Synergistic interactions between ß-lapachone and fluconazole in the inhibition of CaCdr2p and CaMdr1p in Candida albicans.

BACKGROUND: Mortality rate of invasive Candida infections is raising mainly amongst immunocompromised patients. These infections are hard-to-treat mainly due to the increasing incidence of resistance. The overexpression of ATP-binding cassette and major facilitator superfamily transporters is the main responsible for the failure of antifungal therapies. In a Saccharomyces cerevisiae model, ß-lapachone inhibited Pdr5p, a transporter homologous to those found in Candida albicans. AIMS: To determine whether ß-lapachone reverses the resistance phenotype mediated by efflux transporters in C. albicans clinical isolates. METHODS: The antifungal activity of ß-lapachone combined with fluconazole was measured by agarose chemosensitization and microdilution assays. CaCdr2p and CaMdr1p activities were evaluated through fluorescent dyes accumulation. ATPase activity was assessed using transporter-enriched plasma membranes. RESULTS: ß-lapachone reverted antifungal resistance of S. cerevisiae and C. albicans strains overexpressing CaCdr2p and CaMdr1p transporters by inhibiting these proteins activities. CaCdr2p ATPase activity was not impaired by the compound. CONCLUSIONS: ß-lapachone is a promising drug candidate to be used as an adjuvant in the treatment of candidiasis caused by fluconazole-resistant C. albicans strains.

Synergistic interactions between Beta-lapachone and fluconazole in the inhibition of CaCdr2p and CaMdr1p in Candida albicans / Las interacciones sinérgicas entre la beta-lapachona y el fluconazol en la inhibición de CaCdr2p y CaMdr1p en Candida albicans

BACKGROUND: Mortality rate of invasive Candida infections is raising mainly amongst immunocompromised patients. These infections are hard-to-treat mainly due to the increasing incidence of resistance. The overexpression of ATP-binding cassette and major facilitator superfamily transporters is the main responsible for the failure of antifungal therapies. In a Saccharomyces cerevisiae model, Beta-lapachone inhibited Pdr5p, a transporter homologous to those found in Candida albicans. AIMS: To determine whether Beta-lapachone reverses the resistance phenotype mediated by efflux transporters in C. albicans clinical isolates. METHODS: The antifungal activity of Beta-lapachone combined with fluconazole was measured by agarose chemosensitization and microdilution assays. CaCdr2p and CaMdr1p activities were evaluated through fluorescent dyes accumulation. ATPase activity was assessed using transporter-enriched plasma membranes. RESULTS: Beta-lapachone reverted antifungal resistance of S. cerevisiae and C. albicans strains overexpressing CaCdr2p and CaMdr1p transporters by inhibiting these proteins activities. CaCdr2p ATPase activity was not impaired by the compound. CONCLUSIONS: Beta-lapachone is a promising drug candidate to be used as an adjuvant in the treatment of candidiasis caused by fluconazole-resistant C. albicans strains

ANTECEDENTES: Las tasas de mortalidad de infecciones invasivas causadas por Candida están en aumento, principalmente entre los pacientes inmunocomprometidos. Estas infecciones son difíciles de tratar debido a la creciente incidencia de resistencia a los antifúngicos. La sobreexpresión de los transportadores dependientes de ATP y los de la superfamilia de facilitadores principales es el mayor responsable del fracaso de las terapias antimicóticas. En un modelo de Saccharomyces cerevisiae, la beta-lapachona inhibió Pdr5p, un transportador homólogo a los encontrados en Candida albicans. OBJETIVOS: Determinar si la beta-lapachona revierte el fenotipo de resistencia mediado por transportadores de eflujo en aislamientos clínicos de C. albicans. MÉTODOS: Se midió la actividad antifúngica de la beta-lapachona combinada con fluconazol mediante ensayos de quimiosensibilización con agarosa y microdilución. Las actividades CaCdr2p y CaMdr1p se evaluaron mediante la acumulación de colorantes fluorescentes, y la actividad de ATPasa se evaluó usando membranas plasmáticas enriquecidas con transportador. RESULTADOS: La beta-lapachona revirtió la resistencia antifúngica de las cepas de S. cerevisiae y C. albicans que sobreexpresaban los transportadores CaCdr2p y CaMdr1p al inhibir sus actividades. El compuesto no afectó la actividad ATPasa de CaCdr2p. CONCLUSIONES: La beta-lapachona es una candidata prometedora para ser utilizada como adyuvante en el tratamiento de la candidiasis causada por cepas de C. albicans resistentes al fluconazol

Salivary pH and colonization by oral Candida in children and adolescents submitted to haemodialysis.

BACKGROUND: Fungal infections are a serious problem among haemodialysis patients. AIM: The aims of this study were to estimate the frequency of oral Candida species among children and adolescents undergoing haemodialysis (HD), to identify the isolated species, and to study the relationship between haemodialysis duration, amounts of colony-forming units, and salivary pH. DESIGN: A matched sample of 52 patients undergoing HD and 52 healthy individuals were selected. The samples were obtained from the dorsum of the tongue, and the colonies were identified through a substrate assimilation test. Stimulated whole saliva was collected from each patient for evaluation of salivary pH. RESULTS: The frequency of oral Candida species was 34.6% (18/52) and 46.20% (24/52) in the HD and control groups (P = 0.23), respectively. Candida parapsilosis complex was the most frequently isolated fungi species in the HD group (P = 0.03). A HD therapy duration of more than 1 year was statistically correlated with a higher number of colony-forming units (P = 0.03) but was not statistically related to salivary pH. CONCLUSIONS: Candida parapsilosis complex was the most frequently isolated fungal species in the young HD patients, and the duration therapy was associated with higher oral colonization.

Effect of the secretory leucocyte proteinase inhibitor (SLPI) on Candida albicans biological processes: a therapeutic alternative?

OBJECTIVES: The aim of this study was to evaluate the effect of SLPI on the growth and biological processes of Candida albicans. METHODS: Two C. albicans strains were used in this study, a clinical isolate resistant to fluconazole (PRI) and a reference strain ATCC 24433. The minimal inhibitory concentration (MIC) was determined according to the CLSI methodology. The influence of SLPI on secreted serine proteinase activities (SSP) was measured by the cleavage of specific substrate, and surface hydrophobicity was determined by the aqueous-hydrocarbon biphasic separation method. Flow cytometry was performed to investigate receptors for SLPI and variations in the cell wall mannoprotein expression. Interaction between yeast and epithelium was assessed using the MA-104 cells lineage. Ultrastructure was analyzed by transmission electron microscopy (TEM). RESULTS: MIC values were calculated as 18 and 18.9µM for the PRI and ATCC 24433, respectively. SSP activity was reduced by 48.8% by 18µM of SLPI and cell surface hydrophobicity increased by 11.1%. Flow cytometry suggest the existence of SLPI binding sites on the surface of the yeast. Results showed a reduction in the expression of mannoproteins in 20.8% by the cells treated with 80µM of SLPI, and 18µM reduced the adhesion of yeasts to mammalian cells in 60.1%. TEM revealed ultrastructural changes in cells treated with 80µM of SLPI, such as the presence of membrane-like structures within the cytoplasm. CONCLUSIONS: SLPI exerts a significant influence on C. albicans viability and biological processes. Considering its constitutive and physiologic features, SLPI may become a promising tool for the development of new methodologies for the treatment and control of candidiasis.

Salivary lactoferrin in HIV-infected children: correlation with Candida albicans carriage, oral manifestations, HIV infection and its antifungal activity.

OBJECTIVES: This study aimed to evaluate the concentration of lactoferrin in the saliva of HIV infected and healthy children and analyze the associations between lactoferrin levels, Candida sp. colonization, oral manifestations and medical data. Also, the antifungal ability of lactoferrin to inhibit the growth of Candida albicans isolated from saliva of these children was investigated in vitro. SUBJECTS AND METHODS: Saliva was collected from 70 HIV-infected and 50 healthy children, followed by oral manifestation assessments. The salivary lactoferrin was quantified using an ELISA Kit. The salivary samples were cultured and the Candida spp. colonies counted and then identified by sugar assimilation and fermentation. The antifungal activity of lactoferrin was analyzed in vitro with 10 isolates of C. albicans from each group. RESULTS: The HIV infected children (mean age 9.8 ± 2.8) had higher lactoferrin levels (median 6.13 µg/ml (3.58-7.89)) and were colonized three times more by Candida sp. than the control group (mean age 9.4 ± 2.4) (median 5.74 µg/ml (3.12-6.86)) (p=0.003). Statistical associations were found considering the salivary lactoferrin levels and Candida sp. and oral manifestations between the groups. No associations between lactoferrin concentrations and oral manifestations, immunosuppression, presence of AIDS and use of HAART were observed in the HIV group. The percentage of dead C. albicans due to lactoferrin was inversely proportional to C. albicans cell density for both groups (p<0.001). CONCLUSIONS: HIV-infected children have higher concentrations of lactoferrin and it was associated with Candida sp. colonization but no association with medical data was found. Also, both groups showed similar lactoferrin antifungal activity.

Lower dental caries prevalence associated to chronic kidney disease: a systematic review.

BACKGROUND: The aim of this systematic review was to determine whether there is any evidence in the literature referring to a lower prevalence of dental caries in children and adolescents with chronic kidney disease (CKD) compared to healthy individuals. METHODS: A search of the PubMed Medline, Ovid Medline and Cochrane Library databases was performed using the MeSH terms "dental caries" and "chronic renal failure". To be eligible for entry in our study, controlled observational studies had to present a decayed, missing and filled index for primary teeth (dmft) and/or for permanent teeth (DMFT) in children and adolescents with CKD. RESULTS: After evaluation of title, keywords and abstracts of the articles selected, six articles met the inclusion criteria. Three of these six articles included studies which showed susceptibility to bias and possible confounding factors. A subsequent assessment of the six studies revealed that the mean caries indices in both primary (dmf) and permanent (DMF) teeth were lower in the children and adolescents with CKD compared with healthy individuals. CONCLUSION: Data in the literature weakly support a lower prevalence of caries in children and adolescents with CKD than in their healthy counterparts. There is still a lack of well-designed studies that provide better scientific evidence in support of this conclusion.

Conventional therapy and promising plant-derived compounds against trypanosomatid parasites.

Leishmaniasis and trypanosomiasis are two neglected and potentially lethal diseases that affect mostly the poor and marginal populations of developing countries around the world and consequently have an important impact on public health. Clinical manifestations such as cutaneous, mucocutaneous, and visceral disorders are the most frequent forms of leishmaniasis, a group of diseases caused by several Leishmania spp. American trypanosomiasis, or Chagas disease, is caused by Trypanosoma cruzi, a parasite that causes progressive damage to different organs, particularly the heart, esophagus, and lower intestine. African trypanosomiasis, or sleeping sickness, is caused by Trypanosoma brucei and is characterized by first presenting as an acute form that affects blood clotting and then becoming a chronic meningoencephalitis. The limited number, low efficacy, and side effects of conventional anti-leishmania and anti-trypanosomal drugs and the resistance developed by parasites are the major factors responsible for the growth in mortality rates. Recent research focused on plants has shown an ingenious way to obtain a solid and potentially rich source of drug candidates against various infectious diseases. Bioactive phytocompounds present in the crude extracts and essential oils of medicinal plants are components of an important strategy linked to the discovery of new medicines. These compounds have proven to be a good source of therapeutic agents for the treatment of leishmaniasis and trypanosomiasis. This work highlights some chemotherapeutic agents while emphasizing the importance of plants as a source of new and powerful drugs against these widespread diseases.

Effects of direct electric current on Herpetomonas samuelpessoai: An ultrastructural study.

The literature shows that the effects of direct electric currents on biological material are numerous, including bactericidal, fungicidal, parasiticidal, and anti-tumoral, among others. Non-pathogenic trypanosomatids, such as Herpetomonas samuelpessoai, have emerged as important models for the study of basic biological processes performed by a eukaryotic cell. The present study reports a dose-dependent anti-protozoan effect of direct electric treatment with both cathodic and anodic current flows on H. samuelpessoai cells. The damaging effects can be attributable to the electrolysis products generated during electric stimulation. The pH of the cell suspension was progressively augmented from 7.4 to 10.5 after the cathodic treatment. In contrast, the anodic treatment caused a pH decrease varying from 7.4 to 6.5. Transmission electron microscopy analyses revealed profound alterations in vital cellular structures (e.g., mitochondrion, kinetoplast, flagellum, flagellar pocket, nucleus, and plasma membrane) after exposure to both cathodic and anodic current flows. Specifically, cathodic current flow treatment induced the appearance of autophagic-like structures on parasite cells, while those submitted to an anodic current flow presented marked disorganization of plasma membrane and necrotic appearance. However, parasites treated in the intermediary chamber (without contact with the electrodes) did not present significant changes in viability or morphology, and no pH variation was detected in this system. The use of H. samuelpessoai as a biological model and the direct electric current experimental approach used in our study provide important information for understanding the mechanisms involved in the cytotoxic effects of this physical agent.

Tratamento com bioterápico Candida albicans influencia a adesão de fungos a células Ma104 in vitro / Treatment with Candida albicans biotherapic influences in vitro fungal adhesion to Ma-104 cells

Oral candidiasis is an opportunist fungal infection in humans, mainly caused by Candida albicans. It occurs when the host presents an imbalance in the immune system and Candida spp., normally found in human flora, become able to develop the infection [1]. This disease is very common in HIV patients, and in all individuals that present immunossupression, such as patients treated with chemotherapy. Considering this scenario, the development of new medicines to treat oral candidiasis is mandatory.These results showed that the biotherapic did not present any citotoxicity, but was able to modify the morphological aspects of Ma-104 cells. Additionally, the interaction between host cells and ethilogic agent is directly influenced by biotherapic treatment, suggesting a promising antifungal potential of this medicine.(AU)

Inhibitory activity of root canal irrigants against Candida albicans, Enterococcus faecalis and Staphylococcus aureus.

The present study evaluated the antimicrobial activity of three root canal irrigants against Enterococcus faecalis, Candida albicans, and Staphylococcus aureus. These microorganisms were incubated in the presence of citric acid (6 and 10%), EDTA (17%), and NaOCl (0.5, 1.0, 2.5, and 5.25%). Agar diffusion tests were performed and redox indicator resazurin was used to evaluate the inhibitory effect of the irrigants on the metabolic activity of these microorganisms. The mean diameters of the inhibition zones for the C. albicans cultures were 11.6 mm (17% EDTA), 5.5 mm (0.5% NaOCl), 12.9 mm (1% NaOCl), 22.1 mm (2.5% NaOCl), and 28.5 mm (5.25% NaOCl). The mean diameters of the inhibition zones for E. faecalis were 2.8 mm (1% NaOCl), 5.4 mm (2.5% NaOCl), and 8.3 mm (5.25% NaOCl). For S. aureus, the mean values were 8.0 mm (17% EDTA), 3.0 mm (1% NaOCl), 8.8 mm (2.5% NaOCl), and 10.0 mm (5.25% NaOCl). Most of the irrigant solutions presented effective antimicrobial activity against C. albicans. A high inhibitory effect on the metabolic activity of E. faecalis was detected when the microorganisms were incubated with 17% EDTA. The same result was reached when S. aureus was incubated in the presence of > 2.5% NaOCl. Altogether, these results indicate that 2.5% and 5.25% NaOCl are microbicides against S. aureus while 0.5% and 1% NaOCl are only microbiostatic against the tested bacteria. The 6% and 10% citric acid as well as 17% EDTA did not affect the viability of any of the assayed microorganisms.

Inhibitory activity of root canal irrigants against Candida albicans, Enterococcus faecalis and Staphylococcus aureus

The present study evaluated the antimicrobial activity of three root canal irrigants against Enterococcus faecalis, Candida albicans, and Staphylococcus aureus. These microorganisms were incubated in the presence of citric acid (6 and 10 percent), EDTA (17 percent), and NaOCl (0.5, 1.0, 2.5, and 5.25 percent). Agar diffusion tests were performed and redox indicator resazurin was used to evaluate the inhibitory effect of the irrigants on the metabolic activity of these microorganisms. The mean diameters of the inhibition zones for the C. albicans cultures were 11.6 mm (17 percent EDTA), 5.5 mm (0.5 percent NaOCl), 12.9 mm (1 percent NaOCl), 22.1 mm (2.5 percent NaOCl), and 28.5 mm (5.25 percent NaOCl). The mean diameters of the inhibition zones for E. faecalis were 2.8 mm (1 percent NaOCl), 5.4 mm (2.5 percent NaOCl), and 8.3 mm (5.25 percent NaOCl). For S. aureus, the mean values were 8.0 mm (17 percent EDTA), 3.0 mm (1 percent NaOCl), 8.8 mm (2.5 percent NaOCl), and 10.0 mm (5.25 percent NaOCl). Most of the irrigant solutions presented effective antimicrobial activity against C. albicans. A high inhibitory effect on the metabolic activity of E. faecalis was detected when the microorganisms were incubated with 17 percent EDTA. The same result was reached when S. aureus was incubated in the presence of > 2.5 percent NaOCl. Altogether, these results indicate that 2.5 percent and 5.25 percent NaOCl are microbicides against S. aureus while 0.5 percent and 1 percent NaOCl are only microbiostatic against the tested bacteria. The 6 percent and 10 percent citric acid as well as 17 percent EDTA did not affect the viability of any of the assayed microorganisms.

Atividade antifúngica da clorexidina em biofilmes de Candida spp / Antifungal activity of chlorhexidine on Candida spp. biofilm

Candidíase oral é a infecção oportunista mais comum em pacientes imunocomprometidos, sendo a clorexidina um importante antimicrobiano auxiliar no seu tratamento. O objetivo do presente estudo foi avaliar o efeito antifúngico de diferentes soluções de clorexidina (Periogard®, NoPlak Max®, Noplak®, Perioxidin®, Clorexidina 0.06%, Paradontax® e Clorexidina 1%) sobre biofilmes artificiais de Candida spp.: C. albicans (ATCC36801); C. parapsilosis (ATCC22019); C. krusei (ATCC6258); C. glabrata (ATCC2001) e C. tropicalis (ATCC750). As cepas foram cultivadas em meio de cultura BHI ágar sobre fragmentos de esmalte bovino por 72 horas a 37 °C. Após o crescimento, cada fragmento de esmalte bovino foi imerso nas diferentes soluções de Clorexidina por 3 minutos. Nistatina e solução salina foram utilizadas como controle negativo e positivo, respectivamente. Para remoção das células não aderidas, os fragmentos foram então imersos em solução salina por 10 minutos e agitados em vortex. Alíquotas de 100 µL foram inoculadas em placas contendo BHI ágar por 24 horas a 37 °C para contagem de unidades formadoras de colônias (UFC). Observamos que o número de UFC de C. albicans e C. parapsilosis, apresentou um percentual de redução variando de 79 a 99% quando do uso das diferentes soluções (p < 0,001), o mesmo não foi observado para o NoPlak Max® (2,94 e 1,3%, respectivamente); Para C. krusei e C. glabrata, a solução menos efetiva foi a Nistatina (23 e 3,4%, respectivamente) enquanto que para C. tropicalis, todas as soluções apresentaram um alto percentual de redução (99 a 100%). As soluções de clorexidina foram capazes de reduzir significativamente o número de UFC provenientes de biofilme de Candida spp. in vitro.

Oral candidiasis is the most common opportunistic infection in immunocompromised patients and chlorhexidine is an important antimicrobial for its treatment. The antifungal effect of different CHX solutions (Periogard®, NoPlak Max®, Noplak®, Perioxidin®, Chlorhexidine 0.06%, Paradontax® and Chlorhexidine 1%) was evaluated on artificial biofilms of Candida spps: C. albicans (ATCC36801), C. parapsilosis (ATCC22019), C. krusei (ATCC6258), C. glabrata (ATCC2001) and Candida tropicalis (ATCC750). The strains were grown, in a BHI agar medium on bovine teeth enamel for 72 hours at 37 °C. After growth, the fragments were immersed in the CHX solutions for 3 minutes. Nystatin and saline solutions were used as positive and negative controls respectively. To remove the non-adhered cells, the fragments were inoculated in saline solution for 10 minutes, transferred to Falcon tubes containing saline solution and mixed in a vortex. Aliquots of 100 µL were inoculated on BHI agar for 24 hours at 37 °C to count the number of colony forming units (CFU). We observed that the number of (CFU) of C. albicans and C. parapsilosis, showed a reduction rate ranging from 79 to 99% with the use of different solutions (p < 0.001), except for NoPlak Max® (2.94 and 1.3%, respectively). For C. krusei and C. glabrata, nystatin was the least effective solution (23 and 3.4%, respectively); and for C. tropicalis, all the substances presented a high reduction percentage (99-100%). The chlorhexidine solutions were able to reduce the colony forming units of Candida biofilm.

Avaliacao in vitro da microdureza de cimentos de ionomero de vidro modificados por resina submetidos a biofilme de Candida albicans / In vitro evaluation of microhardness of resin-modifi ed glass ionomer cements subjected to a candida albicans biofilm

Objetivo: Analisar in vitro a influencia da presenca de um biofilme de Candida albicans na microdureza superficial de duas marcas de cimentos de ionomero de vidro modificados por resina encontrados no mercado, o Vitremer© e o Vitro Fill© LC. Metodo: Nove amostras de cada material foram confeccionadas utilizando-se moldes de plasticos, previamente padronizados. Os nove especimes de cada marca foram divididos em tresgrupos: G1 solucao salina; G2 BHI liquido sem C. albicans; G3 suspensao celular com 105 de leveduras/mL em BHI liquido e a inducao da formacao de biofilme ocorreu apos incubacao das placas a 37§C por 48 horas. Os especimes foram esterilizados atraves de oxido de etileno. Foi avaliada a microdureza Knoop (50g, 15s). Os resultados foram submeti dos a Analise de Variancia (ANOVA) e teste de Tukey (5%). Resultados: Os grupos G1 e G2 obtiveram valores medios de dureza (KHN) maiores que G3 para ambos os materiais testados. Comparando os grupos G2 e G3, os especimes de G3 apresentaram menor resistencia as indentacoes. Conclusao: Nas condicoes do estudo in vitro, o biofilme de Candida albicans apresentou potencial de reduzir a dureza superficial de dois ionomeros de vidro modificados por resina encontrados no mercado (Vitremer© e Vitro Fill LC©).

Objective: To assess in vitro the influence of the presence of a Candida albicans biofilm on the surface microhardness of two commercial brands of resin-modified glass ionomer cements, Vitremer© and Vitro Fill© LC. Method: Nine samples of each material were fabricated using standardized plastic molds. The nine specimens of each brand were assigned to three groups: G1: saline; G2: BHI broth without C. albicans; G3: cell suspension with 105 yeasts/mL in BHI broth. Induction of biofilm formation occurred after incubation of the plates at 37§C for 48 hours and the specimens were sterilized with ethylene oxide. Knoop microhardness was measured (50 g, 15 s). Data were subjected to ANOVA and Tukey test at 5% significance level. Results: G1 and G2 presented higher mean hardness values (KHN) than G3 for both tested materials. Comparing G2 and G3, G3?s specimens presented lower resistance to theindentations. Conclusion: Under the conditions of this in vitro study, the Candida albicans biofilm reduced the surface hardness of two commercially available resin-modified glass ionomer cements (Vitremer© and Vitro Fill LC©).

Comparison of techniques to evaluate the quantification of Candida spp. in HIV-infected children.

This study sought to compare techniques used to make a quantified evaluation of Candida spp. in children infected with HIV. Twenty-four HIV-infected children (age 3 to 13) were selected. Three sterilized swabs were used for each child: one for the dorsum of the tongue, one for the hard palate mucosa, and one for the right jugal mucosa; each swab was rubbed for 10 seconds and transferred to sterilized test tubes containing 1 mL of 0.9% saline solution. Candida spp. growth was observed in 95.8% of all samples, including 95.7% of tongue samples (Group T), 87.0% of saliva samples, 56.6% of hard palate mucosa samples (Group P), and 47.8% of right jugal mucosa samples (Group J). There was no statistical difference in Candida spp. growth between saliva samples and Group T samples, although both had higher growth compared to Groups P and J (p < 0.05; chi(2)). Regarding the sensitivity of each site for positive Candida spp. growth, Group T samples showed 69.5%, while saliva samples showed 52.2%, Group P samples showed 21.7%, and Group J samples showed 13.04%, with no significant statistical difference between Group T and saliva; however, both were more sensitive than Groups J and P (p < 0.05, chi(2)). It was concluded that whole stimulated saliva and swabbing the tongue were considered satisfactory for measuring Candida spp. in HIV-infected children.

Biofilm formation by Candida species on silicone surfaces and latex pacifier nipples: an in vitro study.

The present study assessed the growth and development of biofilm formation by isolates of C. albicans, C. glabrata and C. parapsilosis on silicone and latex pacifier nipples. The silicone and latex surfaces were evaluated by scanning electronic microscopy (SEM). The plastic component of the nipple also seems to be an important factor regarding the biofilm formation by Candida spp. The biofilm growth was measured using the MTT reduction reaction. C. albicans was found to have a slightly greater capacity of forming biofilm compared to the other Candida species. Analysis of the pattern of biofilm development by C. albicans, C. glabrata and C. parapsilosis on latex and silicon pacifier shields showed an increased biofilm formation regarding the latter substrate. Silicone was shown to be more resistant to fungal colonization, particularly in the case of C. parapsilosis, despite the lack of any statistically significant differences (P > 0.05). In addition, silicone has a smoother surface compared to latex, whose surface was found to be rugose and irregular.

Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae.

Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite-host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS-PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc-chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae.

Effect of sphingosine and phorbol-12-myristate-13-acetate on the growth and dimethylsulfoxide-induced differentiation in the insect trypanosomatid Herpetomonas samuelpessoai.

We investigated the effect of two modulators of protein kinase C, sphingosine and phorbol-12-myristate-13-acetate (PMA), on the growth and dimethylsulfoxide (DMSO)-induced differentiation in Herpetomonas samuelpessoai. Sphingosine did not stimulate the transformation of undifferentiated-promastigotes in differentiated-paramastigotes. PMA alone or in association with DMSO increased the number of paramastigotes in comparison to control cells. DMSO inhibited the parasite growth (35%) and several unusual morphological features resembling aberrant cell division were observed. Sphingosine did not significantly reduce the growth in contrast to PMA. Collectively, our results demonstrated that the reduction of the proliferation translates in an increase of the differentiation rate in the insect trypanosomatid H. samuelpessoai.

Effect of sphingosine and phorbol-12-myristate-13-acetate on the growth and dimethylsulfoxide-induced differentiation in the insect trypanosomatid Herpetomonas samuelpessoai

We investigated the effect of two modulators of protein kinase C, sphingosine and phorbol-12-myristate-13-acetate (PMA), on the growth and dimethylsulfoxide (DMSO)-induced differentiation in Herpetomonas samuelpessoai. Sphingosine did not stimulate the transformation of undifferentiated-promastigotes in differentiated-paramastigotes. PMA alone or in association with DMSO increased the number of paramastigotes in comparison to control cells. DMSO inhibited the parasite growth (35 percent) and several unusual morphological features resembling aberrant cell division were observed. Sphingosine did not significantly reduce the growth in contrast to PMA. Collectively, our results demonstrated that the reduction of the proliferation translates in an increase of the differentiation rate in the insect trypanosomatid H. samuelpessoai.

Examining dentinal carious lesions as a predisposing factor for the oral prevalence of Candida spp in HIV-infected children.

PURPOSE: The aims of this study were to verify the oral prevalence of Candida spp in HIV-infected children, and investigate the association between Candida colonization and dentinal caries lesions. METHODS: Whole stimulated saliva was collected from 62 HIV-infected children (group 1) and 40 seronegative siblings (group 2), followed by oral examination to determine: dmft/dmfs scores, DMFT/DMFS scores, the number of dentinal carious teeth (D+) and the presence of oral candidiasis. The salivary samples were cultured, and plates with positive isolation (G+) were classified as mild growth, moderate growth, and strong growth. Data was analyzed using chi-square, Mann-Whitney, and Spearman tests for correlations. RESULTS: The patients' mean age was 8.8 for group 1 and 8.0 years for group 2. In group 1, 61% of the subjects had AIDS. Eighty percent of HIV-infected children (N=50) were positive for Candida growth, having a mean CD4% of 22, those who were Candida-free (N=12) presented a mean CD4% of 21. Correlation was observed between the mean D+ and G+ in groups 1 and 2 (P<.05, Mann-Whitney test), but not between the mean dmft/dmfs-DMFT/DMFS in group 1 (P>.05, Mann-Whitney test). Association of G+ and the D+ was noted in group 1 (P<.05; chi-square test). Positive correlation between high Candida counts and an increase in D+ was demonstrated in groups 1 and 2 (P<.05). CONCLUSIONS: Dentinal carious lesions may be associated with Candida spp colonization in HIV-infected children.

Candida guilliermondii isolated from HIV-infected human secretes a 50 kDa serine proteinase that cleaves a broad spectrum of proteinaceous substrates.

Non-albicans Candida species cause 35-65% of all candidemias in the general population, especially in immunosuppressed individuals. Here, we describe a case of a 19-year-old HIV-infected man with pneumonia due to a yeast-like organism. This clinical yeast isolate was identified as Candida guilliermondii through mycological tests. C. guilliermondii was cultivated in brain heart infusion medium for 48 h at 37 degrees C. After sequential centrifugation and concentration steps, the free-cell culture supernatant was obtained and extracellular proteolytic activity was assayed firstly using gelatin-SDS-PAGE. A 50 kDa proteolytic enzyme was detected with activity at physiological pH. This activity was completely blocked by 10 mM phenylmethylsulphonyl fluoride (PMSF), a serine proteinase inhibitor, suggesting that this extracellular proteinase belongs to the serine proteinase class. E-64, a strong cysteine proteinase inhibitor, and pepstatin A, a specific aspartic proteolytic inhibitor, did not interfere with the 50 kDa proteinase. Conversely, a zinc-metalloproteinase inhibitor (1,10-phenanthroline) restrained the proteinase activity released by C. guilliermondii by approximately 50%. Proteinases are a well-known class of enzymes that participate in a vast context of yeast-host interactions. In an effort to establish a functional implication for this extracellular serine-type enzyme, we investigated its capacity to hydrolyze some serum proteins and extracellular matrix components. We demonstrated that the 50 kDa exocellular serine proteinase cleaved human serum albumin, non-immune human immunoglobulin G, human fibronectin and human placental laminin, generating low molecular mass polypeptides. Collectively, these results showed for the first time the ability of an extracellular proteolytic enzyme other than aspartic-type proteinases in destroying a broad spectrum of relevant host proteins by a clinical species of non-albicans Candida.