Claudete Rodrigues Paula

A new fluorescent viability test for fungi cells

The fluorescein diacetate-ethidium bromide (FDA-EB) fluorescence method, primarily used to determine viability of mammalian cells, was applied to several fungi species. Living fungi cells produced fluorochromasia, i.e., an intracellular accumulation of fluorescein which could be easily visualized as a green color under the U.V. microscope. Dead cells showed a red bright color due to ethidium bromide penetration. The FDA-EB test can be successfuly employed to assay yeast and yeast like cells viability since a good correlation was observed between this assay and the colony count technique. The main advantages of FDA-EB test are its speed, high sensitivity and simplicity.

Cryptococcus neoformans shows a remarkable genotypic diversity in Brazil

ABSTRACT The genotypic diversity of Brazilian Cryptococcus neoformans strains was analyzed. The majority of the samples were αA (65%), followed by αB (17.5%), αD (9%), αAaD hybrids (5%), and αC (3.5%). A considerable genotypic diversity occurred within C. neoformans var. grubii, and a new amplified fragment length polymorphism genotype, 1B, was recognized.

Susceptibility profile of vaginal yeast isolates from Brazil.

Vaginal specimens for culture were obtained from two hundred and five immunocompetent, non-hospitalized patients selected among all women attending the Gynecology and Obstetric Ambulatory Clinic of the Universityof Espírito Santo, Brazil, during a 2-year period (From 1998 to 1999). Patients were checked for signs and symptoms of vulvovaginitis and previous use of topical and systemic antifungal drugs. Yeast isolates were identified by classical methods and the antifungal susceptibility profile was determined according to NCCLS microbroth assay.The prevalence of vaginal yeast isolates from asymptomatic women was 25%(30/121) and 60% (50/84) among patients with symptoms of vulvovaginitis.Candida albicans was the most frequently isolated species in both groups (46% and 90%, respectively), followed by C. glabrata (13% and 6%, respectively). All isolates were susceptible to amphotericin B. Only ten isolates had dose dependent susceptibility (DDS) or resistance to azoles; and seven of these were non-albicans species.Based on our results we suggest that species identification and antifungal susceptibility testing need not be routinely performed in immunocompetent women, and may be reasonable only for the minority of patients with complicated vulvovaginal candidiasis that fail to respond to therapy.

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.

Yeasts isolated from blood and catheter in children from a Public Hospital of São Paulo, Brazil

The great majority of nosocomial fungal infections, especially fungemias, are caused by yeasts, mostly of the genus Candida. In addition, such infections may be associated with intravascular catheters. In this study, 80 yeast strains were isolated from hospitalized children, being 59 from blood cultures and 21 from vascular catheter cultures. The prevalent species in both blood and catheter was C. parapsilosis (32.2% and 48.9%, respectively), followed by C. albicans (16.9% and 28.6%, respectively). Concerning enzyme production, 78.8% of the 80 isolates presented strong proteolytic activity but 78.8% showed no phospholipase activity. We also detected two prevalent “killer” biotypes: 511 and 888. Additionally, in five patients, it was possible to observe that the yeast species, “killer” biotype and proteolytic and phospholipase activity of blood and catheter were similar. In view of this, we suggest a transmission of nosocomial yeast infection from catheter to blood.

Postharvest and stored corn in Brazil: Mycoflora interaction, abiotic factors and mycotoxin occurrence

The mycoflora of 130 samples of postharvest and stored corn was analysed throughout one year. The sample originated from Riberirão Preto, State of São Paulo, Brazil. The influences of abiotic factors (moisture content, relative humidity, temperature, rainfall) and mycotoxin occurrence were also verified. The isolation of the fungi was performed with Potato Dextrose Agar. Fungi were identified by using standard techniques. The determination of mycotoxins (aflatoxins, ochratoxin A, sterigmatocystin, zearalenone, deoxynivalenol and fumonisin B1) was carried out by thin-layer chromatography. The Fusarium spp. was the most dominant fungi (83.8%) followed by Penicillium spp. (55.3%), Aspergillus spp. (40.7%) and 11 other filamentous fungi. All of these were isolated from grains with moisture contents of 12.3-17.8%, an average temperature of 18.4-24.1 degrees C, a relative humidity between 64.0 and 97.5% and rainfall of up to 337 mm. With regard to the number of colony forming units (cfu), Fusarium spp. was the main contaminant, varying from 545 x 10(3) to 1.5 x 10(3). The Simple linear correlation analysis showed significant positive correlation of the Fusarium genus with moisture content of grains, and a significant negative correlation in relation to the minimum and medium temperatures, rainfall and relative humidity. The linear correlation of the Penicillium and Aspergillus genera with the abiotic factors was not significant. In the samples analysed only one contained aflatoxin B1.

Environmental straits of Cryptococcus neoformans variety grubii in the city of Santos, SP, Brazil

This study involved a total of 116 samples, 79 taken from pigeon droppings and 37 of atmospheric air taken close to accumulations of excrement. Cryptococcus neoformans var. grubii was isolated from 11 (13.9%) of these samples. Other species of Cryptococcus were also isolated from these samples, such as C. albidus (12.6%) and C. laurentii (8.9%). C. neoformans was not isolated from the air samples, though C. albidus (5.4%) was. All the strains of C. neoformans were found to belong to the A serotype (C. neoformans var. grubii). In regard to the studies with the antifungal agents 5-fluorocytosine, fluconazole, itraconazole, amphotericin B and voriconazole, by means of the microdilution method (EUCAST), we point out that one sample demonstrated resistance to fluconazole, this being especially significant because this is an environmental strain.

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.

Treatment with probiotics in experimental oral colonization by Candida albicans in murine model (DBA/2)

The aim of this study is to evaluate the oral colonization by Candida albicans in experimental murine immunosuppressed DBA/2 and treatment with probiotic bacteria. To achieve these objectives, 152 DBA/2-immunosuppressed mice were orally inoculated with a suspension of C. albicans containing 10(8) viable yeast cells, the animals were treated with nystatin or with the probiotics (Lactobacillus acidophilus and Lactobacillus rhamnosus). Evaluations were performed by Candida count from oral mucosa swabbing. The oral mucosa colonization by C. albicans started at day 1 after inoculation, remained maximal from day 3 until day 7, and then decreased significantly. Probiotics reduced the C. albicans colonization significantly on the oral mucosa in comparison with the untreated animal group. In the group treated with L. rhamnosus, the reduction in yeast colonization was significantly higher compared with that of the group receiving nystatin. Immunosuppressed animal model DBA/2 is a relevant model for experimental Candida oral colonization, and the treatment with probiotics in this model may be an effective alternative to prevent it.