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Aline Raquel Voltan,1 Guillermo Quindós,2 Kaila P Medina Alarcón,3 Ana Marisa Fusco-Almeida,3 Maria José Soares Mendes-Giannini,3 Marlus Chorilli1 1Department of Drugs and Medicines, Faculty of Pharmaceutical Sciences, Univ. Estadual Paulista, Araraquara, Sao Paulo, Brazil; 2Immunology, Microbiology, and Parasitology Department, Facultad de Medicina y Odontología, Universidad del País Vasco, Bilbao, Spain; 3Department of Clinical Analysis, Faculdade de Ciências Farmacêuticas, Univ. Estadual Paulista, Araraquara, Sao Paulo, Brazil Abstract: Invasive mycoses are a major problem for immunocompromised individuals and patients in intensive care units. Morbidity and mortality rates of these infections are high because of late diagnosis and delayed treatment. Moreover, the number of available antifungal agents is low, and there are problems with toxicity and resistance. Alternatives for treating invasive fungal infections are necessary. Nanostructured systems could be excellent carriers for antifungal drugs, reducing toxicity and targeting their action. The use of nanostructured systems for antifungal therapy began in the 1990s, with the appearance of lipid formulations of amphotericin B. This review encompasses different antifungal drug delivery systems, such as liposomes, carriers based on solid lipids and nanostructure lipids, polymeric nanoparticles, dendrimers, and others. All these delivery systems have advantages and disadvantages. Main advantages are the improvement in the antifungal properties, such as bioavailability, reduction in toxicity, and target tissue, which facilitates innovative therapeutic techniques. Conversely, a major disadvantage is the high cost of production. In the near future, the use of nanosystems for drug delivery strategies can be used for delivering peptides, including mucoadhesive systems for the treatment of oral and vaginal candidiasis. Keywords: fungal diseases, antifungal agents, amphotericin B, azoles, nanoparticles, nanotech­nology

In vitro activity of caspofungin and vori conazole against 184 clinical isolates of Candida and other medically important yeasts in comparison with that of fluconazole, ketoconazole, itraconazole and amphotericin B was determined by using a disk diffusion method (Neo-Sensitabs) standardized according to the recommendations of the CLSI documents M44-A and M44–S1 (same medium: Mueller-Hinton plus methylene blue; inoculum and minimal inhibitory concentration/zone breakpoints). Seventy-two percent of clinical isolates were susceptible to caspofungin, 23.6% showed an intermediate susceptibility (most of them were Candida parapsilosis) and 4.3% were resistant (values for Candida spp. were 71.2, 23.8 and 5%, respectively). For voriconazole, 96.7% of clinical isolates were susceptible and 3.3% were resistant (Candida spp.: 96 and 3.8%, respectively). Both caspofungin and voriconazole showed high activity against a wide variety of clinically important yeasts. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Sensititre® is a colorimetric microdilution method for in vitro antifungal susceptibility testing based on the M27-A document (National Committee for Clinical Laboratory Standards) for yeasts. Difference between both methods is the presence of Alamar-blue® and RPMI 1640 (glucose 2%) as culture medium. Antifungal susceptibility to amphotericin B, fluconazole, itraconazole, ketoconazole and flucytosine, 100 opportunistic filamentous fungi ( Aspergillus spp., Fusarium spp., Scedosporium spp.) obtained from pathological samples was determined by the Sensititre method. Induction to conidium and sporangiospore formation at 35°C was used to get inoculum and plates were covered by 1 ml of saline and suspensions were made by gently probing by a sterile loop. Optical densities of the conidial suspensions were adjusted to 80–82% transmittance for Aspergillus spp. and 68–70% for the rest of strains tested. Final inoculum concentration size was 0.4 × 104–5 × 104 CFU ml−1. Readings were made at 72 h of incubation at 35°C; amphotericin B and itraconazole was active against Aspergillus fumigatus with CMI90 1 and 0.5 μg ml−1, respectively, opposite to Scedosporium prolificans and Scedosporium apiospermum. As it was expected, a CMI90 of 256 μg ml−1 for fluconazole and CMI90 for flucytosine amounting to 64 g ml−1 were obtained. Sensititre Yeast OneTM is a useful method and an alternative to reference methods to determine antifungal susceptibility of filamentous fungi for clinical laboratory routine. Correlation with microdilution results is studied. New triazole derivatives should be included as soon as their clinical use will be feasible. [ABSTRACT FROM AUTHOR]

Sensititre® is a colorimetric microdilution method for in vitro antifungal susceptibility testing based on the M27-A document (National Committee for Clinical Laboratory Standards) for yeasts. Difference between both methods is the presence of Alamar-blue® and RPMI 1640 (glucose 2%) as culture medium. Antifungal susceptibility to amphotericin B, fluconazole, itraconazole, ketoconazole and flucytosine, 100 opportunistic filamentous fungi ( Aspergillus spp., Fusarium spp., Scedosporium spp.) obtained from pathological samples was determined by the Sensititre method. Induction to conidium and sporangiospore formation at 35°C was used to get inoculum and plates were covered by 1 ml of saline and suspensions were made by gently probing by a sterile loop. Optical densities of the conidial suspensions were adjusted to 80–82% transmittance for Aspergillus spp. and 68–70% for the rest of strains tested. Final inoculum concentration size was 0.4 × 104–5 × 104 CFU ml−1. Readings were made at 72 h of incubation at 35°C; amphotericin B and itraconazole was active against Aspergillus fumigatus with CMI90 1 and 0.5 μg ml−1, respectively, opposite to Scedosporium prolificans and Scedosporium apiospermum. As it was expected, a CMI90 of 256 μg ml−1 for fluconazole and CMI90 for flucytosine amounting to 64 g ml−1 were obtained. Sensititre Yeast OneTM is a useful method and an alternative to reference methods to determine antifungal susceptibility of filamentous fungi for clinical laboratory routine. Correlation with microdilution results is studied. New triazole derivatives should be included as soon as their clinical use will be feasible. [ABSTRACT FROM AUTHOR]

BacticardTM Candida was compared with the germ tube test for the rapid, presumptive identification of Candida albicans. This test kit detects the enzymatic activities l-proline aminopeptidase and beta-galactosaminidase in yeast colonies grown on culture media. Candida albicans produces both enzymes whereas other yeasts produce only one or neither of the enzymes. We evaluated 536 isolates including eight genera and 33 species of medically important yeasts, including 228 C. albicans and 36 C. dubliniensis. Both tests did not discriminate between C. albicans and C. dubliniensis isolates. The sensitivity and specificity for the BacticardTM Candida test were 97.8 and 96.5%, respectively. BacticardTM Candida and germ tube tests detected 246 (93.2%), and 256 (97%) C. albicans plus C. dubliniensis isolates. There were eight false-positive results with BactiCardTM Candida kit and four false-positive results with the germ tube test. Positive and negative predictive values for BacticardTM Candida enzymatic test were 95.3 and 98.4%, respectively, while 97.4 and 98.1% for the germ tube test, its specificity being 98.1% and efficiency 97% (97.7% for germ tube) . We have observed slightly lower values of sensitivity and specificity than those reported by others using the BactiCardTM test kit. BacticardTM Candida provides a rapid and accurate alternative to the germ tube test for the presumptive identification of C. albicans. [ABSTRACT FROM AUTHOR]

Salivary secretory IgA reacts with a group of heat-shock mannoproteins preferentially expressed on Candida albicans yeast cells and germ tubes grown at 37°C. Since other environmental factors can also modulate the expression of those antigens, we have investigated the influence of the pH of the culture medium on the expression of the antigens reacting with human salivary IgA by C. albicans. By indirect immunofluorescence, yeast cells grown in Sabouraud glucose broth at 37°C showed a statistically significant increase in reactivity with salivary IgA (p < 0.0001) when compared with cells grown at 25°C at the 4 pH values studied (3.3, 5.9, 7.5, and 9.5), the highest reactivity and the major heat-shock effect being observed at pH 5.9. The decrease in reactivity with salivary IgA observed in C. albicans cells grown at pH values of 3.3 and 9.5 was confirmed by Western blotting. Salivary IgA reacted with polydispersed materials from the cell walls of molecular masses > 55 kDa, which were more expressed at neutral pH than at acidic or alkaline pH values. A similar reactivity was observed when the antigenic extracts were stained with an antiserum directed against oligosaccharides present in antigen 6 of C. albicans serotype A. The differences in reactivity presented by salivary IgA may be related to a decrease in the expression of polysaccharides present on the surfaces of the yeast cells of C. albicans grown at acidic or alkaline pH values. The low reactivity of salivary IgA with C. albicans cells grown at acidic pH values may help to explain the association between acidic saliva and the carriage of Candida in the oral cavity, as well as with oral candidiasis. [ABSTRACT FROM AUTHOR]

In this report we compare the activity of two new antifungal agents, voriconazole (UK-109,496) and LY303366 with the activities of other antifungals including fluconazole, itraconazole, 5-fluorocytosine (5FC) and amphotericin B against 219 oral Candida spp. isolates from HIV-infected patients. We used the broth microdilution method following the guidelines of the NCCLS. The in-vitro activity of voriconazole (UK-109,496) (MIC(90) 0.12 mg/L) and LY303366 (CMI(90) 0.25 mg/L) against clinical isolates of Candida spp. was excellent and comparable with that of amphotericin B (MIC(90) 0.5 mg/L), and better than those of fluconazole (MIC(90) > or = 64 mg/L), itraconazole (MIC(90) 4 mg/L) and 5FC (MIC(90) 1 mg/L). [ABSTRACT FROM AUTHOR]

The in-vitro susceptibilities of 120 clinical isolates of yeasts to liposomal nystatin were compared with those to amphotericin B lipid complex (ABLC), liposomal amphotericin B (LAB), amphotericin B cholesteryl sulphate (ABCD), amphotericin B desoxycholate, nystatin, fluconazole and itraconazole. Yeast isolates examined included strains of Candida albicans, Candida parapsilosis, Candida glabrata, Candida krusei, Candida guilliermondii, Candida tropicalis, Candida kefyr, Candida viswanathii, Candida famata, Candida rugosa, Rhodotorula rubra, Trichosporon spp., Cryptococcus laurentii and Cryptococcus neoformans. The mean MICs for all strains examined were: liposomal nystatin 0.96 mg/L; nystatin 0.54 mg/L; ABLC 0.65 mg/L; LAB 1.07 mg/L; ABCD 0.75 mg/L; amphotericin B 0.43 mg/L; fluconazole 5.53 mg/L; and itraconazole 0.33 mg/L. No significant differences were seen between the activity of liposomal nystatin and the polyene drugs or itraconazole, but liposomal nystatin was more active than fluconazole. MICs were lower than the reported blood concentrations following therapeutic doses of this drug, indicating the potential for a therapeutic use of liposomal nystatin in humans. These results indicate good activity in vitro against medically important yeasts, which compares favourably with the activities of other currently available antifungal drugs. Liposomal nystatin may have a role in the treatment of disseminated and systemic mycoses. [ABSTRACT FROM AUTHOR]

The Etest strip is a promising tool of broad application in clinical microbiology. The method provides MIC readings and is easier to perform than broth microdilution. We carried out a study to compare the MICs of fluconazole and itraconazole obtained by the Etest with those obtained by broth microdilution, performed according to the guidelines of the NCCLS document M27-A, with 402 clinical isolates (360 Candida albicans, 17 Candida tropicalis, nine Candida krusei, nine Candida glabrata and seven Candida parapsilosis) and seven control isolates. The agreement between MICs by the two methods (at +/- 2 dilutions) was 74.5% for fluconazole and 61.4% for itraconazole. These results suggest that further development is necessary to standardize the medium and incubation conditions before introduction of the Etest as a routine method in the clinical microbiology laboratory for fluconazole and itraconazole susceptibility testing. [ABSTRACT FROM AUTHOR]

Salivary secretory IgA (sIgA) has been shown to react with a group of heat shock mannoproteins preferentially expressed on yeast cells grown at 37°C. Since at this temperature C. albicans can induce germ tubes, we explored the role of germ tube induction on human salivary sIgA reactivity in both germinative and agerminative C. albicans strains, in an attempt to investigate whether the germ tube expressed the heat shock mannoproteins reactive with sIgA. The reactivity with sIgA of the agerminative strain, grown at 25 and 37°C for different times, was measured spectrofluorometrically and was fairly constant with time. Yeast cells grown at 37°C tended to be more reactive than those grown at 25°C. In contrast, when compared with the yeast cells of the germinative strain grown at 25°C, there was a statistically significant decrease in reactivity with sIgA during germ tube formation. Serum IgA and IgG did not show statistically significant changes in reactivity with C. albicans during germination, suggesting differences in reactivity with C. albicans cell wall antigens between mucosal and systemic humoral responses. Cell wall mannoproteins of molecular masses > 60 kDa were characterized by Western blotting as responsible for the decrease in sIgA reactivity observed in the germ tube, and the fall in sIgA reactivity was related to the release of cell wall mannoproteins into the culture medium. The release of these mannoproteins may be a mechanism whereby C. albicans avoids the action of sIgA, and it may play an important role in the host-parasite relationship in oral candidiasis. [ABSTRACT FROM AUTHOR]

CHROMagar Candida (CHROMagar, France) was evaluated as a medium for the presumptive identification and isolation of yeasts using 1,537 isolates of medically important yeasts, including 970 Candida albicans, 165 Candida parapsilosis, 131 Candida glabrata, 62 Candida guilliermondii, 35 Candida krusei, 32 Candida tropicalis, 31 Rhodotorula rubra, 23 Trichosporon spp. (17 Trichosporon beigelii), 17 Candida famata, 16 Candida pelliculosa, 10 Pichia etchelsii, 10 Saccharomyces cerevisiae, 8 Candida lusitaniae, 7 Cryptococcus spp., and 20 isolates of other Candida spp. After 48 h of incubation at 37°C, the sensitivity and specificity were, respectively, 99% and 100% for Candida albicans, 93.8% and 99.1 % for Candida tropicalis, and 100% and 100% for Candida krusei. In addition to colony color, other colony characteristics were important for identification of some species, such as rough colonies in Candida krusei isolates or the halo around the colonies of Candida tropicalis. A great variety of colors was observed among species other than Candida albicans, Candida tropicalis, and Candida krusei. For identification purposes, CHROMagar Candida medium has an accuracy similar to that of germ-tube tests and chlamydospore development tests for Candida albicans and to that of the ATB ID32C kit (API, bioMérieux, France) for Candida tropicalis and Candida krusei. [ABSTRACT FROM AUTHOR]

In vitro testing of the activity of 15 antibacterial agents against 522 clinical isolates of Aeromonas species demonstrated some species-associated trends. Amoxicillin plus clavulanic acid was effective against approximately 45% of Aeromonas caviae and Aeromonas hydrophila, but all Aeromonas sobria isolates were resistant. Aztreonam, piperacillin and mezlocillin were highly active against all the strains of Aeromonas tested. Ticarcillin was equally effective against Aeromonas caviae and Aeromonas hydrophila, but more than 50% of Aeromonas sobria isolates were resistant. The latter species was more susceptible to cephalosporins than Aeromonas hydrophila and Aeromonas caviae. Chloramphenicol, tetracycline and trimethoprim-sulfamethoxazole were extremely active against all three Aeromonas species, likewise ofloxacin and ciprofloxacin. Aztreonam, third-generation cephalosporins, chloramphenicol and the quinolones can thus be considered for therapy of infections when Aeromonas is implicated. [ABSTRACT FROM AUTHOR]

To test the value of detection of anti- Candida albicans germ tube antibodies by indirect immunofluorescence assay in the diagnosis of systemic candidosis, a retrospective study was done using 126 sera from 27 patients with presumptive systemic candidosis (13 immunocompromised), 165 sera from 45 patients with aspergillosis (29 immunocompromised), 35 sera from eight patients with cryptococcosis (6 immunocompromised), and 101 sera from 101 blood donors. While 21 of 27 patients with systemic candidosis (77.8%) had anti-germ tube antibodies, these antibodies were absent in all patients with cryptococcosis and in all blood donors. They were however detected in 5 of 45 patients with aspergillosis (11.1%). Ten of 13 (76.9%) immunocompromised patients with candidosis had anti-germ tube antibodies; similar results were obtained in immunocompetent patients with candidosis (78.6%). The specificity was 96.8%, indicating a high degree of discrimination was possible between systemic candidosis and other invasive mycoses in the patients studied. Anti-germ tube responses did not appear to be significantly reduced in immunocompromised patients. [ABSTRACT FROM AUTHOR]

Sera from 109 subjects were tested for the presence of tantiCandida albicans antibodies by an indirect immunofluorescence assay. Aliquots of the sera were adsorbed with heat-killed blastospores to remove the antibodies against the surface of the yeast-phase cell wall and tested for anti-germ tube cell wall antibodies. Unadsorbed sera stained the entire cell wall of yeast and germ-tubes. Immunoglobulin G (IgG) antibodies were found in all patients with systemic candidiasis and in 81.2% of patients with Candida albicans isolated from skin and mucous membranes. IgA and IgG were found in 67.4 and 57.1 %, respectively, of controls without evidence of candidiasis. After the adsorption only sera from patients with systemic candidiasis showed antibodies, predominantly IgA, against germ tube cell wall. Adsorption of the sera thus increased the specificity, efficiency, and positive and negative predictive values of the test. The test achieved the highest sensitivity in adsorbed sera for the combination of IgA and IgG. [ABSTRACT FROM AUTHOR]

Abstract Background Candida-associated denture stomatitis is a frequent infectious disease. Treatment of this oral condition is difficult because failures and recurrences are common. The aim of this study was to test the in vitro antifungal activity of pure constituents of essentials oils. Methods Eight terpenic derivatives (carvacrol, farnesol, geraniol, linalool, menthol, menthone, terpinen-4-ol, and α-terpineol), a phenylpropanoid (eugenol), a phenethyl alcohol (tyrosol) and fluconazole were evaluated against 38 Candida isolated from denture-wearers and 10 collection Candida strains by the CLSI M27-A3 broth microdilution method. Results Almost all the tested compounds showed antifungal activity with MIC ranges of 0.03-0.25% for eugenol and linalool, 0.03-0.12% for geraniol, 0.06-0.5% for menthol, α-terpineol and terpinen-4-ol, 0.03-0.5% for carvacrol, and 0.06-4% for menthone. These compounds, with the exception of farnesol, menthone and tyrosol, showed important in vitro activities against the fluconazole-resistant and susceptible-dose dependent Candida isolates. Conclusions Carvacrol, eugenol, geraniol, linalool and terpinen-4-ol were very active in vitro against oral Candida isolates. Their fungistatic and fungicidal activities might convert them into promising alternatives for the topic treatment of oral candidiasis and denture stomatitis.

Abstract Background Poor outcomes of invasive candidiasis (IC) are associated with the difficulty in establishing the microbiological diagnosis at an early stage. New scores and laboratory tests have been developed in order to make an early therapeutic intervention in an attempt to reduce the high mortality associated with invasive fungal infections. Candida albicans IFA IgG has been recently commercialized for germ tube antibody detection (CAGTA). This test provides a rapid and simple diagnosis of IC (84.4% sensitivity and 94.7% specificity). The aim of this study is to identify the patients who could be benefited by the use of CAGTA test in critical care setting. Methods A prospective, cohort, observational multicentre study was carried out in six medical/surgical Intensive care units (ICU) of tertiary-care Spanish hospitals. Candida albicans Germ Tube Antibody test was performed twice a week if predetermined risk factors were present, and serologically demonstrated candidiasis was considered if the testing serum dilution was ≥ 1:160 in at least one sample and no other microbiological evidence of invasive candidiasis was found. Results Fifty-three critically ill non-neutropenic patients (37.7% post surgery) were included. Twenty-two patients (41.5%) had CAGTA-positive results, none of them with positive blood culture for Candida. Neither corrected colonization index nor antifungal treatment had influence on CAGTA results. This finding could corroborate that the CAGTA may be an important biomarker to distinguish between colonization and infection in these patients. The presence of acute renal failure at the beginning of the study was more frequent in CAGTA-negative patients. Previous surgery was statistically more frequent in CAGTA-positive patients. Conclusions This study identified previous surgery as the principal clinical factor associated with CAGTA-positive results and emphasises the utility of this promising technique, which was not influenced by high Candida colonization or antifungal treatment. Our results suggest that detection of CAGTA may be important for the diagnosis of invasive candidiasis in surgical patients admitted in ICU.