fluconazol 400 mg por 6 semanas endovenoso y 2 semanas vía enteral, evolu cionando favorablemente con mejoría clínica e image nológica continuando seguimiento clínico ambulatorio. Este reporte resalta la importancia del diagnóstico y tratamiento de esta complicación infrecuente en enfer medades complejas como la diabetes.

Abstract Renal abscesses are a rare complication of urinary tract infections and may be associated with increased morbidity and mortality. Most cases occur in patients with predisposing factors such as immunosuppression. Diagnosis requires high clinical suspicion and its treat ment consists in the use of parenteral antibiotics and antifungals associated or not with surgical interventions such as nephrostomy and nephrectomy. Few cases have been published in the medical literature of multifocal bilateral renal abscesses and even fewer due to Candida albicans. We present the case of a 20-year-old woman with type 1 diabetes mellitus, diagnosed at age 8, multiple hospitalizations for diabetic ketoacidosis, and recent hospitalization for candidemia (Candida albicans) treated with fluconazole for 23 days. Eighteen days after her discharge, she consulted for dull flank pain and gen eral symptoms. Contrast enhanced abdominal tomography showed bilateral multifocal abscesses and Candida albicans was isolated in one of the samples obtained from lesions. She received fluconazole 400 mg, 6 weeks i.v. and 2 weeks via enteral route, evolving favorably with clinical and imag ing improvement, continuing outpatient clinical monitoring. This report highlights the importance of diagnosis and treatment of this rare complication in complex diseases such as diabetes mellitus.

Elimination of E. faecalis and C. albicans Biofilm: A comparison between single and multiple file systems in an Ex Vivo study

To evaluate whether the WaveOne Gold and Reciproc single file instrumentation systems, are effective in reducing the microbial load of a mixed biofilm and the cleaning of apical third compared to the Twisted File Adaptive system (multiple- file system). Seventy mesial roots of the first and second molars were included and randomly divided into three experimental groups (n=20, n=10 controls). Biofilms were formed inside canals over 31 days. After instrumentation with the unique file systems, WaveOne Gold and Reciproc and the multiple file system Twisted File Adaptive, using 2.25% sodium hypochlorite as an irrigant in all cases, a count of colony forming units was performed using serial dilutions, cleaning of the apical third was evaluated using scanning electron microscopy. Comparisons amongst groups were made by using parametric and non-parametric statistics, according to a normal or non-normal data distribution, respectively. No significant differences in the reduction of the microbial load after employing a single-file system in comparison to the multiple-file system were found; in addition, the cleaning of the apical third was similar for the three different instrumentation systems. The single-file system is equal in effectiveness compared with the multiple-file system in reducing the microbial load.

Evaluar si los sistemas de instrumentación de lima única, como WaveOne Gold y Reciproc son efectivos para reducir la carga microbiana de un biofilm mixto y la limpieza del tercio apical, comparado con los sistemas de limas múltiples, como Twisted File Adaptive. Setenta raíces mesiales de primeros y segundos molares fueron incluidos y divididos de forma aleatoria en tres grupos experimentales (n=20, n=10 controles). El biofilm fue formado en el interior de los conductos durante 31 días. Después se instrumentó con los sistemas de lima única (WaveOne Gold y Reciproc) y el sistema de limas múltiples Twisted File Adaptive, usando hipoclorito de sodio al 2.5% en todos los casos. El conteo de unidades formadoras de colonias se realizó usando diluciones seriales, la limpieza del tercio apical se evaluó empleando el microscopio electrónico de barrido. La comparación entre grupos se realizó con pruebas paramétricas y no paramétricas, de acuerdo con la distribución normal y no normal de los datos, respectivamente. No hubo una diferencia significativa en la reducción de la carga microbiana después de emplear los sistemas de lima única en comparación a los de limas múltiples, además, la limpieza del tercio apical fue similar en los 3 diferentes sistemas de instrumentación. Los sistemas de lima única son igual de efectivos para reducir la carga microbiana comparados con los sistemas de limas múltiples.

gDNA extraction from Candida albicans and Candida dubliniensis in subgingival samples in Argentina. Evaluation of different methods / Evaluación de diferentes métodos de extracción de gADN en Candida albicans y Candida dubliniensis en muestras subgingivales en Argentina

ABSTRACT The oral cavity constitutes a unique ecosystem with highly variable ecological niches that harbor a great variety of microorganisms, including yeasts. Molecular methods are currently considered the gold standard for identifying species, although they involve limitations associated with the disruption of yeast cell walls to release the genomic DNA (gDNA) for amplification. Aim: The aim of this study was to compare the performance of different methods for extracting gDNA from Candida albicans and Candida dubliniensis, subsequently amplifying DNA by PCR. Materials and Method: Fifty-two isolates (16 C. albicans and 36 C. dubliniensis) were obtained from subgingival biofilm of HIV+ patients with clinical signs of periodontal disease. The study evaluated 6 gDNA extraction methods and two PCR amplification methods. Furthermore, the presence of alleles of HWP1 gene was determined in C. albicans. Results: Comparisons of six methods show statistically significant differences (p<0.001) except for C. albicans in two of them. For C. dubliniensis, statistical differences were observed in all comparisons. Commercial methods were more efficient for concentrating gDNA than in-house methods, and both PCRs were effective. Ten heterozygous C. albicans isolates for this allele were positive for the HWP1-1 / HWP1-2 allele, one was homozygous for Wild Type HWP1-1 allele, and 5 were homozygous for novel/rare HWP1-2 allele. Conclusions: This study aims to provide simple, inexpensive strategies for phenotypic identification and molecular confirmation of Candida albicans and Candida dubliniensis for non-reference laboratories with low complexity and/or low budgets.

RESUMEN La cavidad oral constituye un ecosistema único con nichos ecológicos muy variables, capaz de albergar una gran variedad de microorganismos, incluidas las levaduras. Los métodos moleculares son considerados actualmente los métodos de identificación definitivos ya que a diferencia de los anteriores, nos brindan una correcta sensibilidad y especificidad. Sin embargo, existen limitaciones asociadas con la ruptura de las paredes celulares de estas levaduras para liberar el ADN genómico (gADN) necesario para la amplificación. Objetivo: El objetivo de este estudio fue comparar el rendimiento de diferentes métodos de extracción de gADN de Candida albicans y Candida dubliniensis, amplificando posteriormente por PCR. Materiales y Método: Se estudiaron 52 aislamientos, 16/52 de Candida albicans y 36/52 de Candida dubliniensis obtenidos de biofilm subgingival de pacientes VIH+ con signos clínicos de enfermedad periodontal. Se evaluaron seis métodos de extracción de gADN y la posterior amplificación se realizó por dos técnicas de PCR. Además en C. albicans se determinó la presencia de alelos para el gen HWP1. Resultados: Las comparaciones de seis métodos son estadísticamente significativas (p<0,001) excepto para C. albicans en dos de ellos. Para C. dubliniensis se observaron diferencias estadísticas en todas las comparaciones. Los métodos comerciales mostraron una mayor eficiencia en la concentración de gADN que los métodos caseros y ambos fueron efectivos en las dos PCR. 10 aislados de C. albicans resultaron positivos para el alelo HWP1-1/HWP1-2, siendo heterocigotos para este alelo. Solo un aislamiento fue homocigoto para el alelo HWP1-1 de tipo salvaje y 5 eran homocigotos para el alelo HWP1-2 nuevo/raro. Conclusiones: Este estudio tiene como objetivo proporcionar estrategias simples y económicas para la identificación fenotípica y confirmación molecular de Candida albicans y Candida dubliniensis para laboratorios de no referencia con baja complejidad y/o bajo presupuesto económico.

Diagnostic utility of skin prick test in fungal asthma

Background: Allergic bronchopulmonary mycosis (ABPM) is a clinical syndrome associated with immune sensitivity to various fungi. Aspergillus spp. predominates in colonizing the airways of asthmatics. Early and accurate identification of fungus in such cases can prevent worsening of asthma. Also, can help in retarding the progression of ABPM. Objectives of this study were to evaluate different fungal allergens associated with clinically diagnosed Asthma patients by Skin Prick testing (SPT), to study total IgE in asthmatic patients by serological testing and to characterize the fungal isolate associated with SPT+ cases by conventional mycological culture. Methods: A prospective study of known asthma cases was done. Their sensitivity to fungal allergens was tested by SPT. The total IgE levels were measured by ELISA. Sputum collected from SPT+ cases were subjected for fungal identification. Results: Out of 175 patients, 25 (14.2%) showed positive reaction against fungal antigens in which fungal growth was seen in 21 (84%) sputum specimens. Aspergillus fumigatus was isolated from 16 (76%) specimens followed by Candida albicans in 3 (14%) and Penicillium spp in 2 (9.5%) cases. Out of 25 SPT+ asthmatics, 21 patients with fungal growth had total IgE levels >600 IU/ml and 4 patients with negative culture had IgE levels 400-500 IU/ml. Conclusions: A significant prevalence of fungal asthma is seen among asthmatics. Thus, it is essential to screen asthma patients for fungal allergy. SPT seems to be a good screening test. SPT is easy to perform, less time consuming and inexpensive however needs to be performed under pulmonologist’s supervision.

An in vitro study of probiotic activity exhibited by Lactobacillus acidophilus and Lactobacillus rhamnosus on oral isolates of Streptococcus mutans and Candida albicans

Background: Oral infections caused by microorganisms have led to increased risk of oral health problems like dental caries (DC). Streptococcus mutans and Candida albicans are the organisms responsible for DC. The goal of the presented study was to investigate the potential of probiotics to prevent and treat DC. An in vitro assay was developed to investigate several probiotic strains for their ability to inhibit the aforementioned oral pathogens. Methods: 40 oral isolates of Streptococcus mutans and 51 oral isolates of Candida albicans were tested for probiotic activity against Lactobacillus acidophilus and Lactobacillus rhamnosus using agar overlay interference technique as prescribed by Fleming et al. Results: The zone of inhibition shown by L. acidophilus was higher than L. rhamnosus against Streptococcus mutans and Candida albicans. Conclusions: In conclusion the two probiotic strains L. acidophilus and L. rhamnosus exhibited inhibitory activity on S. mutans and C. albicans respectively in vitro.

Diagnostic utility of skin prick test in fungal asthma

Background: Allergic bronchopulmonary mycosis (ABPM) is a clinical syndrome associated with immune sensitivity to various fungi. Aspergillus spp. predominates in colonizing the airways of asthmatics. Early and accurate identification of fungus in such cases can prevent worsening of asthma. Also, can help in retarding the progression of ABPM. Objectives of this study were to evaluate different fungal allergens associated with clinically diagnosed Asthma patients by Skin Prick testing (SPT), to study total IgE in asthmatic patients by serological testing and to characterize the fungal isolate associated with SPT+ cases by conventional mycological culture. Methods: A prospective study of known asthma cases was done. Their sensitivity to fungal allergens was tested by SPT. The total IgE levels were measured by ELISA. Sputum collected from SPT+ cases were subjected for fungal identification. Results: Out of 175 patients, 25 (14.2%) showed positive reaction against fungal antigens in which fungal growth was seen in 21 (84%) sputum specimens. Aspergillus fumigatus was isolated from 16 (76%) specimens followed by Candida albicans in 3 (14%) and Penicillium spp in 2 (9.5%) cases. Out of 25 SPT+ asthmatics, 21 patients with fungal growth had total IgE levels >600 IU/ml and 4 patients with negative culture had IgE levels 400-500 IU/ml. Conclusions: A significant prevalence of fungal asthma is seen among asthmatics. Thus, it is essential to screen asthma patients for fungal allergy. SPT seems to be a good screening test. SPT is easy to perform, less time consuming and inexpensive however needs to be performed under pulmonologist’s supervision.

An in vitro study of probiotic activity exhibited by Lactobacillus acidophilus and Lactobacillus rhamnosus on oral isolates of Streptococcus mutans and Candida albicans

Background: Oral infections caused by microorganisms have led to increased risk of oral health problems like dental caries (DC). Streptococcus mutans and Candida albicans are the organisms responsible for DC. The goal of the presented study was to investigate the potential of probiotics to prevent and treat DC. An in vitro assay was developed to investigate several probiotic strains for their ability to inhibit the aforementioned oral pathogens. Methods: 40 oral isolates of Streptococcus mutans and 51 oral isolates of Candida albicans were tested for probiotic activity against Lactobacillus acidophilus and Lactobacillus rhamnosus using agar overlay interference technique as prescribed by Fleming et al. Results: The zone of inhibition shown by L. acidophilus was higher than L. rhamnosus against Streptococcus mutans and Candida albicans. Conclusions: In conclusion the two probiotic strains L. acidophilus and L. rhamnosus exhibited inhibitory activity on S. mutans and C. albicans respectively in vitro.

Epithelial responses and Candida albicans pathogenicity are enhanced in the presence of oral streptococci

Abstract Experimental models that consider host-pathogen interactions are relevant for improving knowledge about oral candidiasis. The aim of this study was to assess the epithelial immune responses, Candida penetration of cell monolayers, and virulence during mixed species culture infections. Single species cultures of Candida albicans and mixed cultures (C. albicans, Streptococcus mutans, and Streptococcus sanguinis) were used to infect monolayers of HaCaT and FaDu ATCC HTB-43 cells for 12 h. After infection, IL-18 and IL-34 gene expression was measured to assess epithelial cell immune responses, and lactate dehydrogenase (LDH) activity was measured as an indicator of cell damage. Microscopy determined C. albicans morphology and penetration of fungal cells through the keratinocyte monolayer. Monolayers devoid of infection served as controls. Data were analyzed by an ANOVA one-way test followed by Tukey's post-hoc test (α = 0.05). The results found that IL-18 and IL-34 gene expression and LDH activity were significantly (p < 0.05) upregulated for both cell lines exposed to mixed species cultures compared with C. albicans alone. Candida albicans yeast and hyphae were evident in C. albicans only infections. In contrast, monolayers infected by C. albicans, S. mutans, and S. sanguinis exhibited higher microbial invasion with several hyphal aggregates detected. The presence of streptococci in C. albicans infection enhances the virulence and pathogenicity of the fungus with associated increased immune responses and tissue damage. Extrapolation of these findings to oral infection would indicate the added potential benefit of managing bacterial components of biofilms during treatment.

Resumo O objetivo deste estudo foi avaliar a resposta epithelial imune, a colonização da Candida albicans em monocamadas celulares e sua virulência em resposta a infecções de culturas de biofilme multiespécie. Culturas de biofilme monoespécie de C. albicans e culturas mistas (C. albicans, Streptococcus mutans e Streptococcus sanguinis) foram utilizadas para infectar monocamadas de células HaCaT e FaDu por 12 h. Após a infecção, a expressão dos genes IL-18 e IL-34 foi medida para avaliar as respostas imunes das células epiteliais. A atividade da lactato desidrogenase (LDH) foi medida como um indicador de dano celular. A microscopia determinou a morfologia de C. albicans e a penetração das células fúngicas através da monocamada de queratinócitos. Monocamadas em que não houve infecção serviram como controles. Os dados foram analisados por um teste ANOVA one-way seguido pelo teste post-hoc de Tukey (α = 0,05). Os resultados demonstraram que a expressão gênica de IL-18 e IL-34 e a atividade de LDH foram (p < 0,05) reguladas positivamente para ambas as linhagens de células expostas a culturas de espécies mistas em comparação com C. albicans isoladamente. Leveduras de C.albicans e hifas foram evidentes em infecções apenas por C. albicans. Entretanto, monocamadas infectadas por C. albicans, S. mutans e S. sanguinis exibiram maior invasão microbiana com vários agregados de hifas detectados. Dessa maneira, a presença de estreptococos na infecção por C. albicans aumentou a virulência e a patogenicidade do fungo com respostas imunes aumentadas associadas a danos nos tecidos. A extrapolação desses achados para a infecção oral indicaria o potencial benéfico do controle dos componentes bacterianos em biofilmes durante a terapia da candidíase