Anadenanthera Colubrina vell Brenan: anti-Candida and antibiofilm activities, toxicity and therapeutical action

Abstract: We evaluated the antifungal and antibiofilm potential of the hydroalcoholic extract of bark from Anadenanthera colubrina (vell.) Brenan, known as Angico, against Candida spp. Antifungal activity was evaluated using the microdilution technique through the Minimum Inhibitory and Fungicide Concentrations (MIC and MFC). The antibiofilm potential was tested in mature biofilms formed by Candida species and analyzed through the counting of CFU/mL and scanning electron micrograph (SEM). In vivo toxicity and therapeutic action was evaluated in the Galleria mellonella model. The treatment with the extract, in low doses, was able to reduce the growth of planktonic cells of Candida species. MIC values range between 19.5 and 39 µg/mL and MFC values range between 79 and 625 µg/mL. In addition was able to reduce the number of CFU/mL in biofilms and to cause structural alteration and cellular destruction, observed via SEM. A. colubrina showed low toxicity in the in vivo assay, having not affected the viability of the larvae at doses below 100mg/kg and high potential in the treatment of C. albicans infection. Considering its high antifungal potential, its low toxicity and potential to treatment of infections in in vivo model, A. colubrina extract is a strong candidate for development of a new agent for the treatment of oral candidiasis.

Interactions between Terpinen-4-ol and Nystatin on biofilm of Candida albicans and Candida tropicalis

Abstract The aim of this study was to evaluate the antifungal activity of Terpinen-4-ol associated with nystatin, on single and mixed species biofilms formed by Candida albicans and Candida tropicalis, as well as the effect of terpinen-4-ol on adhesion in oral cells and the enzymatic activity. The minimum inhibitory concentrations and minimum fungicide concentrations of terpinen-4-ol and nystatin on Candida albicans and Candida tropicalis were determined using the microdilution broth method, along with their synergistic activity ("checkerboard" method). Single and mixed species biofilms were prepared using the static microtiter plate model and quantified by colony forming units (CFU/mL). The effect of Terpinen-4-ol in adhesion of Candida albicans and Candida tropicalis in coculture with oral keratinocytes (NOK Si) was evaluated, as well as the enzymatic activity by measuring the size of the precipitation zone, after the growth agar to phospholipase, protease and hemolysin. Terpinen-4-ol (4.53 mg mL-1) and nystatin (0.008 mg mL-1) were able to inhibit biofilms growth, and a synergistic antifungal effect was showed with the drug association, reducing the inhibitory concentration of nystatin up to 8 times in single biofilm of Candida albicans, and 2 times in mixed species biofilm. A small decrease in the adhesion of Candida tropicalis in NOK Si cells was showed after treatment with terpinen-4-ol, and nystatin had a greater effect for both species. For enzymatic activity, the drugs showed no action. The effect potentiated by the combination of terpinen-4-ol and nystatin and the reduction of adhesion provide evidence of its potential as an anti-fungal agent.

Resumo O objetivo desse estudo foi avaliar a atividade antifúngica do Terpinen4-ol associado à nistatina em biofilmes simples e misto, formados por Candida albicans e Candida tropicalis, bem como o efeito do terpinen-4-ol na adesão em células orais e atividade enzimática. As concentrações inibitórias mínimas e as concentrações fungicidas mínimas do terpinen-4-ol e da nistatina em Candida albicans e Candida tropicalis foram determinadas pelo método de microdiluição em caldo, juntamente com a atividade sinérgica (método do tabuleiro de "xadrez"). Biofilmes simples e misto foram preparados usando o modelo de placa de microtitulação estática e quantificados por unidades formadoras de colônias (CFU/mL). O efeito do Terpinen-4-ol na adesão de Candida albicans e Candida tropicalis em co-cultura com queratinócitos orais (NOK Si) foi avaliado, bem como a atividade enzimática, medindo o tamanho da zona de precipitação, após o crescimento em ágar fosfolipase, protease e hemolisina. O terpinen-4-ol (4.53 mg mL-1) e a nistatina (0,008 mg mL-1) conseguiram inibir o crescimento de biofilmes e um efeito antifúngico sinérgico foi demonstrado com a associação de fármaco, reduzindo a concentração inibidora de nistatina até 8 vezes em biofilme simpes de Candida albicans e 2 vezes em biofilme misto. Uma pequena diminuição na adesão de Candida tropicalis em células NOK Si foi mostrada após o tratamento com terpinen-4-ol e a nistatina teve um efeito maior para ambas as espécies. Para a atividade enzimática, as drogas não apresentaram ação. O efeito potencializado pela combinação de terpinen-4-ol e nistatina e a redução de adesão evidenciam seu potencial como agente anti-fúngico.

Activity of the aqueous extract of Schinus terebinthifolius Raddi on strains of the Candida genus / Atividade do extrato aquoso de Schinus terebinthifolius Raddi sobre cepas do gênero Candida

ABSTRACT Objectives: To evaluate the antifungal susceptibility profile of the aqueous extract of the bark of Schinus terebinthifolius Raddi against the strains of the genus Candida. Methods: By using the disk diffusion method, 50 samples of the genus Candida (Candida albicans; Candida krusei; Candida glabrata; and Candida tropicalis), isolated from patients receiving treatment at Hospital Santa Casa de Misericórdia de São Paulo, and 1 American Type Culture Collection (ATCC) sample of each species were tested against: the isolated aqueous extract of the bark of Schinus terebinthifolius Raddi, isolated nystatin, and the association of nystatin and the aqueous extract of Schinus terebinthifolius Raddi. Results: There were no significant differences regarding the different strains of Candida tested. In the presence of the aqueous extract of Schinus terebinthifolius Raddi, no inhibition halo was visible. Isolated nystatin formed an inhibition halo measuring respectively 18.50 mm and 19.50 mm for the Candida albicans species and the others referred to as non-Candida albicans (Candida krusei; Candida glabrata; and Candida tropicalis). The association of nystatin and the aqueous extract of Schinus terebinthifolius Raddi resulted in inhibition halos measuring 14.25 mm and 16.50 mm respectively. The comparisons of these results are statistically significant (p < 0,001). Conclusion: The aqueous extract of Schinus terebinthifolius Raddi showed no antifun-gal activity in vitro against the strains tested, whereas the association of nystatin and the aqueous extract of Schinus terebinthifolius Raddi caused a decrease in the inhibition halo when compared with isolated nystatin.

RESUMO Objetivos: Avaliar o perfil de susceptibilidade antifúngica do extrato aquoso das cascas de Schinus terebinthifolius Raddi frente às cepas do gênero Candida. Métodos: Por meio do método de difusão em disco, 50 amostras do gênero Candida (Candida albicans, Candida krusei, Candida glabrata e Candida tropicalis) provenientes de pacientes do Hospital da Santa Casa de Misericórdia de São Paulo, e 1 amostra American Type Culture Collection (ATCC) de cada espécie foram testadas frente ao extrato aquoso das cascas de Schinus terebinthifolius Raddi isolado, nistatina isolada, e a associação da nistatina ao extrato aquoso de Schinus terebinthifolius Raddi. Resultados: Não houve diferenças significantes em relação às diferentes espécies de cepas de Candida testadas. O extrato aquoso de Schinus terebinthifolius Raddi não formou halo de inibição. A nistatina isolada formou halo de inibição de 18,50 mm e 19,50 mm respectivamente para as espécies Candida albicans e as demais nomeadas como não Candida albicans (Candida krusei, Candida glabrata e Candida tropicalis). A associação da nistatina ao extrato aquoso de Schinus terebinthifolius Raddi resultou no halo de inibição de 14,25 mm e 16,50 mm respectivamente, sendo que as comparações destes resultados são estatisticamente significantes (p < 0,001). Conclusão: O extrato aquoso de Schinus terebinthifolius Raddi não demonstrou propriedade antifúngica in vitro frente às cepas testadas, e a associação da nistatina ao extrato aquoso de Schinus terebinthifolius Raddi causou a diminuição do halo de inibição quando comparado à nistatina isolada.

Efeito da incorporação de agentes antifúngicos na resistência à tração e porosidade de materiais resilientes temporários para base de próteses / Effect of incorporation of antifungal agents on the ultimate tensile strength and porosity of temporary soft denture liners

Este estudo investigou a resistência à tração (ou limite de resistência à tração- LRT) e a porosidade de reembasadores resilientes temporários modificados por concentrações inibitórias mínimas (CIMs) de agentes antifúngicos para o biofilme Candida albicans (SC5314). Para os testes de LRT, corpos de prova em forma de halteres (n=7) com uma área transversal de 33 mm x 6 mm x 3 mm foram produzidos para os materiais resilientes (Trusoft e Softone) sem (controle) ou com incorporação de cinco fármacos em suas CIMs: nistatina- 0,032 g; diacetato de clorexidina- 0,064; cetoconazol- 0,128 g; miconazol- 0,256 g; itraconazol-0,256 g (grama de fármaco por grama de pó de material resiliente). Após a plastificação, as amostras foram imersas em água destilada a 37°C durante 24 h, 7 e 14 dias e, então, testadas em tensão em uma máquina universal de ensaios (EMIC DL-500 MF) a 40 mm/min. A porosidade foi mensurada por absorção de água, com base na exclusão do efeito plastificante. Inicialmente, determinou-se por isotermas de sorção, que a solução de armazenagem adequada para os corpos de prova (65 mm x 10 mm x 3,3 mm) de ambos os materiais foi o cloreto de cálcio anidro a 50% (S50). Assim, o fator de porosidade (FP) foi calculado para os grupos de estudo (n=10) formados por espécimes sem (controle) ou com incorporação de fármaco em suas CIMs (nistatina, clorexidina ou cetoconazol) após a armazenagem em água destilada ou S50 por 24 h, 7 e 14 dias. Os dados de resistência à tração (MPa) e percentagem de alongamento (%) foram submetidos à ANOVA de 3 fatores seguida pelo teste de Tukey (=0,05). Os dados de porosidade foram analisados estatisticamente por ANOVA de medidas repetidas para 4 fatores e teste de Tukey (=0,05). Ao final de 14 dias, a resistência à tração para ambos os materiais foi significativamente menor nos grupos modificados pelo miconazol e itraconazol em relação aos outros grupos (P<0,0001), que não mostraram diferenças significativas entre si (P>0,05). Após 7 e 14 dias em água, o miconazol e itraconazol adicionados a ambos os materiais resultaram em percentagens significativamente menores de alongamento em comparação com os outros fármacos e ao controle (P<0,0001), que foram semelhantes entre si (P>0,05). O cetoconazol não resultou em alterações significativas no FP para ambos os materiais resilientes em água ao longo de 14 dias (P>0,05). Em comparação aos controles, houve aumento dos FPs do Softone e Trusoft aos 14 dias de imersão em água somente após a adição de nistatina e clorexidina e de clorexidina, respectivamente (P<0,05). Ambos os materiais não apresentaram alterações significativas no FP em até 14 dias de imersão na S50, em comparação aos controles (P>0,05). Em todas as condições experimentais, os FPs do Softone e Trusoft foram significativamente menores quando imersos em S50 em comparação com a água destilada (P<0,05). Concluiu-se que a adição de nistatina, clorexidina e cetoconazol nas CIMs para o biofilme de C. albicans não resultou em efeitos deletérios na resistência à tração e na percentagem de alongamento dos materiais resilientes temporários para base de prótese até o período de 14 dias. A adição de antifúngicos nas CIMs não resultou em efeitos adversos à porosidade de ambos os materiais resilientes temporários em diferentes períodos de imersão em água, com exceção da clorexidina e nistatina no Softone e clorexidina no Trusoft aos 14 dias. Não foram observados efeitos deletérios para a porosidade de ambos os materiais resilientes modificados com as CIMs dos fármacos durante os 14 dias de imersão na S50.(AU)

This study investigated the tensile strength (ultimate tensile strength- UTS) and porosity of temporary soft denture liners modified by minimum inhibitory concentrations (MICs) of antifungal agents for Candida albicans biofilm (SC5314). For UTS tests, dumbbell-shaped specimens (n=7) with a central cross-sectional area of 33 mm x 6 mm x 3 mm were produced by resilient materials (Trusoft and Softone) without (control) or with incorporation of five drugs at MICs: nystatin- 0.032 g; chlorhexidine diacetate-0.064 g; ketoconazole- 0.128 g; miconazole- 0.256 g; itraconazole- 0.256 g (each per gram of soft liner powder). After plasticization, specimens were immersed in distilled water at 37°C for 24 h, 7 and 14 days, and then tested in tension in a universal testing machine (EMIC DL-500 MF) at 40 mm/min. The porosity was measured by water absorption, based on exclusion of the plasticizer effect. Initially, it was determined by sorption isotherms that the adequate storage solution for specimens (65 mm x 10 mm x 3.3 mm) of both materials was 50% anhydrous calcium chloride (S50). Then, the porosity factor (PF) was calculated for the study groups (n=10) formed by specimens without (control) or with drug incorporation at MICs (nystatin, chlorhexidine or ketoconazole) after storage in distilled water or S50 for 24 h, 7 and 14 days. Data of tensile strength (MPa) and elongation percentage (%) were submitted to 3-way ANOVA followed by Tukey's test (=0.05). Data of porosity were statistically analyzed by 4-way repeated measures ANOVA and Tukeys test (=0.05). At the end of 14 days, the tensile strength for both materials was significantly lower in the groups modified by miconazole and itraconazole compared to the other groups (P<0.0001), which showed no significant difference between them (P>0.05). After 7 and 14 days in water, miconazole and itraconazole added into both materials result in significant lower elongation percentages compared to the other drugs and control (P<.0001), which were similar to each other (P>0.05). Ketoconazole resulted in no significant changes in PF for both liners in water over 14 days (P>0.05). Compared to the controls, Softone and Trusoft PFs were increased at 14-day water immersion only after addition of nystatin and chlorhexidine, and chlorhexidine, respectively (P<0.05). Both materials showed no significant changes in PF in up to 14 days of S50 immersion, compared to the controls (P>0.05). In all experimental conditions, Softone and Trusoft PFs were significantly lower when immersed in S50 compared to distilled water (P<0.05). It was concluded that the addition of the nystatin, chlorhexidine and ketoconazole at MICs for C. albicans biofilm resulted in no harmful effects on the ultimate tensile strength and elongation percentage of the temporary soft denture liners up to 14-day period. The addition of antifungals at MICs resulted in no detrimental effects for the porosity of both temporary soft liners in different periods of water immersion, except for chlorhexidine and nystatin in Softone and chlorhexidine in Trusoft at 14 days. No deleterious effect was observed for the porosity of both soft liners modified by the drugs at MICs over 14 days of S50 immersion.(AU)

Impact of brief exposure to antifungal agents on the post-antifungal effect and hemolysin activity of oral Candida albicans

AbstractPost-antifungal effect (PAFE) of Candida and its production of hemolysin are determinants of candidal pathogenicity. Candida albicans is the foremost aetiological agent of oral candidosis, which can be treated with polyene, azole, and echinocandin antifungals. However, once administered, the intraoral concentrations of these drugs tend to be subtherapeutic and transient due to the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intra-orally, Candidamay undergo a brief exposure to antifungal drugs.Objective Therefore, the PAFE and hemolysin production of oral C. albicans isolates following brief exposure to sublethal concentrations of the foregoing antifungals were evaluated.Material and Methods A total of 50 C. albicans oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to sublethal concentrations of nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole for 60 min. Thereafter, the drugs were removed and the PAFE and hemolysin production were determined by previously described turbidometric and plate assays, respectively.Results Nystatin, amphotericin B, caspofungin and ketoconazole induced mean PAFE (hours) of 2.2, 2.18, 2.2 and 0.62, respectively. Fluconazole failed to produce a PAFE. Hemolysin production of these isolates was suppressed with a percentage reduction of 12.27, 13.47, 13.33, 8.53 and 4.93 following exposure to nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole, respectively.Conclusions Brief exposure to sublethal concentrations of antifungal drugs appears to exert an antifungal effect by interfering with the growth as well as hemolysin production of C. albicans.

Evaluation of antimicrobial activity and cell viability of Aloe vera sponges

Background: Aloe vera L., member of the Liliaceae family, has been shown to stimulate cell proliferation and contribute to healing and angiogenesis, has anti-bacterial, anti-fungal and anti-inflammatory activity. In addition, Aloe vera can be used as a support for drug transport. Our objective is to evaluate antimicrobial activity and cytotoxicity of sponges of Aloe vera L. for use as a carrying support of drugs. Results: In this work, sponge of free Aloe vera (AV) loaded with amoxicillin (AMX) or nystatin (NYS) at 1% w/w, were prepared and physico-chemically characterized via X-ray diffraction, Fourier Transform Infrared Spectroscopy and thermal analysis. Antimicrobial potency of AV sponge alone, loaded with AMX or NYS, against strains of Streptococcus mutans, Staphylococcus aureus, Aggregatibacter actinomycetemcomitans, Enterococcus faecalis and Candida albicans was determined. Osteoblasts and human gingival fibroblasts were cultivated on AV, Aloe vera loaded with amoxicillin (AV/AMX) and Aloe vera loaded with nystatin (AV/NYS) and cellular viability was assessed. The physico-chemical characterization performed suggested that the loaded drugs were dispersed in the sponge and those interactions between the AV sponge and the loaded drugs were weak. Furthermore, AV loaded with AMX or NYS demonstrated antimicrobial potency and osteoblasts and fibroblasts were viable after 24 hrs on free AV, and AV loaded with AMX or NYS. Conclusions: Our results indicate that sponges of free AV, loaded with AMX or NYS, are biocompatible and exhibit antimicrobial activity.

Exopolysaccharide matrix of developed candida albicans biofilms after exposure to antifungal agents

This study aimed to evaluate the effects of fluconazole or nystatin exposure on developed Candida albicans biofilms regarding their exopolysaccharide matrix. The minimal inhibitory concentration (MIC) against fluconazole or nystatin was determined for C. albicans reference strain (ATCC 90028). Poly(methlymethacrylate) resin (PMMA) specimens were fabricated according to the manufacturer's instructions and had their surface roughness measured. Biofilms were developed on specimens surfaces for 48 h and after that were exposed during 24 h to fluconazole or nystatin prepared in a medium at MIC, 10 x MIC or 100 x MIC. Metabolic activity was evaluated using an XTT assay. Production of soluble and insoluble exopolysaccharide and intracellular polysaccharides was evaluated by the phenol-sulfuric method. Confocal laser scanning microscope was used to evaluate biofilm architecture and percentage of dead/live cells. Data were analyzed statistically by ANOVA and Tukey's test at 5% significance level. The presence of fluconazole or nystatin at concentrations higher than MIC results in a great reduction of metabolic activity (p<0.001). At MIC or 10 x MIC, fluconazole showed high amounts of intracellular polysaccharides (p<0.05), but did not affect the exopolysaccharide matrix (p>0.05). The exposure to nystatin also did not alter the exopolysaccharide matrix at all the tested concentrations (p>0.05). Biofilm architecture was not affected by either of the antifungal agents (p>0.05). Nystatin promoted higher proportion of dead cells (p<0.05). It may be concluded that fluconazole and nystatin above the MIC concentration reduced the metabolic activity of C. albicans biofilms; however, they were not able to alter the exopolysaccharide matrix and biofilm architecture.

Este estudo avaliou o efeito da exposição de fluconazol ou nistatina a biofilmes de Candida albicans desenvolvidos, considerando a matriz de polissacarídeos extracelulares. Inicialmente uma cepa referência de C. albicans (ATCC 90028) foi submetida ao teste de concentração inibitória mínima (CIM) utilizando-se o fluconazol ou nistatina como agentes antifúngicos. Após, espécimes foram confeccionados em resina acrílica de polimetilmetacrilato (PMMA) de acordo com as recomendações do fabricante e tiveram sua rugosidade de superfície padronizada. Após, biofilmes de C. albicans foram desenvolvidos na superfície dos espécimes durante 48 h. Em seguida, os biofilmes foram expostos a fluconazol ou nistatina nas concentrações de CIM, 10 x CIM ou 100 x CIM, por 24 h. A atividade metabólica dos biofilmes foi avaliada pelo teste de XTT. A produção de polissacarídeos extracelulares solúveis e insolúveis, bem como dos polissacarídeos intracelulares foi avaliada pelo método fenol-sulfúrico. A arquitetura dos biofilmes e proporção de células vivas e mortas foi investigada utilizando-se microscopia confocal a laser. Os resultados foram analisados por ANOVA seguido do teste de Tukey, utilizando-se o nível de significância de 5%. A presença do fluconazol ou nistatina em concentrações maiores que CIM resultaram em uma redução significativa da atividade metabólica (p<0,001). Nas concentrações de CIM e 10 x CIM, biofilmes expostos ao fluconazol apresentaram quantidades significativas de polissacarídeos intracelulares (p<0,05), enquanto não houve alterações na quantidade de polissacarídeos extracelulares (p>0,05). A presença de nistatina também não alterou a matriz de polissacarídeos extracelulares em todas as concentrações investigadas (p>0,05). A arquitetura dos biofilmes não foi afetada por ambos os agentes antifúngicos, em qualquer concentração testada (p>0,05). A nistatina apresentou maior proporção de células mortas (p<0,05). Conclui-se que tanto para o fluconazol quanto para a nistatina, concentrações maiores que CIM reduziram a atividade metabólica dos biofilmes de C. albicans; no entanto, tais concentrações não alteraram a matriz de polissacarídeos extracelulares nem a arquitetura dos biofilmes.

In vitro antifungal susceptibility of Candida spp. oral isolates from HIV-positive patients and control individuals

Oropharyngeal candidiasis is the most common fungal infection among HIV-positive patients. This condition can be treated with either systemic or topical antifungal agents; treatments are usually indicated empirically on the basis of clinical data. The knowledge of in vitro antifungal susceptibility is important to determine correct therapeutic guides for the treatment of fungal infections. Therefore, the objective of this study was to determine the antifungal susceptibility profile of oral Candida isolates from HIV-positive patients and control individuals. Amphotericin B, fluconazole, flucytosine, nystatin and ketoconazole were tested according to the methodology of microdilution proposed by the Clinical and Laboratory Standards Institute (CLSI); results were recorded in values of minimal inhibitory concentration (MIC). A total of 71 Candida isolates from HIV-positive patients were examined with the following species represented: C. albicans (59), C. tropicalis (9), C. glabrata (1), C. guilliermondii (1) and C. krusei (1). A total of 15 Candida isolates were evaluated from control individuals comprised of 11 C. albicans and 4 C. tropicalis samples. Our results demonstrated that the tested antifungal agents showed good activity for most isolates from both groups; however, variability in MIC values among isolates was observed.

Susceptibilidad a la nistatina de aislamientos bucales de Candida y su correlación con la respuesta al tratamiento / Susceptibility to nystatin of oral Candida isolates and its correlation with the response to treatment

INTRODUCCIÓN: la candidiasis bucofaríngea es un marcador precoz de progresión a sida en los pacientes seropositivos al VIH sin tratamiento antirretroviral, así como un indicador de no adherencia al tratamiento antirretroviral o su posible fracaso. OBJETIVO: determinar la susceptibilidad frente a nistatina de las cepas de Candida aisladas de la cavidad bucal de pacientes seropositivos al VIH, y correlacionar los resultados con la respuesta al tratamiento. MÉTODOS: se realizó un estudio de susceptibilidad in vitro frente a nistatina para 104 cepas aisladas de lesiones de candidiasis bucofaríngea de 97 pacientes VIH/sida que habían participado, como voluntarios, en un ensayo clínico para evaluar la respuesta a cuatro drogas. De los 104 aislamientos, 58 fueron obtenidos antes de iniciar los esquemas de tratamientos y 46 una vez finalizado estos. Se procedió según el micrométodo el documento M27-A3 del Clinical and Laboratory Standards Institute. RESULTADOS: todas las cepas se comportaron como sensibles a la nistatina, con valores de concentración mínima inhibitoria (CMI) menores que 16 µg/mL, independientemente del momento de su aislamiento y de la droga administrada a los pacientes. En el grupo de pacientes tratados con nistatina, los rangos de CMI80 (1-2 µg/mL), las medias geométricas y los valores acumulativos de la droga frente a los aislamientos de Candida albicans obtenidos antes de iniciar el tratamiento, resultaron similares a los obtenidos frente a las cepas recobradas al finalizar este. De todas las especies identificadas, C. lusitaniae fue la que alcanzó el valor absoluto de CMI más elevado, 4 µg/mL. CONCLUSIONES: la correlación entre la evolución clínica de los pacientes y los resultados de los estudios de susceptibilidad de sus aislamientos in vitro, fue buena para los pacientes con evolución favorable, pero no para predecir posibles fracasos terapéuticos.

INTRODUCTION: oropharyngeal candidiasis is an early marker of progression to AIDS in HIV-positive patients and an indicator of non-adherence of treatment or possible failure in patients undergoing anti-retroviral therapy. OBJECTIVE: to determine the in vitro susceptibility to nystatin in Candida strains isolated from the oral cavity of HIV-positive patients, and to correlate the results with the response to treatment. METHODS: a study of in vitro susceptibility to nystatin was conducted in 104 oral isolates from 97 HIV/AIDS patients, who participated as volunteers in a clinical trial to evaluate the response to four antifungal drugs. Fifty-eight of the 104 isolates were obtained before starting treatment and the remaining 46 at the end of therapy. Broth microdilution method was performed according to the document M27-A3 of the Clinical and Laboratory Standards Institute. RESULTS: one hundred percent of the strains were susceptible to nystatin, with minimum inhibitory concentration (MIC) values of <16 µg/mL, regardless of the time of isolation and of the drug administered to patients. In the group of patients treated with nystatin, the MIC80 ranges (1-2 µg/mL), geometric mean and cumulative values of the drug against Candida albicans isolates before starting treatment were similar to those obtained against strains recovered at the end. Among the identified species, C. lusitaniae reached the highest absolute MIC value (4 µg/mL). CONCLUSIONS: the correlation between the clinical evolution and the results of in vitro susceptibility tests was good in patients with a favorable outcome; however, it did not allow predicting possible treatment failure.

Alterations in growth and branching of Neurospora crassa caused by sub-inhibitory concentrations of antifungal agents / Alteraciones de crecimiento y ramificación en Neurospora crassa provocadas por concentraciones subinhibitorias de agentes antimicóticos

Six antifungal agents at subinhibitory concentrations were used for investigating their ability to affect the growth and branching in Neurospora crassa. Among the antifungals herein used, the azole agent ketoconazole at 0.5 μg/ml inhibited radial growth more than fluconazole at 5.0 μg/ml while amphotericin B at 0.05 μg/ml was more effective than nystatin at 0.05 μg/ml. Morphological alterations in hyphae were observed in the presence of griseofulvin, ketoconazole and terbinafine at the established concentrations. The antifungal agents were more effective on vegetative growth than on conidial germination. Terbinafine markedly reduced growth unit length (GU) by 54.89%, and caused mycelia to become hyperbranched. In all cases, there was a high correlation between hyphal length and number of tips (r > 0.9). All our results showed highly significant differences by ANOVA, (p < 0.001, α = 0.05). Considering that the hyphal tip is the main interface between the fungus and its environment /through which enzymes and toxins are secreted and nutrients absorbed, it would not be desirable to obtain a hyperbranched mycelia with inefficient doses of antifungal drugs.

Se investigó el efecto de seis agentes antimicóticos en concentraciones subinhibitorias sobre el crecimiento y la ramificación en Neurospora crassa. El agente azólico ketoconazol a la concentración de 0,5 μg/ml inhibió el crecimiento radial más que el fluconazol a 5,0 μg/ml, y la anfotericina B a 0,05 μg/ ml fue más eficiente que 0,05 μg/ml de nistatina, entre los agentes poliénicos usados. En presencia de griseofulvina, ketoconazol y terbinafina a las concentraciones establecidas se observaron alteraciones morfológicas en las hifas. Los agentes antimicóticos fueron más eficientes sobre el crecimiento vegetativo que sobre la germinación conidial. La terbinafina redujo marcadamente (54,89%) la longitud de la unidad de crecimiento y provocó la hiperramificación del micelio. En todos los casos, existió gran correlación entre la longitud y el número de ápices de las hifas (r > 0,9). Todos los resultados mostraron diferencias altamente significativas de acuerdo con ANOVA (p < 0,001, α = 0,05). Considerando que el ápice de la hifa es la principal interfase entre el hongo y su ambiente, a través de la cual las enzimas y las toxinas son secretadas y los nutrientes son absorbidos, un micelio hiperramificado resultante de dosis ineficientes de agentes antimicóticos sería perjudicial.

Estomatitis subprótesis: estudio clínico y microbiológico de candida / Denture stomatitis: clinical and microbiological study of candida

La estomatitis subprótesis es una patología asociada al uso de prótesis dentales removibles. Su diagnóstico es fundamentalmente clínico y se basa en el reconocimiento de sus lesiones, siendo una de la clasificación de Newton una de las más aceptadas. Además, el diagnóstico debe ser confirmado por la observación microscópica de Candida en las muestras orales. El objetivo de este estudio fue determinar las especies de Candida más frecuentes en la mucosa del paladar y determinar la susceptibilidad in vitro de estas cepas a Nistatina y a Fluconazol. Se examinaron un total de 100 pacientes portadores de prótesis removibles determinando la presencia o ausencia de ES según la clasificación de Newton. A cada paciente se le tomó una muestra de la zona palatina y se le realizó el examen microbiológico. El 75 por ciento de los pacientes presentó alteraciones en la mucosa palatina compatibles con ES (33 por ciento presentó ES tipo I, 42,7 por ciento tipo II y 24 por ciento tipo III; mientras que un 25 por ciento presentó una mucosa sana. EL 53,3 por ciento de los pacientes con ES presentó cultivo positivo para Candida, mientras que un16 por ciento en la mucosa normal. Las especies de Candida albicans fueron las más frecuentemente aislada (75 por ciento), seguida por la C. tropicallis (15 por ciento) y en el resto de los cultivos se presentaron ambas especies (10 por ciento). El 100 por ciento de las cepas estudiadas fueron sensibles a Nistatina y Fluconazol, lo que se evidenció por la formación de un halo de inhibición amplio entre las levaduras. Existe una correlación positiva entre la ES y la presencia del género Candida, siendo la especie C. albicans quien se aisló conmayor frecuencia desde la mucosa palatina de pacientes con ES y sin ella. Todas las levaduras encontradas fueron susceptibles a Nistatina y Fluconazol in vitro, demostrando su efectividad fungicida ante especies del género Candida.

Denture stomatitis (DS) is a condition associated with the use of removable dentures. Its diagnosis is mainly clinical and based in recognition of their injuries, being the Newton’s one of the most accepted classifications. It stresses that the diagnosis should be confirmed by microscopic observation of Candida in oral samples. The aim of this study was to determine the species of Candida more frequent in the palatal mucosa and determine in vitro susceptibility to Nystatin and Fluconazole of these isolated strains. A total of 100 patients were examined by identifying the removable prosthesis presence or absence of DS according to the classification of Newton. Each patient had a complete sample of the area and palate was carried out microbiological examination. 75 percent of patients had abnormalities in the palate mucosa compatible with DS (33 percent presented DS type I, type II 42.7 percent and 24 percent Type III), while 25 percent filed a healthy mucosa. In 53.3 percent of patients with DS presented positive culture for Candida, only 16% had positive culture in the normal mucosa. The species of Candida albicans were the most frequently isolated (75 percent ), followed by C. tropicallis (15 percent ) and the rest of the crops were presented both species (10 percent ). 100 percent of the strains were sensitive to Nistatina and Fluconazole, which was evidenced by the formation of inhibition area in yeast. There is a positive correlation between the DS and the presence of Candida, being C. albicans the kind who was isolated most frequently from the mucosa of patients with DS palate and healthy patients. All yeast were found susceptible to Nystatin and Fluconazole in vitro, demonstrating its effectiveness in Candida.

In vitro effect of nystatin, gentian violet and garlic extract on candida albicans isolates from oral cavity

To evaluate in-vitro anticandidal activity of Nystatin. Gentian Violet and Garlic extract on Candida albicans isolates from oral cavity. For screening of antifungal activity of Nystatin. Gentian Violet and Garlic Extract, 100 clinical isolates were collected from the department of Radiotherapy. Jinnah Postgraduate Medical Center and Fatima Jinnah Dental College and Hospital, Karachi. The samples were collected from the oral cavity and the sample site was buccal mucosa, tongue and hard palate. These isolates were finally identified on the basis of morphology and cultural characteristics and confirmed using germ tube method. The antifungal activity of Nystatin, Gentian Violet and Garlic Extract were carried out by disc diffusion method. Result showed that the mean zone of inhibition of Nystatin was 17.9mm, Gentian violet was 16.6mm and Garlic Extract was 16.6mm and all the drugs used were found to be very effective. It can be concluded from the study that all the drugs are very effective against clinical isolates of Candida albicans and secondly the organism had not developed any resistance to these drugs

Susceptibilidad in vitro de aislamientos vaginales de Candida frente a clotrimazol y nistatina

Se determinó la susceptibilidad in vitro frente a clotrimazol y nistatina de 123 aislamientos de Candida, obtenidos mediante exudados vaginales de 404 mujeres que asistieron al Hospital Ginecoobstétrico "Ramón González Coro" de Ciudad de La Habana. De acuerdo con el número de colonias obtenidas en el aislamiento primario, las cepas fueron separadas en 2 categorías: colonización e infección. Para cada cepa se determinó la concentración mínima inhibitoria (CMI) de cada antifúngico, mediante un método de microdilución en caldo casitona. Las medias geométricas de los valores de CMI fueron mayores frente a nistatina (1,08 mg/mL) que frente a clotrimazol (0,22 mg/mL), aunque los rangos fueron similares (£ 0,125-16 mg/mL). Para Candida albicans, que fue la especie aislada con mayor frecuencia (54,5 %); las medias geométricas de los valores de la CMI fueron de 0,17 y 0,16 mg/mL para clotrimazol y de 0,71 y 0,92 mg/mL para nistatina, en ambas categorías. C. glabrata mostró el valor de CMI más elevado (16 mg/mL) frente a ambos antifúngicos. Solo los aislamientos de C. lusitaniae mostraron diferencia significativa (p < 0,01) entre los valores de CMI de nistatina de acuerdo con el número de colonias en el aislamiento primario. En aislamientos procedentes de mujeres tratadas con clotrimazol y/o nistatina en episodios anteriores de candidiasis, se observaron valores más elevados de las medias de CMI frente a nistatina que frente a clotrimazol; aunque esta diferencia no fue estadísticamente significativa.

The susceptibility in vitro of 123 isolates of Candida against clotrimazole and nystatin was determined. The isolates were obtained by vaginal smears from 404 women that attended "Ramón Gonzalez Coro" Gynecoobstetric Hospital, in Havana City According to the number of colonies obtained in the primary isolation, the strains were separated into 2 categories: colonization and infection. The inhibitory minimum concentration was determined for each antigunfal by a method of microdilution in casitone broth. The geometrical means of the IMC values were higher against nystatin (1.08 mg/mL) than against clotrimazole (0.22 mg/mL), eventhough the features were similar ( £ 0.125 -16 mg/mL) For Candida albicans, that was the most frequently isolated species (54.5 %), the geometrical means of the IMC values were 0.17 and 0.16 mg/mL for clotrimazole and 0.71 and 0.92 for nystatin, in both categories. C. glabrata showed the highest IMC values (16 mg/mL) against antifungals. Only the isolates of C. lusitaniae showed a significant difference (p < 0.001) between the IMC values of nystatin according to the number of colonies in the primary isolation. In isolates from women treated with clotrimazole and/or nystatin in previous episodes of candidiasis, there were observed more elevated values of the means of IMC against nystatin than against clotrimazole, although this difference was not statistically remarkable.

Na+i, K+i and Cl-i regulation of exocytosis in guinea-pig antral mucous cells

Effects of intracellular Na+, K+ and Cl- on Ca(2+)-regulated exocytosis activated by 10 microM acetylcholine (ACh) were studied in guinea-pig antral mucous cells which are permeabilized by nystatin treatment. Ca(2+)-regulated exocytotic events were modulated by [Na+]i, [K+]i and [Cl-]i via mediation of PTX-sensitive G proteins. Increases in [Na+]i and PTX inhibit G protein (G(Na)), which suppressed the exocytosis. Increases in [K+]i caused the exchange of G proteins (from G(Na) to G(K)) to increase, and GK evoked activation of the exocytosis and was inhibited by PTX. Increases in [Cl-]i and PTX inhibit G protein (G(Cl)), which stimulates exocytotic events. Based on these observations, the exocytosis in antral mucous cells were modulated by intracellular ions, concentration of which were increased or decreased by cell volume changes caused by Ach.

Efectos de ketoconazol, clotrimazol, nistatina y cicloheximida sobre el crecimiento de phaffia rhodozyma / Effects of ketoconazole, clotrymazole, nystatin and cycloheximide on the growth of phaffia rhodozyma

Debido a la importancia biotecnológica en la producción de pigmentos, se determinaron los niveles de sensibilidad a antifúngicos de 3 cepas de Phaffia rhodozyma, con la finalidad de obtener mutantes resistentes, característica usada como marcador en el análisis genético de diferentes microorganismos. El desarrollo de las tres cepas, fue inhibido por ketoconazol, clotrimazol, nistatina y cicloheximida respectivamente. La cepa UCD 67-210 resultó ser la más sensible al ketoconazol, siendo inhibida por concentraciones de 5 ug/ml de dicho compuesto. Para las cepas UCD 67-383 y UCD 67-385 se requieren concentraciones de 20 ug/ml. El cotrimazol, inhibió el crecimiento de las cepas UCD 67-210 y UCD 76-385, a una concentración de 10 ug/ml y a la cepa UCD 67-383 a 20 ug/ml. La nistatina inhibe el crecimiento de las cepas UCD 67-383 y UCD 67-385 a una concentración de 3 ug/ml y a la cepa UCD 67-210 a 5 ug/ml. La cicloheximida impide el desarrollo de las cepas UCD 67-210 y UCD 67-385 a una concentración de 2 ug/ml. La cepa UCD 67-383 resultó ser la más resistente, requiriéndose concentraciones sobre 6 ug/ml para inhibir su crecimiento

Study of combination effects of nystatin and clotrimazole with some antibiotics on candida albicans

Binary combinations between 2 antifungal agents nystatin and clotrimazole and 4 antibiotics tetracyclines, chloramphenicol, rifampicin and gentamicin were tried using 7 Candida albicans strains isolated from clinical specimen using the checker board method. The best combinations were obtained with nystatin-tetracyclines and clotrimazole- gentamicin. However, synergistic activity between the 2 antifungal agents and these antibiotics was found only at high concentrations of the latter 500 ug - 125 ug/ml, thus limiting its application in vivo

Os caminhos da investigaçäo de novos antifúngicos / The pathways of the investigation of new antifungal drugs

Neste artigo, säo tecidas consideraçöes sobre a necessidade de se pesquisar novas drogas antifúngicas mais efetivas contra o agente agressor e menos tóxicas para o ser humano, bem como drogas para uso profilático. Säo revistos os pontos vulneráveis dos fungos e os mecanismos de açäo dos principais antifúngicos, tanto de uso tópico quanto de administraçäo oral, existentes no mercado. Finalmente, o autor apresenta os caminhos possíveis de investigaçäo deste tipo de quimioterápico, visando atender as necessidades atuais

Chronically inflamed tissue activates plasma mediators of vascular permeability

After inflammation was induced in the foot-pad of rats with nistatin or BCG, injection of "non-activated" homologous plasma at the inflamed site caused a significant increase in the vascular permeability of the lesions (Evans blue test), which was more intense in older lesions, increasing from 7.83 + or - 1.11 to 8.70 + or - 1.18 (nistatin, 4 and 21 days) and 7.30 + or - 0.66 to 7.54 + or - 0.80 (BCG, 4 and 21 days). Steroidal (acetyltriamcinolone, 25 mg/kg) and non-steroidal (indomethacin, 2 mg/kg) anti-inflammatory drugs markedly decreased this effect on 14-day old lesions induced by nistatin plus "non-activated" plasma (2.37 + or - 0.10 for acetyltriamcinolone treatment vs 8.15 + or - 1.22 for untreated animals; 3.34 + or - 0.41 for indomethacin treatment vs 8.15 + or - 1.22 for untreated animals) and BCG plus "non-activated" plasma (1.67 + or - 0.11 for acetyltriamcinolne treatment vs 10.27 + or - 0.52 for untreated animals; 5.87 + or - 0.35 for indomethacin treatment vs 9.14 + or - 0.23 for untreated animals). These data suggest that an increase in exudation in chronic lesions might result in "reactivation" of the process as observed clinically, for example, in rheumatoid arthritis in man

Leveduras isoladas de pacientes com câncer: incidência e sensibilidade a antibióticos poliênicos

Foram estudadas as incidências de leveduras isoladas de pacientes com câncer e submetidos a radioterapia ou a quimioterapia e a sensibilidade destes fungos a antibióticos poliênicos. De 227 desses pacientes, também com suspeita clínica de micose, foram desses pacientes, também com suspeita clínica de micose, foram isoladas 200 cepas que incluiam apenas espécies de Candida. C. albicans foi a espécie de maior incidência (87.5%), seguindo-se C. tropicalis (9.0%), C. parapsilosis (6.0%), C. brusei (4,0%), C. guilliermondii, C. pseudotropicalis (3,0% e C. famata (1,0%). Em concentracöes dos antibióticos consideradas eficazes para amostras sensíveis, todas as leveduras foram inibidas pela nistatina (8 microng/ml) e 97,4% o foram pela anfotericina B (2 microng/ml) e pela pimaricina (10microng/ml). Nessas concentraçöes, 10,6% das leveduras mostraram-se resistentes a açäo letal da nistatina, 32,6% a da anfotericina B e 38,7% a da pimaricina