In vitro susceptibility of Paracoccidioides brasiliensis yeast form to antifungal agents

A study was conducted to determine the susceptibility of P. brasiliensis yeast form to amphotericin B (A), ketoconazole (K), 5-fluorocytosine (5-FC) and rifampin (R). The three isolates tested produced minimal inhibitory concentrations (MICs) (mcg/ml) in the following range: A: 0.09-0.18; K: 0.001-0.007; 5-FC: 62.5-250 and R: 40-80. The minimal fungicidal concentrations (MFC) were several times higher than the corresponding MICs. Precise MFC for 5-FC were not obtained (> 500 mcg/ml). Combination of K plus A proved synergic, with the fractional inhibitory concentration (FIC) indices revealing synergy when the drugs were combined at the 1 to 1 and 1 to 5 MIC ratios. R (40 mcg/ml) appeared to antagonize K. These results indicate promise for the combined use of K plus A as a therapeutical regimen.

In vitro susceptibility to antifungal agents of clinical and environmental Cryptococcus neoformans isolated in Southern of Brazil

The purpose of the present study was to compare the susceptibility to four antifungal agents of 69 Cryptococcus neoformans strains isolated from AIDS patients with that of 13 C. neoformans strains isolated from the environment. Based on the NCCLS M27-A methodology the Minimal Inhibitory Concentrations (MICs) obtained for amphotericin B, itraconazole and ketoconazole were very similar for clinical and environmental isolates. Clinical isolates were less susceptible to fluconazole than environmental isolates. The significance of these findings and aspects concerning the importance, role and difficulties of C. neoformans susceptibility testing are also discussed.

In vitro susceptibility testing of dermatophytes isolated in Goiania, Brazil, against five antifungal agents by broth microdilution method

The antifungal activities of fluconazole, itraconazole, ketoconazole, terbinafine and griseofulvin were tested by broth microdilution technique, against 60 dermatophytes isolated from nail or skin specimens from Goiania city patients, Brazil. In this study, the microtiter plates were incubated at 28 ºC allowing a reading of the minimal inhibitory concentration (MIC) after four days of incubation for Trichophyton mentagrophytes and five days for T. rubrum and Microsporum canis. Most of the dermatophytes had uniform patterns of susceptibility to the antifungal agents tested. Low MIC values as 0.03 µg/mL were found for 33.3%, 31.6% and 15% of isolates for itraconazole, ketoconazole and terbinafine, respectively.

In vitro susceptibility to antifungal agents of environmental Cryptococcus spp. isolated in the city of Ribeirão Preto, São Paulo, Brazil

Infections by Cryptococcus strains other than C. neoformans have been detected in immunocompromised patients. Of these strains, three are considered human pathogens: C. albidus, C. laurenttii, and C. uniguttulatus. This study deals with the in vitro susceptibility of Cryptococcus to drugs such as amphotericin B, itraconazole, fluconazole, and 5-fluorocytosine. Environmental Cryptococcus isolates (50) distributed as follows: C. neoformans var. neoformans (16), C. albidus (17), C. laurentii (14), and C. uniguttulatus (3) were evaluated by the micro and macrodilution techniques, according to EUCAST and NCCLS recommendations, respectively. Considering both methodologies the respective minimal inhibitory concentrations (MIC) were 0.125 and 2 µg/ml for amphotericin B, 0.06 and 8 µg/ml for itraconazole, and 0.5 and more than 64 µg/ml for fluconazole and 5-fluorocytosine. Agreement percentages for the two methodologies were 100% for amphotericin B and fluconazole for all the strains tested. For itraconazole, the agreement percentage was 81.3% in the C. neoformans strain and 100% for all the others. All species had a agreement percentage of 94.1 to 100% when susceptibility to 5-fluorocytosine was tested. It is concluded that environmental isolates of C. neoformans var. neoformans, C. albidus, C. laurentii, and C. uniguttulatus may show high MICs against certain drugs, suggesting in vitro primary resistance to the antifungals tested.

Genotyping of the MTL loci and susceptibility to two antifungal agents of Candida glabrata clinical isolates

The opportunistic fungal pathogen Candida glabrata is the second most common isolate from bloodstream infections worldwide and is naturally less susceptible to the antifungal drug fluconazole than other Candida species. C. glabrata is a haploid yeast that contains three mating-type like loci (MTL), although no sexual cycle has been described. Strains containing both types of mating information at the MTL1 locus are found in clinical isolates, but it is thought that strains containing type a information are more common. Here we investigated if a particular combination of mating type information at each MTLlocus is more prevalent in clinical isolates from hospitalized patients in Mexico and if there is a correlation between mating information and resistance to fluconazole and 5-fluorocytosine. We found that while both types of information at MTL1 are equally represented in a collection of 64 clinical isolates, the vast majority of isolates contain a-type information at MTL2 and α-type at MTL3. We also found no correlation of the particular combination of mating type information at the three MTL loci and resistance to fluconazole.

Sporothrix schenckii COMPLEX: SUSCEPTIBILITIES TO COMBINED ANTIFUNGAL AGENTS AND CHARACTERIZATION OF ENZYMATIC PROFILES

SUMMARY Sporothrix schenckiiwas reclassified as a complex encompassing six cryptic species, which calls for the reassessment of clinical and epidemiological data of these new species. We evaluated the susceptibility of Sporothrix albicans (n = 1) , S. brasiliensis (n = 6) , S. globosa (n = 1), S. mexicana(n = 1) and S. schenckii(n = 36) to terbinafine (TRB) alone and in combination with itraconazole (ITZ), ketoconazole (KTZ), and voriconazole (VRZ) by a checkerboard microdilution method and determined the enzymatic profile of these species with the API-ZYM kit. Most interactions were additive (27.5%, 32.5% and 5%) or indifferent (70%, 50% and 52.5%) for TRB+KTZ, TRB+ITZ and TRB+VRZ, respectively. Antagonisms were observed in 42.5% of isolates for the TRB+VRZ combination. Based on enzymatic profiling, the Sporothrix schenckii strains were categorized into 14 biotypes. Leucine arylamidase (LA) activity was observed only for S. albicans and S. mexicana. The species S. globosaand S. mexicanawere the only species without β-glucosidase (GS) activity. Our results may contribute to a better understanding of virulence and resistance among species of the genus Sporothrixin further studies.

Comparison of in vitro activity of five antifungal agents against dermatophytes, using the agar dilution and broth microdilution methods

The purpose of this study was to compare the agar dilution and broth microdilution methods for determining the minimum inhibitory concentration (MIC) of fluconazole, itraconazole, ketoconazole, griseofulvin and terbinafine for 60 dermatophyte samples belonging to the species Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. The percentage agreement between the two methods, for all the isolates with < 2 dilutions that were tested was 91.6% for ketoconazole and griseofulvin, 88.3% for itraconazole, 81.6% for terbinafine and 73.3% for fluconazole. One hundred percent agreement was obtained for Trichophyton mentagrophytes isolates evaluated with ketoconazole and griseofulvin. Thus, until a reference method for testing the in vitro susceptibility of dermatophytes is standardized, the similarity of the results between the two methods means that the agar dilution method may be useful for susceptibility testing on these filamentous fungi.

Slime production and proteinase activity of Candida species isolated from blood samples and the comparison of these activities with minimum inhibitory concentration values of antifungal agents

Slime and proteinase activity of 54 strains consisting of 19 Candida parapsilosis and 35 C. albicans strains isolated from blood samples were investigated in this study. Ketoconazole, amphothericin B, and fluconazole susceptibility of Candida species were compared with slime production and proteinase activity of these species. For both Candida species, no correlation was detected between the slime activity and minimum inhibitory concentration (MIC) values of the three antifungal agents. For both Candida species no correlation was detected between the proteinase activity and the MIC values of amphothericin B, and fluconazole however, statistically significant difference, was determined between the proteinase activity and MIC values of ketoconazole (p = 0.007). Slime production was determined by using modified Christensen macrotube method and proteinase activity was measured by the method of Staib. Antifungal susceptibility was determined through the guidelines of National Committee for Laboratory Standards (NCCLS M27-A).

Environmental isolation, biochemical identification, and antifungal drug susceptibility of Cryptococcus species

Introduction The incidence of opportunistic fungal infections has increased in recent years and is considered an important public health problem. Among systemic and opportunistic mycoses, cryptococcosis is distinguished by its clinical importance due to the increased risk of infection in individuals infected by human immunodeficiency virus. Methods To determine the occurrence of pathogenic Cryptococcus in pigeon excrement in the City of Araraquara, samples were collected from nine environments, including state and municipal schools, abandoned buildings, parks, and a hospital. The isolates were identified using classical tests, and susceptibility testing for the antifungal drugs (fluconazole, itraconazole, voriconazole, and amphotericin B) independently was also performed. After collection, the excrement samples were plated on Niger agar and incubated at room temperature. Results A total of 87 bird dropping samples were collected, and 66.6% were positive for the genus Cryptococcus. The following species were identified: Cryptococcus neoformans (17.2%), Cryptococcus gattii (5.2%), Cryptococcus ater (3.5%), Cryptococcus laurentti (1.7%), and Cryptococcus luteolus (1.7%). A total of 70.7% of the isolates were not identified to the species level and are referred to as Cryptococcus spp. throughout the manuscript. Conclusions Although none of the isolates demonstrated resistance to antifungal drugs, the identification of infested areas, the proper control of birds, and the disinfection of these environments are essential for the epidemiological control of cryptococcosis.

Dermatophyte susceptibilities to antifungal azole agents tested in vitro by broth macro and microdilution methods

The in vitro susceptibility of dermatophytes to the azole antifungals itraconazole, fluconazole and ketoconazole was evaluated by broth macro and microdilution methods, according to recommendations of the CLSI, with some adaptations. Twenty nail and skin clinical isolates, four of Trichophyton mentagrophytes and 16 of T. rubrum were selected for the tests. Itraconazole minimal inhibitory concentrations (MIC) varied from < 0.03 to 0.25 µg/mL in the macrodilution and from < 0.03 to 0.5 µg/mL in the microdilution methods; for fluconazole, MICs were in the ranges of 0.5 to 64 µg/mL and 0.125 to 16 µg/mL by the macro and microdilution methods, respectively, and from < 0.03 to 0.5 µg/mL by both methods for ketoconazole. Levels of agreement between the two methods (± one dilution) were 70% for itraconazole, 45% for fluconazole and 85% for ketoconazole. It is concluded that the strains selected were inhibited by relatively low concentrations of the antifungals tested and that the two methodologies are in good agreement especially for itraconazole and ketoconazole.

Sporothrix schenckii associated with armadillo hunting in Southern Brazil: epidemiological and antifungal susceptibility profiles

INTRODUCTION: Sporotrichosis is the most common subcutaneous mycosis observed in Brazil and it is generally consequent to a little trauma caused by vegetal particles or spines which inoculate the fungi in the subcutaneous area. Although sporotrichosis had been frequently mentioned with armadillo hunting this form has not been widely reported in Brazil until now. In this study we report ten cases of sporotrichosis evolving the armadillo's hunting diagnosed in some towns located in the central and west regions of Rio Grande do Sul State. METHODS: The cases were established based on clinical and classic mycological laboratorial techniques. The susceptibility tests were conducted by microdilution technique according to M38-A2 CLSI documents. RESULTS: Ten cases of sporotrichosis associated with armadillo hunting detected in the State of Rio Grande do Sul were diagnosed by mycological methods. The susceptibility tests of Sporothrix schenckii isolates to antifungal agents itraconazole, ketoconazole and terbinafine showed that all the isolates were susceptible. CONCLUSIONS: The paper discusses some cultural aspects related to hunting of this wild animal as well as possible causes of this unexpected occurrence in southern Brazil.

Antifungal susceptibility and distribution of Candida spp. isolates from the University Hospital in the municipality of Dourados, State of Mato Grosso do Sul, Brazil

Introduction Hospital infections caused by Candida spp. are a leading cause of morbidity and mortality in hospitalized patients, particularly those that are critically ill or immunocompromised. In this study, the distribution of Candida species in isolates from the University Hospital of the Federal University at Grande Dourados and their in vitro susceptibility to antifungal drugs were analyzed. Methods Yeasts were phenotypically identified using classical methodologies. Antifungal susceptibility tests to amphotericin B and fluconazole were performed using the broth microdilution technique. Results A total of 50 Candida isolates were obtained from hospitalized patients during the study period. We analyzed yeast isolates from urine (n=31; 62%), blood (n=12; 24%), and tracheal secretions (n=7; 14%). The following Candida species were identified: C. tropicalis (n=21; 42%), C. albicans (n=18; 36%), C. glabrata (n=10; 20%), and C. krusei (n=1; 2%). Antifungal susceptibility tests demonstrated that C. albicans was susceptible to both antifungal agents. However, 31.2% of the non-C. albicans Candida isolates displayed dose-dependent susceptibility to fluconazole, and 3.1% were resistant to amphotericin B. Conclusions In contrast to previous reports, our results indicated that C. tropicalis was the most commonly isolated yeast species among the hospital patients. The predominance of non-C. albicans Candida infections confirms the importance of species-level identification for implementing appropriate antifungal therapies.

Candidemia in a Brazilian tertiary care hospital: species distribution and antifungal susceptibility patterns

Recent studies have shown differences in the epidemiology of invasive infections caused by Candida species worldwide. In the period comprising August 2002 to August 2003, we performed a study in Santa Casa Complexo Hospitalar, Brazil, to determine Candida species distribution associated with candidemia and their antifungal susceptibility profiles to amphotericin B, fluconazole and itraconazole. Antifungal susceptibility was tested according to the broth microdilution method described in the NCCLS (M27A-2 method). Only one sample from each patient was analyzed (the first isolate). Most of the episodes had been caused by species other than C. albicans (51.6%), including C. parapsilosis (25.8%), C. tropicalis (13.3%), C. glabrata (3.3%), C. krusei (1.7%), and others (7.5%). Dose-dependent susceptibility to itraconazole was observed in 14.2% of strains, and dose-dependent susceptibility to fluconazole was found in 1.6%. Antifungal resistance was not found, probably related to low use of fluconazole. Further epidemiological surveillance is needed.

Antifungal susceptibilities of clinical and environmental isolates of Cryptococcus neoformans in goiânia city, Goiás, Brasil

We evaluated the antifungal activities of amphotericin B, fluconazole, itraconazole and voriconazole in 70 Cryptococcus neoformans strains obtained from cerebrospinal fluid from AIDS patients and 40 C. neoformans strains isolated from the environment. Four clinical isolates were identified as C. neoformans var. gattii. The susceptibility test was done using a broth microdilution method according to NCCLS M27-A2. Range minimal inhibitory concentrations (MICs) for C. neoformans clinical isolates were 0.06-1.0 µg/mL for amphotericin B, 0.125-8 µg/mL for fluconazole, 0.03-0.5 µg/mL for itraconazole and 0.03-0.25 µg/mL for voriconazole. C. neoformans environmental isolates showed range MICs 0.015-0.125 µg/mL, 0.25-2.0 µg/mL, 0.007-0.125 µg/mL and 0.03-0.25 µg/mL for amphotericin B, fluconazole, itraconazole and voriconazole respectively. The MICs results obtained from clinical and environmental isolates showed similar pattern of susceptibility and no resistance has been found in our isolates.

SUSCEPTIBILITY OF Candida spp. ISOLATED FROM BLOOD CULTURES AS EVALUATED USING THE M27-A3 AND NEW M27-S4 APPROVED BREAKPOINTS

The high mortality rates associated with candidemia episodes and the emergence of resistance to antifungal agents necessitate the monitoring of the susceptibility of fungal isolates to antifungal treatments. The new, recently approved, species-specific clinical breakpoints (SS-CBPs)(M27-S4) for evaluating susceptibility require careful interpretation and comparison with the former proposals made using the M27-A3 breakpoints, both from CLSI. This study evaluated the susceptibility of the different species of Candida that were isolated from candidemias based on these two clinical breakpoints. Four hundred and twenty-two isolates were identified and, among them, C. parapsilosis comprised 46.68%, followed by C. albicans (35.78%), C. tropicalis (9.71%), C. glabrata (3.55%), C. lusitaniae (1.65%), C. guilliermondii (1.65%) and C. krusei (0.94%). In accordance with the M27-A3 criteria, 33 (7.81%) non-susceptible isolates were identified, of which 16 (3.79%) were resistant to antifungal agents. According to SS-CBPs, 80 (18.95%) isolates were non-susceptible, and 10 (2.36%) of these were drug resistant. When the total number of non-susceptible isolates was considered, the new SS-CBPs detected 2.4 times the number of isolates that were detected using the M27-A3 interpretative criteria. In conclusion, the detection of an elevated number of non-susceptible species has highlighted the relevance of evaluating susceptibility tests using new, species-specific clinical breakpoints (SS-CBPs), which could impact the profile of non-susceptible Candida spp. to antifungal agents that require continuous susceptibility monitoring.