Eriques Gonçalves Silva

Light parameters influence cell viability in antifungal photodynamic therapy in a fluence and rate fluence-dependent manner

The aim of this study was to investigate the influence of light parameters on yeast cells. It has been proposed for many years that photodynamic therapy (PDT) can inactivate microbial cells. A number of photosensitizer and light sources were reported in different light parameters and in a range of dye concentrations. However, much more knowledge concerning the importance of fluence, fluence rate and exposure time are required for a better understanding of the photodynamic efficiency. Suspensions (106 CFU/mL) of Candida albicans, Candida krusei, and Cryptococcus neoformans var. grubii were used. Two fluence rates, 100 and 300 mW/cm2 were compared at 3, 6, and 9 min of irradiation, resulting fluences from 18 to 162 J/cm2. The light source was a laser emitting at λ = 660 nm with output power adjusted at 30 and 90 mW. As photosensitizer, one hundred-μM methylene blue was used. Temperature was monitored to verify possible heat effect and reactive oxygen species (ROS) formation was evaluated. The same fluence in different fluence rates showed dissimilar levels of inactivation on yeast cells as well as in ROS formation. In addition, the increase of the fluence rate showed an improvement on cell photoinactivation. PDT was efficient against yeast cells (6 log reduction), and no significant temperature increase was observed. Fluence per se should not be used as an isolate parameter to compare photoinactivation effects on yeast cells. The higher fluence rate was more effective than the lower one. Furthermore, an adequate duration of light exposure cannot be discarded.

Biofilm formation on intrauterine devices in patients with recurrent vulvovaginal candidiasis.

A biofilm is a complex community of surface-associated cells enclosed in a polymer matrix. They attach to solid surfaces and their formation can be affected by growth conditions and co-infection with other pathogens. The presence of biofilm may protect the microorganisms from host defenses, as well as significantly reduce their susceptibility to antifungal agents. Pathogenic microbes can form biofilms on the inert surfaces of implanted devices such as catheters, prosthetic cardiac valves and intrauterine devices (IUDs). The present study was carried out to analyze the presence of biofilm on the surface of intrauterine devices in patients with recurrent vulvovaginal candidiasis, and to determine the susceptibility profile of the isolated yeasts to amphotericin B and fluconazole. Candida albicans was recovered from the IUDs and it was found to be susceptible to the antifungal agents when tested under planktonic growing conditions. These findings indicate the presence of the biofilm on the surface of the IUD as an important risk factor for recurrent vulvovaginal candidiasis.

Cryptococcus neoformans strains isolated from church towers in Rio de Janeiro City, RJ, Brazil

Cryptococcosis has been a significant cause of morbidity and mortality in patients with Aids. Many reservoirs of the agent Cryptococcus neoformans have been reported, but the ecology of this yeast must be elucidated in order to establish surveillance programs and to prevent infections. The objective of this study was to evaluate the presence of C. neoformans in Rio de Janeiro City, RJ, Brazil. Ten churches were selected for sampling and detection of the yeast collecting pigeon dropping, air samples from church towers and neighboring areas during one year. The data demonstrated that C. neoformans has been present in every church selected and was present in 37.8% of 219 pigeon dropping samples. As well as, the yeast was isolated from soil, insects, eggs, pigeon nests and feathers. Fifteen air samples (4.9%) were positive. The growth on C.G.B. medium showed that all strains belonged to C. neoformans var. neoformans, with 98.8% of the strains belonging to serotype A.

Genotyping by RAPD-PCR analyses of Malassezia furfur strains from pityriasis versicolor and seborrhoeic dermatitis patients.

Malassezia furfur is lypophilic yeast commonly associate with dermatological disorders. In the present work, we described the isolation of 47 M. furfur strains from three groups of patients: pityriasis versicolor (21 isolates), seborrhoeic dermatitis (15 isolates) and seborrhoeic dermatitis of the HIV positive patients (11 isolates). To investigate the identity of the strains at molecular level, DNA genomic of M. furfur strains were prepared and used to RAPD-PCR analyses. RAPD assay were carried out using two decamer primers and bands pattern generated were analyzed by an Unweighted Pair-Group Method (UPGMA). Dendrogram established a distinct differentiation between M. furfur isolates from pityriasis versicolor and seborrhoeic dermatitis patients with or without AIDS. We concluded that RAPD typing presented a high discriminatory power between strains studied in this work and can be applied in epidemiological investigation of skin disease causing by M. furfur.

A Multispecies Probiotic Reduces Oral Candida Colonization in Denture Wearers

PURPOSE The prevalence of Candida infections has been rising with an increasingly aging population and a larger population of immunocompromised individuals. The use of probiotics may be an alternative approach to antifungal agents in the prevention and treatment of oral candidiasis. This study aimed to evaluate the short-term effect of probiotics in reducing the infection level of oral Candida in candidiasis-asymptomatic elderly denture wearers. MATERIALS AND METHODS In a double-blind randomized study, 59 denture wearers harboring Candida spp. in the oral cavity with no clinical symptoms were allocated into two groups: probiotic and placebo. All patients were instructed to clean the denture daily. The probiotic group poured a capsule containing lyophilized Lactobacillus rhamnosus HS111, Lactobacillus acidophillus HS101, and Bifidobacterium bifidum daily on the palatal surface of the maxillary denture, whereas the placebo group was submitted to the same regimen using placebo capsules. Candida spp. infection levels were evaluated in palate mucosa samples obtained before and after a 5-week experimental period. RESULTS All patients harbored Candida in the palate mucosa at baseline. Fifty-five individuals completed the experimental period. The detection rate of Candida spp. was 92.0% in the placebo group after the experimental period, whereas it was reduced to 16.7% in the probiotic group. The reduction promoted by the probiotic regimen was independent of baseline characteristics such as Candida infection level and colonizing species, age of denture, and other variables. CONCLUSION The probiotic product was effective in reducing the colonization of the oral cavity with Candida in candidiasis-asymptomatic elderly denture wearers, suggesting that this multispecies probiotic could be used to prevent oral candidiasis. CLINICAL IMPLICATIONS Colonization of oral surfaces by Candida is considered a risk factor for invasive fungal infections. The use of a product with L. rhamnosus, L. acidophilus, and B. bifidum may represent an alternative treatment for reduction of Candida infections in elderly denture wearers.

Extracellular proteolytic activity and molecular analysis of Microsporum canis strains isolated from symptomatic and asymptomatic cats

Resumen Microsporum canis es el principal dermatofito en perros y gatos, siendo agente importante de zoonosis. La literatura informa la existencia de gatos que son portadores asintomaticos de M. canis, debido a la resistencia del portador y a la presencia de cepas con menor virulencia. Este estudio fue realizado para evaluar la actividad queratinolitica, elastinolitica y colagenolitica de M. canis y su relacion con gatos (con y sin sintomas) y para analizar molecularmente las cepas por el metodo de RFLP. Las cepas aisladas de gatos con dermatofitosis tenian actividad queratinasa y elastasa mayores que las cepas aisladas de los gatos asintomaticos (p

The irradiation parameters investigation of photodynamic therapy on yeast cells

It has been proposed that photodynamic therapy (PDT) can inactivate microbial cells. A range of photosensitizers and light sources were reported as well as different fluence parameters and dye concentrations. However, much more knowledge regarding to the role of fluences, irradiation time and irradiance are required for a better understanding of the photodynamic efficiency. The aims of this study were to investigate the role of light parameters on the photoinactivation of yeast cells, and compare cell survivors in different growing phases following PDT. To perform this study, a suspension (106cfu/mL) of Candida albicans ATCC-90028 was used in log and stationary-phase. Three irradiances 100mW/cm2, 200mW/cm2 and 300mW/cm2 were compared under 3min, 6min and 9min of irradiation, resulting in fluences of 18, 36, 54, 72,108 and 162J/cm2. The light source used was a laser emitting at 660nm with output power of 30, 60 and 90mW. As photosensitizer, 100μΜ methylene blue was used. PDT was efficient against yeast cells (6 log reduction) in log and stationary-phase. Neither photosensitizer nor light alone presented any reduction of cell viability. The increase of irradiance and time of irradiation showed a clearly improvement of cell photoinactivation. Interestingly, the same fluences in different irradiances presented dissimilar effects on cell viability. The irradiance and time of irradiation are important in PDT efficiency. Fluence per se is not the best parameter to compare photoinativation effects on yeast cells. The growing-phases presented the same susceptibility under C. albicans photoinactivation.

Photodynamic therapy can kill Cryptococcus neoformans in in vitro and in vivo models

Cryptococcosis is an infection caused by the encapsulated yeast Cryptococcus neoformans and the most afflicted sites are lung, skin and central nervous system. A range of studies had reported that photodynamic therapy (PDT) can inactivate yeast cells; however, the in vivo experimental models of cryptococcosis photoinactivation are not commonly reported. The aim of this study was to investigate the ability of methylene blue (MB) combined with a low-power red laser to inactivate Cryptococcus neoformans in in vitro and in vivo experimental models. To perform the in vitro study, suspension of Cryptococcus neoformans ATCC-90112 (106cfu/mL) was used. The light source was a laser (Photon Lase III, DMC, São Carlos, Brazil) emitting at λ660nm with output power of 90mW for 6 and 9min of irradiation, resulting fluences at 108 and 162J/cm². As photosensitizer, 100μM MB was used. For the in vivo study, 10 BALB/c mice had the left paw inoculated with C. neoformans ATCC-90112 (107cfu). Twenty-four hours after inoculation, PDT was performed using 150μM MB and 100mW red laser with fluence at 180J/cm2. PDT was efficient in vitro against C. neoformans in both parameters used: 3 log reduction with 108J/cm² and 6 log reduction with 162J/cm². In the in vivo experiment, PDT was also effective; however, its effect was less expressive than in the in vitro study (about 1 log reduction). In conclusion, PDT seems to be a helpful alternative to treat dermal cryptococcosis; however, more effective parameters must be found in in vivo studies.

Tissue Changes in Experimental Cryptococcosis in Immunocompetent and Immunodeficient Murine Model

Cryptococcosis is a subacute or chronic disease caused through the inhalation of infectious particles from the opportunistic yeast Cryptococcus neoformans spp. The objective of the present study was to evaluate cryptococcosis in a murine model Immunocompetent (BALB/c), as well as in a model with combined immunodeficiency (SCID) through histopathological analyzes of pulmonary and cerebral tissues. After intravenous inoculation with 3.0 × 105 viable yeast cells the animals were euthanized daily for evaluation. The study period was 15 days. There were no significant changes in lung tissue in immunocompetent murine model (BALB/c). While in brain tissue, it was observed: congested vessel, evolving when C. neoformans was visualized in the meningeal area, and a large area of ischemia, which evolved throughout the studied period culminating on the 15th day of inoculation with visualization of the yeast in the meningeal and parenchyma. In SCID model, twenty-four hours after inoculation were observed in the lung tissue, hemorrhagic areas and a discrete neutrophilic inflammatory infiltrate, presence of discrete congestion in the lung, diffuse hemorrhage, edema and intense quantity of yeast were observed on the wall of the capillary at 11 days after inoculation. In brain tissue discrete area necrosis liquefaction was observed, focal well as the presence of C. neoformans, interspersed with fragments of necrotic cells was observed. On day 11 after inoculation were large areas of liquefaction necrosis associated with the formation of cavities in the parenchyma and an intense quantify of the yeast. Histopathological examination is one of the techniques usually used in the definitive diagnosis of cryptococcosis.

Effect of GXM (Glucuronoxylomannan) on the Inflammatory Response in Lung Infection Caused by Cryptococcus neoformans (Serotype A) in Immunodeficient Murine Model (BALB/c-SCID)

GXM (glucuronoxylomannan) is the major component surrounding the capsule of Cryptococcus neoformans having multiple biological functions, one of them and most important, the reduction in production of inflammatory cytokines. They were evaluated in this study the correlation of the production GXM with TNF, IL-6, IL-10, as well, as survival in murine model (BALB-c) and severe combined immunodeficiency (SCID). The animals were infected intravenously with 0.1 ml of a suspension containing 3.0 × 106 model, BALB-c compared to the model. The high production GXM as well as the induction of viable cells of C. neoformans. There was an increase in the production of GXM, as well as a decrease in survival in (SCID) a severe inflammatory response in this model may be due to a compromised immune system.