Eidi Alvarado-Ramírez

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.

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.

Diferencias en la actividad de la enzima ureasa entre Cryptococcus neoformans y Cryptococcus gattii

Se considera que la capacidad de Cryptococcus neoformans para sintetizar ureasa es uno de sus principales factores de virulencia. Desde 2002 se acepta que la variedad gattii de C. neoformans reune los atributos necesarios para considerarse como una nueva especie, Cryptococcus gattii. No existen estudios experimentales controlados que comparen el grado de actividad ureasa de ambas especies. El objetivo de este trabajo ha sido analizar la produccion de esta enzima en aislamientos de C. neoformans y C. gattii, considerando la distribucion en serotipos de los aislamientos y su origen (clinico o ambiental), utilizando un nuevo metodo semicuantitativo estandarizado. Veinticinco aislamientos de C. neoformans y 19 de C. gattii fueron sembrados en medio liquido de urea de Christensen para determinar la produccion de ureasa por espectrofotometria. Como referencia se determino la actividad de una unidad de ureasa (jack beans urease®, Sigma) sobre el mismo medio de cultivo y se observo que el 50% de la actividad enzimatica correspondia a una densidad optica de A550 = 0,215. Esta absorbancia permitio clasificar la actividad ureasa para cada aislamiento de Cryptococcus. Los resultados mostraron que bajo las mismas condiciones, estas levaduras podian agruparse en dos categorias, bajas productoras de ureasa (A550 < 0,215) y altas productoras (A550 igual o superior a 0,215). A las 72 horas de incubacion, el 76% de los aislamientos de C. neoformans y el 15,8% de los de C. gattii resultaron altos productores de ureasa. El analisis estadistico mostro diferencias significativas entre ambas especies (p = 0,016). Los resultados obtenidos muestran que C. neoformans es mayor productor de ureasa que C. gattii.

Laccase activity in Cryptococcus gattii strains isolated from goats.

Cryptococcosis is a life-threatening infection in humans and animals caused by encapsulated yeasts of the genus Cryptococcus. Cryptococcus neoformans and Cryptococcus gattii are the main agents of this mycosis. Until 2002 C. gattii was classified as a variety of C. neoformans but now is accepted as an independent species. The laccase (phenoloxydase) enzyme produced by these yeasts is considered one of the main pathogenic factors for its ability to induce melanin from dihydroxyphenolic compounds. The vast majority of the studies in laccase and melanin synthesis have been developed using isolates of C. neoformans. The main objective of this study was to evaluate laccase activity in strains of C. gattii, serotype B isolated from immunocompetent goats that died of lung and disseminated cryptococcosis, in several outbreaks occurring in Spain. The laccase activities of these isolates were compared with those of other strains of C. gattii and C. neoformans. After fungal cell rupture, the supernatant of each isolate was analyzed for its laccase activity using as substrate an L-dopa 20 mM solution. The degree of enzymatic activity was assessed according to its absorbance at 450 nm and scored using Enzymatic Units (EU). The maximum values were observed in three strains of C. gattii from goats (EU > 12). The smallest values were observed in one environmental isolate of C. gattii serotype C (EU = 0.7). The highest recorded value for C. neoformans was 6.3 EU in a serotype A isolate from one human case of meningitis. C. gattii serotype B obtained from goats showed different degrees of laccase activity, being the highest in those isolated from severe outbreaks of cryptococcosis. This enzyme appears to represent a major, though nonexclusive, pathogenic factor for Cryptococcus gattii.

Müeller–Hinton methylene blue media as an alternative to RPMI 1640 for determining the susceptibility of Cryptococcus neoformans and Cryptococcus gattii to posaconazole with Etest

Mueller–Hinton modified agar (MH‐GMB) was compared with RPMI + 2% glucose–agar to determine the MICs of 80 isolates of Cryptococcus neoformans and C. gattii to posaconazole with Etest. MH‐GMB minimised trailing and agreement between both media was 94%. Agreement of M27‐A2 microbroth reference method was 98% with RPMI and 94% with MB‐GMB.