Josep M. Torres-Rodríguez

First Case of Human Cryptococcosis Due to Cryptococcus neoformans var. gattii in Spain

ABSTRACT We report the first case of human cryptococcosis due to Cryptococcus neoformans var. gattii described in our country, which was presented as brain cryptococcoma in an immunocompetent patient. An extensive sampling of the patients environment was carried out to find the source of infection.

Aspergillus versicolor as cause of onychomycosis: report of 12 cases and susceptibility testing to antifungal drugs

Background Onychomycoses caused by opportunistic moulds are not well understood, and many are due to Scopulariopsis brevicaulis and other species. Aspergillus versicolor is not documented as an etiological agent in most studies. We have found an increasing prevalence of this species which is involved in 5.8% of all fungal infections of toe nails.

In Vitro Susceptibility of Sporothrix schenckii to Six Antifungal Agents Determined Using Three Different Methods

ABSTRACT The in vitro susceptibility of Sporothrix schenckii to antifungal drugs has been determined with three different methods. Nineteen Peruvian clinical isolates of S. schenckii were tested against amphotericin B (AB), flucytosine (FC), fluconazole (FZ), itraconazole (IZ), voriconazole (VZ), and ketoconazole (KZ). Modified NCCLS M38-A, Sensititre YeastOne (SYO), and ATB Fungus 2 (ATBF2) methods were used to determine the MICs. ATCC isolates of Candida parapsilosis, Candida krusei, and Aspergillus flavus were used for quality control. Sporothrix inocula were prepared with the mycelial form growing on potato dextrose agar at 28 ± 2°C. MICs of AB, FC, FZ, and IZ were determined with all three methods, VZ with M38-A and SYO, and KZ with only SYO. The three methods showed high MICs of FZ and FC (MIC90 of 0.5 μg/ml), being homogeneously lower than those of IZ and KZ. The M38-A method showed a variable MIC range of VZ (4.0 to 16 μg/ml); the geometric mean (GM) was 9.3 μg/ml. The MIC range of AB was wide (0.06 to 16 μg/ml), but the GM was 1.2 μg/ml, suggesting that the MIC is strain dependent. Agreement (two log2 dilutions) between commercial techniques and the modified M38-A method was very high with FZ, IZ, and FC. In AB and VZ, the agreement was lower, being related to the antifungal concentrations of each method. The highest activity against S. schenckii was found with IZ and KZ. Lack of activity was observed with FZ, VZ, and FC. When AB is indicated for sporotrichosis, the susceptibility of the strain must be analyzed. Commercial quantitative antifungal methods have a limited usefulness in S. schenckii.

Multicenter Evaluation of ATB Fungus: A Standardized Micromethod for Yeast Susceptibility Testing

The micromethod for yeast susceptibility testing, ATB Fungus, was evaluated with 30 reference strains in three laboratories. Ready-to-use strips with 5-fluorocytosine, amphotericin B, nystatin, miconazole, econazole and ketoconazole were used. The test allowed the categorization of each strain as susceptible, intermediate or resistance to all the antifungals tested, and 5-fluorocytosine and amphotericin B MIC determination. The results were compared with the MIC for each reference strain obtained by a microdilution method on RPMI 1640 buffered with MOPS. The repeatability and intralaboratory and interlaboratory reproducibility were evaluated. ATB Fungus was a reliable and reproducible method with a repeatability of 96.6%, a reproducibility of 95.4% and showed an excellent correlation 91.7%) with reference MICs.

Cryptococcus gattii: in vitro susceptibility to the new antifungal albaconazole versus fluconazole and voriconazole.

Minimal inhibitory concentrations (MIC) and minimal fungicidal activity of albaconazole, voriconazole and fluconazole against 55 strains of Cryptococcus gattii, clinically or environmentally isolated in Spain and some Latin American countries, were assessed. By means of the microbroth method (National Committee for Clinical Laboratory Standards; document M27-A2), the geometric mean value for fluconazole was 5.01 microg/ml; however, MIC for 12.7% of isolates ranged from 16 to 32 microg/ml, suggesting increased resistance against fluconazole. Geometric mean values of 0.02 and 0.03 microg/ml for albaconazole and voriconazole, respectively, were found, indicating not only a higher susceptibility to these new azoles but also a better performance of albaconazole (P = 0.003). Minimal fungicidal concentrations were also very low for albaconazole and voriconazole (P<0.001; geometric mean values of 0.023 microg/ml and 0.07 microg/ml, respectively). Both azoles may be good alternatives for the treatment of C. gattii cryptococcosis.

Prevalence of tinea pedis, tinea unguium of toenails and tinea capitis in school children from Barcelona

OBJECTIVE To evaluate the prevalence of tinea capitis, tinea pedis, and tinea unguium in children from several schools of Barcelona city. METHODS During the period of 2003-2004, a prospective cross-sectional study was carried out in 1,305 children (9% immigrant population) between the ages 3 and 15 in 17 schools in Barcelona. A systematic examination of the feet, (including nails and scalp), was performed to identify lesions compatible with tinea. Cultures of scalp and feet samples were done and analysis of environmental samples was performed for dermatophyte isolation. RESULTS Dermatophytes were isolated in 2.9% of the samples with a prevalence of 2.5% in feet, 0.23% in scalp, and 0.15% in nails of the feet. The predominant etiologic agents in feet were Trichophyton mentagrophytes in 45.7% of the cases and Trichophyton rubrum in 31.4%. In the nails, T. rubrum and Trichophyton tonsurans were isolated, while T. mentagrophytes (2 cases) and Trichophyton violaceum (1 case) were identified in scalp samples. Forty-five per cent of dermatophytes were isolated from healthy feet, the majority of cases in children 13- 15-years-old (p < 0.05). Microsporum gypseum was the only agent identified in the environmental samples, and was also found in one of the cases of tinea pedis. CONCLUSION The results of this study demonstrate a low prevalence of tinea capitis and tinea unguium in school children of Barcelona. On the contrary, high prevalence of dermatophytes in feet was found. It highlights the high prevalence of healthy carriers of dermatophytes in feet.

Identification of the perfect state of Cryptococcus neoformans from 195 clinical isolates including 84 from AIDS patients.

Filobasidiella neoformans is the teleomorphic state ofCryptococcus neoformans and it is a heterothalic. The purpose of this study was to establish the proportions of each mating types (a, α) from among 195 strains ofC. neoformans isolated from clinical material. The culture medium used was sunflower agar. Cultures were incubated at 20–22 °C for 15 days and observed periodically for one month. Non-reactive strains were mated several times with different reactive strains. Under these conditions 96.8% of the strains were found to be reactors. Among both varieties ofC. neoformans, mating type α was found to have the highest frequency of 95% in the varietyneoformans and 84% in the varietygattii. These results showed a higher reactivity in comparison with other investigators. This difference could be due to the medium used or to repeated mating with different reactive tested strains.

Non-traumatic topical treatment of onychomycosis with urea associated with bifonazole

Summary. The need for a topical, non‐traumatic treatment for onychomycoses of different etiologies has led to a search for increased effectiveness by associating products which have different and complementary effects, as is the case of the keratoplastic and broad‐spectrum antifungal agents. Using a 40% urea and 1% bifonazole cream, 42 nails with fungal infections (28 patients) were treated until the infected area softened and could be removed, after which 1% bifonazole cream was applied. After a 6 to 12 month follow‐up, the treatment success rate has been 93.8% for fingernails and 88.5% for toenails. The cultures became negative within 3 weeks after starting therapy with urea and bifonazole. Tolerance was good and it was not necessary to stop treatment in any patient. The topical, combined use of urea and bifonazole is a good alternative in the treatment of onychomycoses of different etiologies, provided that strict compliance by the patient is ensured, and that the application instructions are followed correctly.

MICs and minimum fungicidal concentrations of posaconazole, voriconazole and fluconazole for Cryptococcus neoformans and Cryptococcus gattii

Sir, The major aetiological species of cryptococcosis is Cryptococcus neoformans, which is distributed especially in association with pigeon droppings, and the most common infection is in the CNS of immunocompromised patients. Cryptococcus gattii, previously considered a biovariety of C. neoformans, is the second agent of cryptococcosis; four basic serotypes have been described: A and D for C. neoformans and B and C for C. gattii. Although its geographical distribution is restricted, C. gattii is being reported in new areas and has produced epidemic outbreaks in humans and animals. Unlike C. neoformans, C. gattii can infect immunocompetent subjects. The majority of the isolates from both species are susceptible to azoles in vitro, although most reports do not discriminate between Cryptococcus species and serotypes. The main goal of this study was to determine the MICs and minimum fungicidal concentrations (MFCs) of the new antifungal drug posaconazole in comparison with those of voriconazole and fluconazole for C. neoformans and C. gattii isolates from various sources. A total of 80 isolates of Cryptococcus from the collection of the Research Unit on Infectious Diseases and Mycology (Barcelona, Spain) were studied. Seventy-five were isolated from the CSF of patients infected with HIV, and five isolates were cultured from environmental samples. Fifty strains were C. neoformans: 25 serotype A (variety grubii) and 25 serotype D (variety neoformans). The remaining 30 isolates were C. gattii strains: 25 serotype B and 5 serotype C. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used for quality control. Posaconazole was provided by Schering-Plough (Kenilworth, NJ, USA), and voriconazole and fluconazole were provided by Pfizer Pharmaceuticals (Groton, CT, USA). Stock solutions of azoles and microplates were prepared and processed, as described in CLSI (formerly NCCLS) document M27-A2. Yeast inocula were diluted to a final concentration of 0.5–2.5 10 cfu/mL. From optically clear wells, 10 mL was withdrawn and plated on Sabouraud dextrose agar for the determination of MFC. Plates were incubated at 358C for 72 h. MFC was defined as the lowest drug concentration that yielded less than three colonies, a killing activity of 99%. For statistical analysis, the Wilcoxon rank-sum test was used using SPSS 14.0 for Windows (SPSS Inc., Chicago, IL, USA), with significance being set at P , 0.05. Results of the study confirmed that MICs for the two quality control Candida isolates were within the limits described in the M27-A2 document. Table 1 shows MIC50 and MFC50 values, geometric means and ranges of the three azoles. Statistical analysis revealed no significant differences between MICs of posaconazole and voriconazole. However, highly significant differences were found between fluconazole MICs and those of the other two azoles (P , 0.001). The MICs for the two Cryptococcus species were compared; they were found to be significantly higher for C. gattii than for C. neoformans (P 1⁄4 0.007), especially the MICs of fluconazole. The MFCs of fluconazole and voriconazole were higher for C. gattii serotype B. In contrast, the MFCs of posaconazole were lower, only 2 mg/L for one C. neoformans serotype A isolate and one C. gattii serotype B isolate. The MFCs of voriconazole were higher: 1 mg/L for 4 C. neoformans isolates and 2 mg/L for 11 C. gattii serotype B isolates (44%). The highest MFCs were 2 mg/L for posaconazole, 4 mg/L for voriconazole and 16 mg/L for fluconazole. Several cases of C. neoformans isolates exhibiting marked reduction in susceptibility to fluconazole have been reported in AIDS patients. Diverse authors attributed the increase in resistance to the widespread use of maintenance therapy with fluconazole. Other studies have reported the opposite trend. The antifungal susceptibility of 70 Spanish C. neoformans clinical isolates did not change significantly between 1994–96 and 1997–2005. The fluconazole MIC50 values remained stable, and the authors concluded that the in vitro resistance to fluconazole decreased over the 11 years. Pfaller et al. examined a large series of strains from 100 medical institutions and reported an accumulative percentage of 99% for isolates inhibited by voriconazole or posaconazole: 99% of the isolates being susceptible at MIC 1 mg/L. Although the authors did not discriminate between C. neoformans and C. gattii, presumably the majority of isolates were C. neoformans. We have found a very low level of resistance of Cryptococcus species to azoles. The highest MICs were obtained for C. gattii serotype B; this species appeared to be less susceptible to the azoles than both serotypes (A and D) of C. neoformans (P 1⁄4 0.007 for MIC and P 1⁄4 0.020 for MFC). MFCs could be better predictors of clinical response to antifungal therapy; however, standard methods have not been developed. Most investigators follow the proposals of Espinel-Ingroff et al. Greater differences in MFCs than MICs were seen; 2 isolates of C. gattii had MFCs of fluconazole 16 mg/L and 13 isolates had MFCs of voriconazole 2 mg/L. Journal of Antimicrobial Chemotherapy (2008) 62, 205–210 doi:10.1093/jac/dkn132 Advance Access publication 29 March 2008

MICs and minimum fungicidal concentrations of amphotericin B, itraconazole, posaconazole and terbinafine in Sporothrix schenckii

The in vitro susceptibility of 62 isolates of Sporothrix schenckii in its mycelial form, from Latin-American countries (Peru, Venezuela, Brazil and Uruguay) and Spain, to amphotericin B (AB), itraconazole (IZ), posaconazole (PZ) and terbinafine (TB) was determined by measuring the MICs and minimum fungicidal concentrations (MFCs) using a standardized Clinical and Laboratory Standards Institute method. In general, TB was the most active drug, with the lowest geometric mean (GM) MIC and MFC values amongst isolates from the five countries tested. IZ and PZ showed almost the same activity against all strains tested, except for isolates from Uruguay where IZ gave the highest GM MIC (10.68 mg l(-1)). AB showed the widest MIC range (0.03-16.0 mg l(-1)); however, this drug was less active against 79 % of isolates (MICs above 1 mg l(-1)). MFCs were 5 to 20 times higher than the MICs, but the lowest GM MFC and range values were found for TB. IZ and PZ gave the highest GM MFC. MFC may be a better predictor of therapeutic response than MIC, especially in immunosuppressed patients, making the use of IZ and PZ an inappropriate treatment. There were some differences in susceptibility according to the geographical source of the isolates, with the MIC being lower for TB in Venezuelan strains (P=0.066) and the MFC higher for PZ in Peruvian strains (P=0.02). Thus, geographical origin may be important for appropriate treatment, and may relate to the identification of species of the S. schenckii complex.