Acácio Gonçalves Rodrigues

A validated 1H NMR method for the determination of the degree of deacetylation of chitosan

A method for the determination of the degree of deacetylation (DDA) of chitosan by 1H NMR spectroscopy has been formally validated. Chitosans with DDA ranging from 48 to 100% have been used for the validation. The method is found to be simple, rapid and more precise than other known techniques like IR or titration for %DDA measurements. The precision, ruggedness, robustness, specificity, stability and accuracy of the technique are discussed in this paper.

Facts and myths on recurrent vulvovaginal candidosis-a review on epidemiology, clinical manifestations, diagnosis, pathogenesis and therapy.

Approximately three-quarters of all women will experience an episode of vulvovaginal candidosis at least once in their life and 5-10% of them will have more than one attack. Women suffering from three to four attacks within 12 months will be diagnosed with recurrent vulvovaginal candidosis (RVVC). This review covers the large number of proposed aetiological factors for RVVC. The diagnosis of the condition made by conventional means by health providers is often false and is also often misdiagnosed by the affected woman herself. The review covers various methods of diagnosing RVVC and the current knowledge on potential pathogenetic mechanisms proposed for genital candida infections. Treatment of RVVC, including local and systemic antimicrobial therapy and behaviour modification to decrease the risk of recurrences, are discussed. Recent knowledge on drug resistance in candida is also included.

New Microsatellite Multiplex PCR for Candida albicans Strain Typing Reveals Microevolutionary Changes

ABSTRACT Five new microsatellite loci were described and characterized for use as molecular markers for the identification and genetic differentiation of Candida albicans strains. Following the typing of 72 unrelated clinical isolates, the analysis revealed that they were all polymorphic, presenting from 5 to 30 alleles and 8 to 46 different genotypes. The discriminatory power obtained by combining the information generated by three microsatellites used in a multiplex PCR amplification strategy was 0.99, the highest ever reported. The multiplex PCR was later used to test a total of 114 C. albicans strains, including multiple isolates from the same patient collected from different body locations and along episodes of vulvovaginal infections. Three different scenarios for strain relatedness were identified: (i) different isolates that were revealed to be the same strain, (ii) isolates that were the same strain but that apparently underwent a process of microevolution, and (iii) isolates that corresponded to different strains. Analysis of the microevolutionary changes between isolates from recurrent infections indicated that the genotype alterations observed could be the result of events that lead to the loss of heterozygosity (LOH). In one case of recurrent infection, LOH was observed at the CAI locus, and this could have been related to exposure to fluconazole, since such strains were exposed to this antifungal during treatment. The analysis of microsatellites by a multiplex PCR strategy was found to be a highly efficient tool for the rapid and accurate differentiation of C. albicans strains and adequate for the identification of fine microevolutionary events that could be related to strain microevolution in response to environmental stress conditions.

Prevalence, Distribution, and Antifungal Susceptibility Profiles of Candida parapsilosis, C. orthopsilosis, and C. metapsilosis in a Tertiary Care Hospital

ABSTRACT Candida parapsilosis, an emergent agent of nosocomial infections, was previously made up of a complex of three genetically distinct groups (groups I, II, and III). Recently, the C. parapsilosis groups have been renamed as distinct species: C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis. In Portugal, no data pertaining to the distribution and antifungal susceptibility of these Candida species are yet available. In the present report, we describe the incidence and distribution of C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis among 175 clinical and environmental isolates previously identified by conventional methods as C. parapsilosis. We also evaluated the in vitro susceptibilities of the isolates to fluconazole, voriconazole, posaconazole, amphotericin B, and two echinocandins, caspofungin and anidulafungin. Of the 175 isolates tested, 160 (91.4%) were identified as C. parapsilosis sensu stricto, 4 (2.3%) were identified as C. orthopsilosis, and 5 (2.9%) were identified as C. metapsilosis. Six isolates corresponded to species other than the C. parapsilosis group. Interestingly, all isolates from blood cultures corresponded to C. parapsilosis sensu stricto. Evaluation of the antifungal susceptibility profile showed that only nine (5.6%) C. parapsilosis sensu stricto strains were susceptible-dose dependent or resistant to fluconazole, and a single strain displayed a multiazole-resistant phenotype; two (1.3%) C. parapsilosis sensu stricto strains were amphotericin B resistant. All C. orthopsilosis and C. metapsilosis isolates were susceptible to azoles and amphotericin B. A high number of strains were nonsusceptible to the echinocandins (caspofungin and anidulafungin).

Variability of Germinative Potential among Pathogenic Species of Aspergillus

ABSTRACT The objective of our study was to evaluate parameters influencing the germination of Aspergillus conidia. Inoculum concentration and age significantly influenced germination. Different incubation temperatures revealed significant differences among Aspergillus species. The internal human milieu provides the ideal conditions for the development of invasive disease by Aspergillus fumigatus but restricts invasion by Aspergillus flavus and Aspergillus niger.

Antifungal activity of ibuprofen alone and in combination with fluconazole against Candida species.

Ibuprofen, a non-steroidal anti-inflammatory drug, exhibited antimicrobial activity against Candida albicans and non-albicans strains. At 10 mg/ml, ibuprofen showed a rapid cidal activity against exponential growth phase C. albicans, accompanied by rapid and extensive leakage of intracellular K+, permeation to propidium iodide, lysis of spheroplasts and severe membrane ultrastructural alterations. These results indicate that the killing of Candida cells is due to direct damage to the cytoplasmic membrane. At 5 mg/ml, ibuprofen inhibited growth; however, it did not kill the yeasts and did not directly affect the cytoplasmic membrane. Evaluation of yeast metabolic vitality with the fluorescent probe FUN-1 showed that growth inhibition induced by the fungistatic drug concentration was due to metabolic alterations. The combination of ibuprofen with fluconazole resulted in synergic activity with eight of the 12 Candida strains studied, including four of the five fluconazole-resistant strains. The MICs of fluconazole for the fluconazole-resistant strains decreased 2-128-fold when the drug was associated with ibuprofen. When in combination with fluconazole, MICs for ibuprofen decreased by up to 64-fold for all the 12 strains studied. These results point to the practicability of using ibuprofen, alone or in combination with azoles, in the treatment of candidosis, particularly when applied topically, taking advantage of the drugs antifungal and anti-inflammatory properties.

Antifungal activity of local anesthetics against Candida species.

OBJECTIVE: To evaluate the activity of benzydamine, lidocaine, and bupivacaine, three drugs with local anesthetic activity, against Candida albicans and non-albicans strains and to clarify their mechanism of activity. METHODS: The minimal inhibitory concentration (MIC) was determined for 20 Candida strains (18 clinical isolates and two American Type Culture Collection strains). The fungistatic activity was studied with the fluorescent probe FUN-1 and observation under epifluorescence microscopy and flow cytometry. The fungicidal activity of the three drugs was assayed by viability counts. Membrane alterations induced in the yeast cells were evaluated by staining with propidium iodide, by quantitation of intracellular K+ leakage and by transmission electron microscopy of intact yeast cells and prepared spheroplasts. RESULTS: The MIC ranged from 12.5-50.0 microg/mL, 5.0-40.0 mg/mL, and 2.5-10.0 mg/mL for benzydamine, lidocaine, and bupivacaine, respectively. The inhibitory activity of these concentrations could be detected with the fluorescent probe FUN-1 after incubation for 60 minutes. A very fast fungicidal activity was shown by 0.2, 50, and 30 mg/mL of benzydamine, lidocaine, and bupivacaine, respectively. CONCLUSIONS: At lower concentrations, the tested drugs have a fungistatic activity, due to yeast metabolic impairment, while at higher concentrations they are fungicidal, due to direct damage to the cytoplasmic membrane.

Anti-Candida activity of essential oils.

Anti-Candida activity of essential oils has been widely studied and as a consequence they are being investigated as possible alternatives or complementary therapeutic agents for candidosis. We reviewed the most studied essential oils concerning chemical composition and in vitro/in vivo studies under the perspective of their possible clinical use.

Comparison of Two Probes for Testing Susceptibilities of Pathogenic Yeasts to Voriconazole, Itraconazole, and Caspofungin by Flow Cytometry

ABSTRACT A cytometric approach to determine the susceptibilities of Candida spp. and Cryptococcus neoformans to voriconazole, itraconazole, and caspofungin is described. A total of 63 clinical isolates with different susceptibility patterns were exposed for 1, 2, 4, and 6 h to serial concentrations of each antifungal agent, followed by staining with two fluorescent probes: propidium iodide (PI) and FUN-1. FUN-1 was able to identify the susceptibility patterns of the assayed strains to the three agents after 1 h. PI penetrated a maximum of 50% of the cells treated with PI, at the highest concentration of caspofungin, 16 μg/ml, after 6 h of incubation (this percentage varied with the strain and was drug concentration and time of incubation dependent) and did not stain cells treated with high concentrations of either azole after 6 h. The use of FUN-1 appears to be an excellent fast and reliable alternative to the classical dilution method for determining the susceptibility of Candida spp. and C. neoformans to these three antifungal agents.

Adhesion, biofilm formation, cell surface hydrophobicity, and antifungal planktonic susceptibility: relationship among Candida spp.

We have performed the characterization of the adhesion profile, biofilm formation, cell surface hydrophobicity (CSH) and antifungal susceptibility of 184 Candida clinical isolates obtained from different human reservoirs. Adhesion was quantified using a flow cytometric assay and biofilm formation was evaluated using two methodologies: XTT and crystal violet assay. CSH was quantified with the microbial adhesion to hydrocarbons test while planktonic susceptibility was assessed accordingly the CLSI protocol for yeast M27-A3 S4. Yeast cells of non-albicans species exhibit increased ability to adhere and form biofilm. However, the correlation between adhesion and biofilm formation varied according to species and also with the methodology used for biofilm assessment. No association was found between strains site of isolation or planktonic antifungal susceptibility and adhesion or biofilm formation. Finally CSH seemed to be a good predictor for biofilm formation but not for adhesion. Despite the marked variability registered intra and inter species, C. tropicalis and C. parapsilosis were the species exhibiting high adhesion profile. C. tropicalis, C. guilliermondii, and C. krusei revealed higher biofilm formation values in terms of biomass. C. parapsilosis was the species with lower biofilm metabolic activity.