First Study of Naturally Formed Fungal Biofilms on the Surface of Intragastric Balloons.

BACKGROUND: Intragastric balloon (IGB) is a medical device used in the endoscopic treatment of pre-obesity and obesity. The involvement of IGB with biofilms has been previously reported; however, little is still known. We determine the frequency of biofilms naturally formed on the external surface of IGB, as well as some variables related to IGB types and patients features, species of fungi involved, and biofilm evidence. METHODS: A retrospective study was conducted based on endoscopies and medical records of patients with explanted IGB between 2015 and 2018, which had masses strongly adhered to the surface of the balloon, suspecting the presence of a biofilm. From 2018, the samples of those masses were investigated seeking biofilm characterization based on mycological and structural aspects. RESULTS: A total of 149 endoscopies were surveyed; 27 IGBs (18.12%) showed signs suggesting biofilm formation. There was no significant difference between biofilm involvement in IGB and the anthropometric and demographic profile of the patients. On the other hand, there was a significant difference regarding the IGB type, 24.05% of the adjustable IGB were compromised by biofilm, while in non-adjustable IGB, it was 11.43% (p = 0.04; OR 2.45; 95% CI, 0.98-6.12). Candida glabrata was the most isolated fungal species from the well-organized fungal biofilm. CONCLUSIONS: The frequency of fungal biofilm naturally formed on the external surface of IGB was elevated. The risk of biofilm formation was increased for the adjustable IGB, but it did not relate to the demographic data and anthropometric patient profile.

Propolis extract has bioactivity on the wall and cell membrane of Candida albicans.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of natural products such as propolis extract (PE) is a promising alternative when topically administered to replace conventional antifungals, mostly due to its therapeutic applications, ease of access and low toxicity. However, despite being the subject of several mycology studies, they focus primarily on exploiting their antimicrobial activity, lacking information on the mechanisms of action of PE on Candida spp., characterizing its antifungal potential. AIM OF THE STUDY: To elucidate the bioactivity of PE on the cellular structure of Candida albicans. MATERIALS AND METHODS: A total of seven C. albicans clinical isolates plus a reference strain of C. albicans ATCC 90028 were used in this study. The PE was characterized and its effect on C. albicans was determined by susceptibility and growth kinetics assays; interference on C. albicans germination and filamentation; evaluation of the integrity of the C. albicans cell wall and membrane, as well as its mutagenic potential. RESULTS: The PE presented strong inhibitory activity, which showed its greatest antifungal activity at 12 h with dose and time dependent fungistatic characteristics, effectively inhibiting and interfering on C. albicans filamentation. In addition, PE caused membrane and cell wall damage with intracellular content extravasation. Moreover, PE was not mutagenic. CONCLUSIONS: The bioactivity of PE is mainly related to the loss of integrity membrane as well as the integrity of the cell wall and consequent increase in permeability, without mutagenic effects.

Influence of Eugenia uniflora Extract on Adhesion to Human Buccal Epithelial Cells, Biofilm Formation, and Cell Surface Hydrophobicity of Candida spp. from the Oral Cavity of Kidney Transplant Recipients.

This study evaluated the influence of the extract of Eugenia uniflora in adhesion to human buccal epithelial cells (HBEC) biofilm formation and cell surface hydrophobicity (CSH) of Candida spp. isolated from the oral cavity of kidney transplant patients. To evaluate virulence attributes in vitro, nine yeasts were grown in the presence and absence of 1000 µg/mL of the extract. Adhesion was quantified using the number of Candida cells adhered to 150 HBEC determined by optical microscope. Biofilm formation was evaluated using two methodologies: XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and crystal violet assay, and further analyzed by electronic scan microscopy. CSH was quantified with the microbial adhesion to hydrocarbons test. We could detect that the extract of E. uniflora was able to reduce adhesion to HBEC and CSH for both Candida albicans and non-Candida albicansCandida species. We also observed a statistically significant reduced ability to form biofilms in biofilm-producing strains using both methods of quantification. However, two highly biofilm-producing strains of Candida tropicalis had a very large reduction in biofilm formation. This study reinforces the idea that besides growth inhibition, E. uniflora may interfere with the expression of some virulence factors of Candida spp. and may be possibly applied in the future as a novel antifungal agent.

Morphological and structural changes in lung tissue infected by Paracoccidioides brasiliensis: FTIR photoacoustic spectroscopy and histological analysis.

This study evaluated physical, chemical and morphological changes in lungs of mice infected with Paracoccidioides brasiliensis. The animals were inoculated with 0.1 mL of fungal suspension of the P. brasiliensis 18 isolate and were euthanized 1, 2, 4 and 8 weeks after inoculation. The upper left lobe of the lung was isolated, fixed and processed for paraffin embedding. The sections were stained with H&E for histopathological study, with Gomori-Grocott to locate and identify the fungus, and with TUNEL immunostaining to detect the occurrence of programmed cell death. The lower and middle right lobes were analyzed by Fourier Transform Infrared Photoacoustic Spectrocopy (FTIR-PAS) to investigate physical and chemical features of the infected lungs. The results showed that lungs infected by P. brasiliensis underwent structural changes that varied according to the time period analyzed, and that changes in the absorption bands of different chemical groups resulted from these morphological changes. The results suggest that the combination of FTIR-PAS spectroscopy with morphological evaluation is an effective procedure for the study of paracoccidioidomycosis, one of the most important systemic mycoses that can damage the lung architecture and consequently impair the respiratory function.

In vivo activity of Sapindus saponaria against azole-susceptible and -resistant human vaginal Candida species.

BACKGROUND: Study of in vivo antifungal activity of the hydroalcoholic extract (HE) and n-BuOH extract (BUTE) of Sapindus saponaria against azole-susceptible and -resistant human vaginal Candida spp. METHODS: The in vitro antifungal activity of HE, BUTE, fluconazole (FLU), and itraconazole (ITRA) was determined by the broth microdilution method. We obtained values of minimal inhibitory concentration (MIC) and minimum fungicide concentration (MFC) for 46 strains of C. albicans and 10 of C. glabrata isolated from patients with vulvovaginal candidiasis (VVC). VVC was induced in hyperestrogenic Wistar rats with azole-susceptible C. albicans (SCA), azole-resistant C. albicans (RCA), and azole-resistant C. glabrata (RCG). The rats were treated intravaginally with 0.1 mL of HE or BUTE at concentrations of 1%, 2.5% and 5%; 100 µg/mL of FLU (treatment positive control); or distilled water (negative control) at 1, 24, and 48 h after induction of the infection, and the progress of VVC was monitored by culturing and scanning electron microscopy (SEM). The toxicity was evaluated in cervical cells of the HeLa cell line. RESULTS: The extracts showed in vitro inhibitory and fungicidal activity against all the isolates, and the MIC and MFC values for the C. glabrata isolates were slightly higher. In vivo, the SCA, RCA, and RCG infections were eliminated by 21 days post-infection, with up to 5% HE and BUTE, comparable to the activity of FLU. No cytotoxic action was observed for either extract. CONCLUSIONS: Our results demonstrated that HE and BUTE from S. saponaria show inhibitory and fungicidal activity in vitro, in addition to in vivo activity against azole-resistant vaginal isolates of C. glabrata and azole-susceptible and resistant isolates of C. albicans. Also considering the lack of cytotoxicity and the low concentrations of the extracts necessary to eliminate the infection in vivo, HE and BUTE show promise for continued studies with purified antifungal substances in VVC yeast isolates.

Antifungal activity of the extract of Curcuma zedoaria (Christm.) Roscoe, Zingiberaceae, against yeasts of the genus Candida isolated from the oral cavity of patients infected with the human immunodeficiency virus

Oropharyngeal candidiasis is the most common fungal infection among patients infected with the human immunodeficiency virus (HIV), and is treated empirically with topical or systemic antifungals. The objective of the present study was to investigate the possible antifungal action of the hydroalcoholic extract of Curcuma zedoaria (Christm.) Roscoe, Zingiberaceae, on yeasts in this population. Samples were collected from HIV-positive patients who attended the Laboratory for Teaching and Research in Clinical Analysis at the Universidade Estadual de Maringá for routine exams. The isolated yeasts were identified at the genus and species levels through classical methodology. Next, tests of microdilution in broth were carried out to determine the profile of susceptibility of these yeasts towards the hydroalcoholic extract of C. zedoaria, following methodology standardised by the CLSI (2002). A total of 53 yeasts were identified, 49 of them C. albicans, two C. tropicalis and two C. glabrata. These yeasts were inhibited by low concentrations of the extract of C. zedoaria (between 1.95 and 15.63 μg/mL). In addition, 7.82 μg/mL inhibited 90 percent of the yeasts. Our results indicate a potent antifungal action for C. zedoaria and suggest more detailed studies with a view towards the practical application of this phytomedicine in topical pharmaceutical forms for the treatment of oral candidosis or candidiasis.

Examination of potential virulence factors of Candida tropicalis clinical isolates from hospitalized patients.

Candida tropicalis has been reported to be one of the Candida species which is most likely to cause bloodstream and urinary tract infections in hospitalized patients. Accordingly, the aim of this study was to characterize the virulence of C. tropicalis by assessing antifungal susceptibility and comparing the expression of several virulence factors. This study was conducted with seven isolates of C. tropicalis from urine and blood cultures and from central venous catheter. C. tropicalis ATCC 750 was used as reference strain. Yeasts adhered (2 h) to epithelial cells and silicone and 24 h biofilm biomass were determined by crystal violet staining. Pseudohyphae formation ability was determined after growth in fetal bovine serum. Enzymes production (hemolysins, proteases, phospholipases) was assessed by halo formation on agar plates. Susceptibility to antifungal agents was determined by E-test. Regarding adhesion, it can be highlighted that C. tropicalis strains adhered significantly more to epithelium than to silicone. Furthermore, all C. tropicalis strains were able to form biofilms and to express total hemolytic activity. However, protease was only produced by two isolates from urine and by the isolates from catheter and blood. Moreover, only one C. tropicalis (from catheter) was phospholipase positive. All isolates were susceptible to voriconazole, fluconazole and amphotericin B. Four strains were susceptible-dose dependent to itraconazole and one clinical isolate was found to be resistant.

Morphologic organization of pulmonary granulomas in mice infected with Paracoccidioides brasiliensis.

A morphologic study of the lungs was carried out in Swiss mice infected with yeast isolated from Paracoccidioides brasiliensis (Pb18). The lung was processed 1, 2, 4, and 8 weeks after inoculation for histologic staining with hematoxylin and eosin (H&E), methenamine silver nitrate (Gomori-Grocott), and picrosirius to qualitative and quantitative analyses of the granulomas and the presence of fungal lesions. The numbers of CFUs/g counted in the lungs were 189.8 +/- 20.64, 353.6 +/- 46.21, 547.2 +/- 108.1, and 295.2 +/- 89.17 in the first, second, fourth, and eighth weeks, respectively. One week after infection, inflammatory cells and reticular and collagens fibers, the latest typical of fibrosis, were detected. After 2 and 4 weeks, a progressive intensification of the infection and fibrosis was observed, but in week 8 a more organized granuloma was evident, with macrophages, epithelioid cells, and yeasts in the central portion, and intense peripheral basophilia. Pycnotic structures typical of apoptotic bodies were observed in weeks 1 and 8. The different histologic staining used acted as a fundamental tool for the study of the morphologic organization of granuloma formation.

Effect of experimental diabetes on the development and maintenance of vulvovaginal candidiasis in female rats.

OBJECTIVE: The purpose of this study was to develop an experimental model of diabetes in female rats and verify its influence on vulvovaginal candidiasis. STUDY DESIGN: The animals were divided into control and diabetic groups. Diabetes was induced with the use of an intravenous solution of alloxan (42 mg/kg bodyweight). One week after confirmation of hyperglycemia, the inoculation of Candida albicans yeast, previously standardized from a vaginal isolate, in concentrations of about 5 x 10(8), was performed. Infection control was made through vaginal culture, Papanicolaou cytology, and scanning electron microscopy (SCEM). RESULTS: The results pointed to different glycemias between the control (74.8 +/- 2.6) and experimental groups (543.1 +/- 12.1) and a significant bodyweight decrease (227.6 +/- 4.77 and 204 +/- 6.39, respectively). The positive infection was shown by culture, Papanicolaou test, and SCEM in the experimental group. CONCLUSION: Diabetes mellitus causes hyperglycemia, which was favorable to the vaginal colonization and infection by C albicans.

Antifungal activity of ajoene on experimental murine paracoccidioidomycosis.

The natural compound ajoene (4,5,9- trithiadodeca-1,6,11-triene 9-oxide) is capable of controlling infection by Paracoccidioides brasiliensis in experimental models. Swiss mice were inoculated with 5.0 x 10e6 cells of the fungus Paracoccidioides brasiliensis Pb18 by intraperitoneal route and treated with ajoene. In weeks 2, 6, 10 and 13 of treatment, levels of anti-Pb antibodies were measured by the ELISA test and the animals were put down and their lungs, livers and spleens removed for histopathological analysis and determination of the number of viable fungus. The results show that experimental murine paracoccidioidomycosis was well established and that ajoene was capable of controlling the evolution of the disease, as it significantly reduced the levels of antibodies from the 10th week of treatment.

Antifungal activity of the extracts and saponins from Sapindus saponaria L

Extracts from the dried pericarp of Sapindus saponaria L. (Sapindaceae) fruits were investigated for their antifungal activity against clinical isolates of yeasts Candida albicans and C. non-albicans from vaginal secretions of women with Vulvovaginal Candidiasis. Four clinical isolates of C. albicans, a single clinical isolated of each of the species C. parapsilosis, C. glabrata, C. tropicalis, and the strain of C. albicans ATCC 90028 were used. The hydroalcoholic extract was bioactivity-directed against a clinical isolate of C. parapsilosis, and showed strong activity. The n-BuOH extract and one fraction showed strong activity against all isolates tested. Further column-chromatography on silica gel separation of this fraction afforded two pure triterpene acetylated saponins: 3-O-(4-acetyl-beta-D-xylopyranosyl)-(1->3)-alpha-Lrhamnopyranosyl-(1->2)-alpha-L-arabinopyranosyl-hederagenin (1) and 3-O-(3,4-di-acetyl-beta-D-xylopyranosyl)-(1->3)-alpha-L-rhamnopyranosyl-(1->2)-alpha-L-arabynopyranosyl-hederagenin (2). The structures of the compounds were based on spectral data (¹H and 13C NMR, HSQC, HMBC and MS), and on with literature. The saponins isolated showed strong activity against C. parapsilosis.

Extratos do pericarpo de frutos de Sapindus saponaria L. (Sapindaceae) foram testados para a atividade antifúngica sobre isolados clínicos de leveduras de Candida albicans e C. não-albicans obtidos de secreção vaginal de mulheres com Candidíase Vulvovaginal. Foram avaliados quatro isolados clínicos de C. albicans, um de cada uma das espécies C. glabrata, C. parapsilosis, C. tropicalis e uma cepa referência de C. albicans ATCC 90028. O extrato hidroalcoólico foi biomonitorado contra um isolado clínico de C. parapsilosis, apresentando forte atividade. O extrato butanólico e uma fração apresentaram forte atividade contra todos os isolados testados. Posterior análise desta fração via cromatografia em sílica gel (CHCl3:CH3OH, 1:1, v/v) resultou no isolamento de duas saponinas triterpênicas puras mono e diacetiladas, 3-O-(4-O-acetil-O-beta-D-xilopiranosil)-(1 -> 3)-alfa-L-ramnopiranosil-(1 -> 2)-alfa-L-arabinopiranosil-hederagenina (1) e 3-O-(3,4-di-O-acetil-beta-D-xilopiranosil)-(1 -> 3)-alfa-L-ramnopiranosil-(1 -> 2)-alfa-L-rabinopiranosil-hederagenina (2) respectivamente. A elucidação estrutural das substâncias foi baseada em dados espectrais (RMN de ¹H e de 13C, HSQC, HMBC, ESI/MS) e comparados com dados da literatura. As saponinas triterpênicas isoladas (1) e (2) apresentaram forte atividade contra C. parapsilosis.

Obtenção de extratos de própolis sob diferentes condições e avaliação de sua atividade antifúngica / Antifungal activity evaluation of different propolis extracts

A própolis é uma resina coletada das árvores pelas abelhas Apis mellifera L., que contém inúmeras substâncias, dentre elas os flavonóides. Devido a grande variedade de sua composição química, apresenta várias ações farmacológicas, destacando-se as ações antiinflamatória, cicatrizante, antitumoral e antimicrobiana, principalmente a antifúngica. Esta ação foi testada frente a leveduras isoladas de onicomicoses, que são infecções de difícil e longo tratamento que causam efeitos indesejáveis ao paciente. A própolis surge como uma eficiente opção de tratamento, pois é de baixa toxicidade e pode ser de uso tópico. Este trabalho objetivou avaliar a otimização do processo extrativo da própolis através de parâmetros físico-químicos e demonstração da atividade antifúngica.

Propolis is a resin collected by the bees Apis mellifera L., which contains several substances including the flavonoids. Due to a diversified chemical composition propolis presents some pharmacological actions, being distinguished the anti-inflammatory, healing, antitumoral and antimicrobial properties and, in particular, its antifungal action. This action has been tested against yeasts obtained from onychomycosis, which are infections of difficult and long treatment and they can manifest intolerable effects on the patient. The propolis appears as an efficient therapy option, as it has low toxicity and should be of dermal use. The aim of the present study was to evaluate the optimization of the propolis extractive process through physiochemical parameters and antifungal activity demonstration.

Contribuição da citologia de papanicolaou para o diagnóstico de leveduras em secreção vaginal / Contribution of the cytology of papanicolaou for the diagnosis of yeasts in vaginal secretion

Introdução: candidíase vulvovaginal (CVV) é uma infecção causada pelo crescimento anormal de fungos do tipo leveduras na mucosa do trato genital. Ultimamente, tem crescido o interesse na utilização da citologia de Papanicolaou (Pap) no diagnóstico de infecções associadas a patógenos de transmissão sexual, mas o método tem sido pouco avaliado para CVV. Objetivos: avaliar a acurácia diagnóstica da citologia de Papanicolaou para o diagnóstico de fungos vaginais, bem como comparar os resultados da citologia com os da bacterioscopia de secreção vaginal (Gram) e cultura. Métodos: foram coletadas secreções cérvico-vaginais para Gram, Papanicolaou e cultura de leveduras, e as colônias crescidas na cultura foram contadas. As mulheres não foram triadas para sintomatologia de CVV e foram descartadas aquelas com outra infecção agente ou imunodeficiências. Foram realizados cálculos estatísticos de sensibilidade, especificidade, valores preditivo positivo e negativo, e freqüência de falso-positivos e negativos, em três situações: na detecção de fungos independente da quantidade de colônias; nos casos com crescimento de 1-9 colônias; e naqueles com crescimento de ? 100. Resultados: a cultura foi positiva em 35 pacientes (21,70%). Sem considerar o número de colônias, a sensibilidade do Gram e da citologia foram muito parecidas (67,65% e 62,85%), nos casos com crescimento de 1-9 colônias, de 37,50% e 50,00%, e nos casos com crescimento ? 100 colônias, de86,61% e 78,26%. Conclusão: a citologia mostrou-se um método morfológico com eficiência muito semelhante e para pequenos números de colônias melhor do que o Gram para detectar leveduras vaginais, tornando-a perfeitamente aplicável na rotina laboratorial para este diagnóstico.

Introduction: vulvovaginal Candidiasis vulvovaginal (VVC) it is an infection caused by the abnormal growth of fungus of the type yeasts in themucous membrane of the genital treatment. Lately, it has been increasing the interest in the use of the cytology of Papanicolaou (Pap) in the diagnosis of associated infections the pathogens of sexual transmission, but the method has been little appraised for VVC. Objectives: to evaluate the accuracy diagnostic of the cytology of Papanicolaou for the diagnosis of vaginal fungus, as well as to compare the results of the cytology with the one of the bacterioscopy of vaginal secretion (Gram) and culture. Methods: cervico-vaginal secretions were collected for Gram, Papanicolaou and culture of yeasts, and the grown colonies in the culture were counted. The women didn?t go selected to simptomatology of VVC and they were discarded those with other infection agent or immunodeficiency. Statistical calculations of sensibility were accomplished, specificity, values positive and negative predictive, and frequency of false positive and negative, in three situations: in the independent detection of fungus of the amount of colonies; in thecases with growth of 1-9 colonies; and in those with growth of ? 100. Results: the culture of it was positive in 35 patients (21,70%). Without considering the number of colonies, the sensibility of Gram and of the cytology they were very similar (67,65% and 62,85%), in the cases with growth of 1-9 colonies, of 37,50% and 50,00%, and in the cases with growth ? 100 colonies, of 86,61% and 78,26%. Conclusion: the cytology was shown a morphologic method with very similar efficiency and for small numbers of colonies better than Gram to detect vaginal yeasts, turning her perfectly applicable in the routine laboratorial for this diagnosis.

Effect of aqueous extract from Neem (Azadirachta indica A. Juss) on hydrophobicity, biofilm formation and adhesion in composite resin by Candida albicans.

OBJECTIVE: Azadirachta indica, a Meliaceae family tree, has been used in India for many years in the treatment of several diseases in medicine and dentistry. Current research analyses the effects of the leaf aqueous extract from Azadirachta indica (Neem) on the adhesion, cell surface hydrophobicity and biofilm formation, which may affect the colonisation by Candida albicans. METHODS: Azadirachta indica extract was tested in vitro on strains of Candida albicans 12A and 156B. Changes in hydrophobicity were reported in assays of yeast adhesion to hydrocarbons, in biofilm formation with glucose and in the adhesion of the microorganisms on light cured composite resin. Assays involved enumeration of candidal colony-forming units together with scintillation counting of radiolabelled Candida and compared to a solution of chlorhexidine digluconate 0.125% widely used in dentistry. RESULTS: Yeast growth in Neem extract was not inhibited in concentrations ranging from 0.1mg/ml. A statistically significant increase (p<0.05) in cell surface hydrophobicity was evident for the two strain tested and there was also an associated increase in biofilm formation after contact with Neem extract in concentration 0.01 g/ml. Decrease in adhesion capacity of cells to composite resin was also recorded. CONCLUSION: An anti-adhesive mechanism of action by Azadirachta indica is proposed based on the results observed.